local translation and the role of the RNA binding protein

Posted comment on ´The function of RNA-binding proteins at the synapse: implications for neurodegeneration`  by C.F. Sephton and G.Yu and published in Cell. Mol. Life Sci. 2015 vol 72 page 3621 doi 10.1007/s00018-015-1943-x

SUMMARY

Sephton and Yu began their article by describing the important role of protein translation taking place locally in the synapse. This type of translation was explained as necessary to accommodate the changes in this area of the neuron brought about by the constantly changing micro-environment due to neuronal activation. In addition, localisation of mRNA in the synapse and protein synthesis has been shown to be critical for synaptic plasticity and memory formation. Control of nuclear translation can depend on different mechanisms such as upstream open reading frames, secondary structures or regulatory protein binding sites, but in their article, Sephton and Yu concentrate on the specificity of the translation of the mRNA by the RNA binding proteins present. This is the main group of proteins regulating mRNA transport and translation in this area and attention to this group has been brought about because of the assumed link between known genetic mutations (or gene deletions) of particular examples and certain neurodegenerative diseases. For example, sufferers of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) exhibit cognitive impairments and loss of motor neuron function and research has showed that there are strong associations between several RNA binding proteins eg. Fused in Sarcoma binding protein (FUS) and Transactive Response DNA binding protein (TDP-43) in both diseases resulting in dysregulation of synaptic function and initiation of abnormal neurodegeneration.

Local translation in neuronal cells is said to occur because of the distances between the soma (the cell body) and the dendrites which creates, what the authors  termed, a ´supply and demand challenge`  for proteins required in the dendrites for them to function, for example responding to neuronal firing and signal transmission. Therefore, local translation means that all requirements for the neurochemical process have to be located within the dendrites in addition to those in the vicinity of the cell nucleus. The components required include mRNA, ribosomes and translation factors. In the synapse, the mRNA is found to exist within granules. It is associated with the RNA binding proteins in the untranslated regions (5UTR or 3UTR) or coding regions to form messenger ribonucleoprotein complexes (mRNP) which can then assemble into more complex structures known as RNA (or RNP) granules. There are many different types and classification is based on composition, location, response to stimuli and function, eg. regulation of, distribution of, translation of and degradation of mRNA transcripts. The granules are constantly changing, forming different interactions and exchanging mRNPs, cytosolic proteins and polysomes, but research has shown that their presence is responsible for synaptic plasticity and communication in the functioning synapse.

Sephton and Yu continued their article with descriptions of particular examples of multi-mRNP complexes such as transport RNPs (tRNPs), stress granules or processing bodies. The transport tRNP granules contain mRNA and are said to be involved in the storage and transport of mRNA. They can also contain microRNA (miRNA). RNA binding proteins found associated with the tRNP type of granule are, for example Staufen 1 and 2, FUS and TDP-43. However, they are also said to contain at least 40 other proteins including motor transport proteins such as kinesin.  The transport of mRNA to the dendrite appears to occur with the mRNA in a translationally dormant state, but this can undergo alteration in certain circumstances. The authors describe the example of stress where mRNPs are exchanged within the tRNP with stress granules and processing bodies. The mRNAs are protected and once stress removed then the stress granules disassemble and mRNAs are repacked into translationally competent forms and their proteins are synthesised. Alternatively, they are selectively exported to another type of granule, the processing bodies which are responsible for degradation, translational repression and recycling.

The role of gene mutations of RNA binding proteins and the resulting neuropathological diseases were described by Sephton and Yu with reference to two mutations in particular, that of FUS mutations and TDP-43 mutations. The mutations of the specific RNA binding proteins in both cases resulted in negative changes in the cell`s mRNA population and protein translation causing adverse effects on synaptic structure and function and neurodegeneration. In both neuropathological diseases, no changes of the processing bodies were observed, but there were changes in tRNP and stress granule populations. FUS-mutations were found to cause an increase in number and size of the tRNP and stress granules. This was suggested as for example, resulting in more spontaneous assembly of tRNP granules that could have an impact on translation, or that it could make both tRNP and stress granules more insoluble. As a result pathological inclusions would be ´seeded` and this is reported as being the case in both ALS and FTD. Regarding TDP-43 disease mutations, larger, but fewer tRNP were observed and larger, but greater numbers of stress granules. Both were assumed to cause the same physiological effects as that seen with the FUS mutations.

In their article, Sephton and Yu then went on to describe how RNA binding proteins regulate local translation. RNA binding proteins are said to be a component of RNA granules and these transport mRNA to the dendrites in a translationally dormant state. This is supported by the presence of nucleic translation initiation factor 4AIII (eIF4AIII) with dendritic mRNA.  The mechanism by which translation is inhibited by the RNA granules was said not to be known since it has been shown that some granules contain translation components such as ribosomes and endoplasmic reticulum (ER). For example tRNP granules with the largest number of Staufen 1 and 2 pools contain ribosomes and ER and those containing the smaller number had kinesin, but no ribosomes and ER. It was assumed that this was the group of tRNP where translation was repressed. In this case, neural activity could therefore cause either release of mRNA from the tRNP granule to the polyribosomes where active translation would occur, or in some circumstances repression of translation would continue. The protein composition of the granule particularly the presence of RNA binding proteins appeared to dictate which path would be followed.  For example, ZBP1 associates with and transports beta-actin mRNA to synapses. Src phosphorylation causes dissociation between ZBP1 and mRNA, hence synthesis of beta-actin, which is required for both cell migration and neurite growth, is allowed. However, ZBP1 can also repress the joining of ribosomal subunits in the cytoplasm so translational initiation regulation can occur. Other examples described by the authors are the CPEB family of RNA-binding proteins where CPEB1 can act as both repressor and activator of translation and the binding protein, FMRP. FMRP mutations are associated with Fragile X syndrome and studies have suggested that FMRP plays a role in the linking between tRNP granules and polyribosomes. The phosphorylated form of FMRP is associated with stalled ribosomal translocation and the non-phosphorylated form is associated with actively translating ones. FMRP can also regulate transmission when in its phosphorylated form by an association with miRNAs eg. miR-125a and RNA-induced silencing complex (RISC) to repress synthesis of proteins such as PSD-95. Dephosphorylated FRMP can lead to the release of RISC from PSD-95 mRNA so translation can take place.

This link between RNA binding protein deficiency and neurodegenerative diseases was continued with Sephton and Yu looking at how the deficiencies are associated with changes in dendritic synaptic degradation and dendrite morphology, for example as seen in ALS and FTD sufferers.  For example Staufen-1 knockout mice are observed to have reduced numbers of dendrites and synapses in the hippocampus and have a defective dendritic delivery system of beta-actin tRNP granules. Sephton and Yu in their article looked at the cases of deficiencies of the FUS and TDP-43 binding proteins in particular.

FUS is a RNA binding protein responsible for the translation of proteins required for neuronal development and synaptic transmission. It is known to bind to thousands of cellular RNAs via two RNA recognition motifs (a zinc-finger domain and 3 arginine-glycine-glycine boxes). As it appears to exist in different ribonucleoprotein complexes it is thought to be involved in all types of translation processes eg. mRNA stability and miRNA biosynthesis. When the neuronal cell is in a steady state, the majority of FUS is localised in the nucleus of the cell where it exists bound with TDP-43 and SMN along the whole length of the nascent RNA. All three binding proteins in this case function in the maintenance of the spliceosome. It is also thought that FUS is involved in transcriptional elongation as it is closely associated with RNA polymerase II, binds to pre-RNA and mRNA at introns and coding sequences etc. Therefore, FUS mutated cells and FUS-null cells demonstrate dysregulation in mRNA and pre-mRNA splicing.

However, FUS can also be localised to cellular compartments other than the nucleus. It has been found in RNA granules and this localisation is facilitated by the non-classical proline-tyrosine nuclear localisation sequence (PY-NLS) and the nuclear export sequence (NES). The localisation results as a response to various stimuli and Sephton and Yu give in their article the example of hippocampal or cortical slices that had been stimulated by treatment with mGluR1/5 agonists. In the response to the agonist presence and hence, neuronal firing, FUS was found in the tRNP granules in the dendrites and also at the synapses where FUS was associated with NMDA receptors themselves. The role of FUS in the tRNP localised in the dendrites was described as being either repression or facilitation of the required translation and there appears to be examples of both.  FUS can also associate with cytoplasmic stress granules, but in this case, this only leads to translation repression.

As expected with a promotion or repression role in local translation, FUS is linked with neurodegeneration under some circumstances. The majority of familial ALS mutations are associated with the FUS encoding gene at its PY-NLS terminal which affects the localisation of the protein and its aggregation. For example, abnormal accumulation of FUS in the cytoplasm or nucleus of motor neurons in ALS patients is observed. The extent of the mutations appears to correlate to disease severity. Some mutations eg. those at the 3 prime end, can cause increased FUS expression. Mutations at the RGG1 and N-terminal region can also occur with FUS although this type of mutation is normally linked to FTD. In this case, the localisation of FUS is not as prominent as normally observed and abnormal aggregation can occur in some cases. Animal models expressing the various FUS mutations and their effects on neurodegeneration are available to support the research, eg. ALS-FUS mutations in D.melanogaster and C.elegans and examples of transgenic mice (eg. ALS-FUS R521G) which demonstrate motor deficits and motor neuron abnormalities. However, results from the animal models appear not to always follow expectations, eg. FUS mutations in transgenic mice appear to be regulated differently to wild type mice in response to neuronal stimulation, or could have altered dendrites. This is also supported by neuropathological studies which have shown as an example that pathological FUS aggregation does not occur in any animal model.

Animal models, however, are said by the authors to be of more value in studies on the reactions of stress granules to FUS mutations. ALS-FUS PY-NLS mutations show a correlation between disease severity and localisation in the cytoplasm and in the presence of these mutations larger granules are formed independent of the presence of stress. It has also been observed that FUS mutations may delay the assembly of stress granules and irreversibly sequester a variety of RNA binding proteins and mRNAs. No effect on the processing bodies or their associations has been observed. Therefore, suggestions have been made that the ability to bind mRNAs and sequester them into RNA granules may be one factor in disease neuropathology. Researchers have suggested that FUS mutations ´seed` protein aggregates which sequester more FUS and other proteins, hence depleting the cell of essential proteins and leading to cell death. This view however, requires according to the authors further research support.

The other example of RNA binding protein described in detail by Sephton and Yu in their article is TDP-43 which like FUS regulates gene expression. This binding protein can bind to over 4500 species of RNAs via 2 highly conserved motif regions: the RRM1 which is a major domain for binding of RNA and DNA and the region, RRM2. Like FUS, TDP-43 regulates transcription and multiple RNA processing mechanisms and hence, deletion leads to dysregulation of mRNA and pre-mRNA splicing. TDP-43 is expressed and located primarily in the nucleus, but it too can localise to different cellular compartments and RNA granules via classical NLS and NES binding. Various stimuli cause localisation to these other areas. For example, under basal conditions TDP-43 can be found in hippocampal dendrites and is co-located with RNA granules such as processing bodies. It can also co-localise with the post synaptic protein, PSD-95. The RNA granules containing TDP-43 contain RNAs including mRNA for beta-actin and CaMK11alpha, an important enzyme in neuronal cell plasticity. However, on depolarisation then RNA granules with TDP-43 co-localise FMRP and Staufen-1 in tRNP granules in the dendrites. In oxidative stress, TDP-43 localises in the cytoplasm and into stress granules. Again, no association with processing bodies has been reported even though reports of FMRP, Staufen-1 have been reported in these bodies in some circumstances.  There are also reports of TDP-43 being an integral component of different complexes eg. Dicer, Drosha and it is believed to be involved in miRNA biogenesis responsible for neuronal outgrowth.  Therefore, it is thought to act like FMRP in that its presence leads to translational repression via miRNA regulation.

Just like FUS mutations, TDP-43 mutations are also associated with neurodegeneration and neuropathology of some diseases.  There are more than 40 TDP-43 familial ALS mutations documented (mostly with mutations at the C terminal) and TDP-43 mutations have been observed in approx. 50% of FTD patients. Mutations do not normally lie in the NLS or NES regions, but instead mainly lie in the 3 prime end of the TDP-43 mRNA and these lead to increased levels of TDP-43. No impact on localisation occurs, but because the mutations lie in the glycine rich C terminal region then protein interactions are adversely affected. Animal models where the endogenous levels of wild type TDP-43 or expressed ALS-TDP-43 mutations exhibit drastic neuronal effects, aspects of ALS and FTD diseases are produced. For example, the depletion of TDP-43 in D. Melanogaster causes a decreased life span and locomotor defects due to alterations in dendritic branching and synapses, whereas over- expression can cause a loss of motor function and a reduction in the number of dendrites and synapses. Transgenic  TDP-43 mice expressing either wild-type or ALS associated mutations can also exhibit motor defects, which can be reversed if expression is inhibited. Mutations appear to be associated with abnormal neurites and decreased cell viability where depletion of TDP-43 leads to an increase in number of mature spines in hippocampal neurons, an increase in clustering of AMPA R at the dendritic membrane and an increase in neuronal firing. A link to Rac1 was established suggesting that TDP-43 could be an upstream suppressor of this spinogenesis regulator.

TDP-43 and ALS-TDP-43 mutations are shown to be actively recruited in response to stress to the other type of RNA granule that of the stress granule found in the cytoplasm. Both the C terminal glycine rich domain and the N terminal RRM1 region appear to be important for this association and therefore, both RNA binding and protein-protein interactions are required. Large stress granules are formed and like FUS, pathological aggregates of these stress granules occur suggesting a role in seeding for the TDP-43. TDP-43 mutations can also affect tRNP granule formation and migration and TDP-43 has been shown to be associated with RNA granules throughout the dendrites. Aggregation of TDP-43 is assumed to be important for the formation of neuronal tRNP granules and mutations found in ALS-TDP-43 models are shown to increase the size of the tRNP granules in rat hippocampal dendrites under basal conditions. Depolarisation with potassium chloride stimulates TDP43 granule migration into the dendrites with mutated forms demonstrating reduced density as well as slower speed of movement and shorter distances covered than the wild-type. Therefore, TDP-43 mutations can produce effects by eliciting the absence of important RNAs at sites of local translation or by producing dysregulated mRNA products.

Sephton and Yu concluded their article by looking at future perspectives regarding local translation, RNA-binding proteins and neurodegeneration. They state that the topic is gaining interest because of several reasons. There has been an increase in the identification of genetic mutations in genes encoding proteins involved in RNA regulation and associating these to the neuropathology of certain diseases. There also appears to be anomalies between in vivo disease models and in vitro ones and lastly that synaptic dysfunction precedes neurodegeneration and therefore, tRNP granule formation, localisation and protein translation dynamics, affected by RNA binding protein mutations could be the trigger to the neurodegeneration process.  In order to understand how this occurs, the authors investigated how RNA granules and RNA binding proteins are involved in the maintenance of the synapse and gave explanations to their function. They suggested that there is evidence that RNA binding proteins are important in transporting mRNA into granules which remain dormant until the mRNA transcripts are required to be locally translated on polyribosomes. The authors also hypothesised that the RNA binding proteins are also important for the presence of dendritic branches and spines since genetic deletions cause alterations in both. Therefore, Sephton and Yu concluded that the role of RNA binding proteins and granules should be researched more intently especially linked to the topic of local translation and downstream consequences at the synapse. This role was said to be especially important in light of evidence that linked RNA binding proteins and RNA granules with neurodegeneration and the pathology of particular neurological diseases such as ALS and FTD. However, the authors did admit that this area of localised mRNA translation would be difficult to study because RNA binding proteins like FUS and TDP-43 are associated with the regulation of thousands of cellular RNAs. Focussing on the area of neuronal stimulation and synaptic responses would they suggest make study of the topic slightly easier.

COMMENT

What makes this article interesting is that is looks at another site of protein synthesis in the neuron other than the common sites close to the cell nucleus. Researchers have proposed that local translation in neurons can take place and the authors of the article commented on in this Blog post describe the local site as being in the neuronal dendrites. The authors also link in their article the idea of genetic mutations of some of this process`s components to certain neurodegenerative and neuropathological diseases. We will concentrate in the comment here on the process of local translation in the dendrites and we begin with a short summary of the local translation process. This then leads on to questioning whether or not RNA granules indicative of this local translation mechanism are ´true components` and not an artefact of the experimental procedure used in cell component separation and if they are ´true components`  whether or not the neuronal cell actually needs local translation.

In order to ascertain whether or not RNA granules are ´true components` or not, we should begin with a brief simplified summary of protein synthesis and how local translation fits in with the general process in neuronal cells. The general process of protein synthesis in neuronal cells begins with the nuclear DNA which contains the information required for the protein`s structure and final location. DNA is transcripted into mRNA and we assume that this transcription process is the same whether the protein will be finally synthesised close to the nucleus or will be localised elsewhere.  The initial transcripts (the pre-mRNA) still within the nucleus contains introns (codes not translated into proteins) and exons (codes giving rise to the protein) and the introns, which can be regarded as ´guiding/regulating` signals can be spliced out. (Continued inclusion of these at this point could give rise to different end-of-process proteins.) The transcript of the message occurs in the mRNA form and this passes from the nuclear DNA site, through the nuclear membrane to sites where translation of that message into the designated proteins themselves can occur. These sites of translation are either close to the nucleus, or as suggested for neurons at sites well away from the nucleus such as the axons and dendrites. The site of protein synthesis if close to the nucleus is the rough endoplasmic reticulum (ER) which consists of stacks of membranes with ribosomes, the protein synthesis machinery, attached.  The mRNA transcripts carrying the protein-building information binds to the ER and is translated in a 5 prime end to 3 prime end direction. Translation means that the ribosomes bring the necessary amino acids of the final protein in the form of tRNA together to allow biochemical binding to occur to make the designated protein. Alternatively proteins, normally those dictated to finally reside in the cytosol, can be synthesised by free ribosomes which attach to the mRNA ´thread` itself to form complexes called polyribosomes. The proteins are synthesised in the same manner as those formed from ribosomes attached to the ER. Folding of the newly formed proteins occurs in the smooth ER followed by post-translational processing which can occur in the Golgi Apparatus (GA) which also is located close by. This is the site where proteins are sorted and processed accordingly to their ultimate destination.

Other researchers and Sephton and Yu in their article described one stage of the protein synthesis mechanism that of translation which was said could occur in the case of the neuron away from the nuclear site and nearer to where the proteins would actually be needed eg. the neuronal dendrites (or axons). This is termed local translation. In local translation, mRNA transcripts of the DNA material are formed just like with nuclear translation and are transported outside the nucleus into the cytoplasm. Although it is not known how long they exist as free threads or where, the next time they are observed is in the synaptic areas of the axons and dendrites where they appear associated with RNA binding proteins in the untranslated regions (5UTR or 3UTR) or coding regions to form messenger ribonucleoprotein complexes (mRNP). These can then assemble into more complex structures known as RNA (or RNP) granules. The complexity of the RNA granules and the range of locations, response to stimuli and function appear to be due to the RNA binding proteins and other components that make up these granules.

Sephton and Yu described in their article particular examples of RNA granules eg.  transport RNPs (tRNPs), stress granules and processing bodies. Each has a specific function, but the granules are constantly changing, forming different interactions and exchanging mRNPs, cytosolic proteins and polyribosomes. In each case the transcript exists primarily in the mRNA form and not in the pre-mRNA form and therefore, the presence of the polyadenyl translational signal would be absent having been spliced from the DNA transcript before leaving the nucleus. Instead, it is possible that RNA binding proteins take over this role and this will be commented on later. This is not uncommon and can been seen in other systems, eg. prokaryotes have promotor regions for every gene. It is also likely that the RNA transcript is in this stage of protein synthesis rather than for example the later elongation or termination stages since to have this level of control at these later stages would be a waste of resources and time. It is also unlikely that the granules are some manifestation of clumped microRNA (miRNA), which is formed from sections of mRNA folding back on itself and being spliced out. This is ruled out since the RNA content has been sequenced and found not to be complimentary not just to the 3 end of DNA as in the case of microRNA, but also that a small amount of microRNA is actually observed in the granules themselves. Other supporting evidence for the mRNA form in the granule and not the miRNA is that the latter can cause DNA modification, eg. histone modification, DNA methylation which RNA granules have not been reported to do. The mRNA in the granule dictates the proteins to be translated but as said before they are associated with other constituents into the granule form. What can definitively be said is that the functions of the granules are related to their molecular constituents eg. the RNA binding proteins and their presence is responsible for synaptic plasticity and communication in the functioning synapse. This explains why the contents of the granules are constantly changing and the content dictates the different outcomes eg. neurogenesis in hippocampus or memory consolidation in Aplysia.

So, now we have our mRNA transcripts in an area away from the nucleus and these are associated in granules whose function is dependent on their biochemical constituents. Probably from a neuronal firing point of view the most common form of granule is the most important, that of the transport tRNP granule type, which is said to be involved in the storage and transport of the mRNA. This type of granule can contain mRNA and also can contain microRNA (miRNA). RNA binding proteins found associated with the tRNP type of granule are for example, Staufen 1 and 2, FUS and TDP-43. However, the granules are also said to contain at least 40 other proteins and can be divided into two groups: those containing a low pool of Staufen 1 and 2, but contain the motor transport proteins such as kinesin; and those containing a larger pool of Staufen 1 and 2 and can contain ribosomes and ER. The transport of mRNA to the dendrite appears to occur with the mRNA in a translationally dormant state. In response to neuronal stimulation for example, the mRNA is released from the tRNP and forms polyribosomes so that translation can occur. This leads to local production of the necessary proteins in the synaptic area. It is thought that the mRNA that is translated is associated with the tRNP granules with the larger pools of Staufen 1 and 2 and the translationally dormant form is linked to the kinesin containing ones. It is also believed that the binding protein FMRP may provide a link between the tRNP and polyribosomes and its phosphorylative state may dictate whether translocation occurs or not, eg. the non-phosphorylated form of FMRP is linked to active translocation, the phosphorylated form with stalled. Following translation, the mRNPs can assemble back into the tRNP state, can be degraded or assembled into another form of RNA granule, the processing bodies. These are responsible for mRNA degradation, translational repression, and recycling or decay and hence, contain many proteins involved in these processes. Alternatively, neural stimulation can lead to repressed translation. Again the nature of the granule components eg. the RNA binding proteins determine the action ie. if protein translation occurs or whether is is repressed.Under stress conditions, another form of RNA granule is formed, that of stress granules. In this case, mRNPs are exchanged within the tRNP with stress granules. The mRNA transcripts are therefore protected and once the stress is removed then the stress granules disassemble and mRNAs are repacked into translationally competent forms and their proteins are synthesised. Or, alternatively, they are selectively exported to the associated processing bodies and the mRNA is degraded and hence, translation is repressed.

Although researchers are certain that RNA granules exist, the changeable physical nature of the system must lead to the question as to whether RNA granules actually exist in the natural neuronal environment or whether they are in fact just an artefact or a product of the experimental preparation techniques employed to investigate them or other neuronal components. It could be that otherwise free mRNA observed closer to the nucleus is bound with RNA binding proteins released due to cell separation techniques and the mediums employed. For example, the biochemical components may take the form of a granule due to the pH or ionic composition of the medium which could cause unnatural folding and bonds being formed. Aggregation of the granules is observed naturally and is believed to be related to greater degradation, but is not seen in some neuropathological cases or animal models. Alternatively, the experimental preparation process could remove blocking proteins and fatty acids and allow unnatural ribosomal binding to occur whether it is at the stage of the initial 30S subunit or later where the subunits actively combine. The result of this could be that unnatural translation would occur. The evidence however, does lead to the conclusion that although dynamic and dependent on the role of the cell at that time, the granules are genuine physical structures and the system is a justified mechanism for RNA translation.

Now that we have established that the RNA granule and local translation are real biochemical components and part of a genuine biochemical process we have to look why they are necessary when established mechanisms are already in play. One advantage of this type of translation as discussed by Sephton and Yu is that local translation would provide a quick protein production and transport mechanism to the sites where the proteins are actually needed, eg. the synaptic membrane so that the cell can respond quickly to the functional demands placed on it due to signal transmission. However, it must be said that in some cases of neuronal functioning this is not applicable and there is no difference in length between neuronal cells and other cells. Also, quick responses are believed to be required only in the transmission of the firing signal and longer term responses like memory occur over an extended period of time. Therefore, it is not necessary to have  a separate translating system from the more standard 2 nuclear systems. However, another advantage of such a system is that there would also be a reduced set of needs on such as system such as post-translational processing by the GA which dictates the proteins location since the mRNA is already there in the vicinity where it functions and hence, speed of synthesis and complexity of the protein support mechanism would not be required. It should be noted however, that this implies that the protein structure itself of a protein translated locally may have constraints. It may either have to be functional alone with minimal post-translational processing, or it will be part of a ´super complex` with other molecules required to form the viable active protein.

Another possible advantage of the local translation process to that of the nuclear process is that that RNA granules provide the equivalent of a ´protection system`  for free mRNA in a location away from either the smooth ER and GA. The cytosol is rich in ions which can be detrimental to protein structure and therefore, it is biochemically not advantageous to have mRNA floating around in it for any length of time. Also neuronal cytoplasm due to the mechanism of firing suffers  extreme changes in ionic composition and electrical charge during the course of depolarisation and re-establishment of electrical normality. Therefore, mRNA would have to be protected if it has to travel any distance in this environment from its nuclear source. Binding it with other components eg. RNA binding proteins to form granules would protect the mRNA thread from the possibly destructive biochemical environment of the synapse.

Therefore, although localised translation in areas well away from the nuclear site can be advantageous in terms of speed of reaction and protection, is it necessary to have another separate system in order to carry out this function? The cell already has two types of translation in place: that on the ER and then with GA modification; and that using free mRNA with the ribosomes attaching themselves in the cytoplasm onto the mRNA thread. Local translation is a separate system and involves the mRNA being physically associated with many other components into ´granules`  (eg. RNA granules) in for example, the dendrites. It is likely that this ´granule` system is based on a system already in place to transfer proteins and molecules in other cells. This transport system uses vesicles and the molecular motor systems associated with microtubules and microfilaments. Vesicles can hold enzymes for synthesis and degradation of molecules and is a quick and efficient intercellular transport system. Therefore, it can be considered that the RNA granules described in the dendrites are the mRNA equivalent to the normal vesicle system observed for other biochemical compounds. However, there are some differences and a problem. Whereas most vesicles are considered ´active` eg. lysosomes, the mRNA carried in RNA granules may not be. In tRNP granules, mRNA appears to be transported in a translationally dormant state and the granule acts as a carrier and provider of conditions for a change to occur when the mRNA is required to become translationally active.  This presents a problem because mRNA becomes active when a signal is received, but because of the constant breakdown of the proteins and other components in the granule which is normal for any biological molecule or system, the mRNA cannot be held in a ´ready` state too long. Therefore, there is a constant need for renewal if the aim of having this type of system is to maintain the cell in a continued state of readiness and this requires the biochemical machinery and energy to do this. Also it appears that the RNA granule transport is one directional and that is away from the nucleus unlike the vesicular system which is bi-directional. This implies that the mRNA is not brought back to the nucleus in order to influence the DNA as is observed in the case of some viruses.

Now that we have established that local translation is a viable mechanism and is dependent on RNA granules of different types we will conclude this comment by taking a quick look at one of the components of the granules which is said to be important for neuronal function and cellular plasticity. Sephton and Yu showed in their article the RNA binding proteins content of the RNA granules present at any one time can dictate whether protein translation is repressed or initiated and the type of granule observed eg. tRNP has binding proteins,  Staufen 1 and 2,  FUS and TDP-43 plus mRNA in transitionally dormant state.  RNA binding proteins can exist at the untranslated end regions or in coding regions of the mRNA and as expected such binding will affect maintenance and translation. The presence of either RNA polymerase I or II makes a difference as to whether the region is translated or not and therefore, RNA binding proteins could dictate which polymerase is allowed to bind. (This of course assumes that RNA granules require the same conditions for mRNA translation as that closer to the nucleus.) The RNA binding proteins could also revert the mRNA form back into a local pre-mRNA form so that the initiation of translation is prevented. Pre-mRNA has specific  5 and 3 ends (5 end – cap structure added, 3 end  – poly A tail) which are required for aiding binding of the mRNA to the ribosome, protecting from premature destruction by ribonucleotidases and as a signal for transport to the cytoplasm. Therefore, specific RNA proteins could take on these roles. In nuclear translation splicing occurs to remove these additions and to remove introns so that exons become joined together. This is also likely to be the case in RNA granules when the signal is given to translate the transcripts. This implies that the signal is in some form that can remove RNA binding proteins from the relevant sites. Other RNA binding proteins determine how mRNA interacts with its environment eg. the binding protein FMRP and mRNA and ribosomes. This implies that the binding of RNA binding proteins promotes conformational changes to the mRNA transcript that opens the attachment sites of perhaps the ribosomes. Phosphorylation of FMRP means no translation whereas removal of the phosphate group means that translation occurs. This mechanism is also seen with miRNA and is a common mechanism in other molecules since addition or removal of molecular groups eg. phosphate groups, or disulphide bridges will create changes in tertiary  and quartenary molecular shapes that promote or decrease function.

The number of RNA binding proteins and the number of different roles that they play make them ideal targets for manipulation whether natural eg. gene mutations leading to decreased numbers of the binding proteins or experimental, eg. FUS knock-out transgenic mice. Sephton and Yu described in their article some of the RNA binding proteins and their links to neurodegenerative diseases eg. FUS mutations observed in ALS, TDP-43 mutations in frontotemporal dementia. It is clear that anything that affects mRNA formation, transport and translation will have an effect on the functioning of the neuronal synapse. These effects may be short-term or long-term. The question that has to be answered is whether long-term changes in neuronal function and plasticity are caused only by permanent changes in local translation of the nature described above (ie. caused only by gene mutations), or not. It is possible that temporary ´blips` in protein synthesis in the area of synapse will have immediate effects that alter current neuronal firing, but may not be sufficient to cause permanent effects which are only elicited through the system located closer to the nucleus. Only further research will answer this question, but it is an interesting topic and again indicates how complicated neuronal cell mechanisms are.

Since we`re talking about the topic……………………….

…….if we could carry out RNA sequencing at the 5 prime end region of mRNA from free mRNA, ER-bound mRNA or RNA granule-bound mRNA would we see differences in genetic code dependent on the source or will they all have the same promotor sequences and would these differ in content as expected to the promoter regions of prokaryote mRNA?

…….if we use cortical slices and subject the cells to different conditions eg oxidative stress, drugs, then separate out the mRNA from the RNA granules of those cells and subject them to Northern Blots will we be able to see alterations in the mRNA transcripts necessary for the cell adaptations required to cope with each condition?

……can we assume that the mRNA transcript length as measured by agarose gel electrophoresis is the same whether the mRNA transcript undergoes ER/GA translation close to the nucleus or at a site away from it?

……since mutations of some RNA binding proteins are associated with increased neurodegradation can we investigate whether the apopteric enzymes necessary for degradation eg. annexin V are actually translated from mRNA associated close to the nucleus or are they locally translated? If RNA granules are removed would the absence of annexin V labelled with fluoresceinisothiocyanate (FITC) be proof that local translation is responsible for the production of apopteric enzymes?

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neuronal firing relating to pain and the influence of anxiety

Posted comment on ´Determining the neural substrate for encoding a memory of human pain and the influence of anxiety` written by M. Tseng, Y. Kong, F. Eippert and I. Tracey and published in Journal of Neuroscience December 2017 vol. 37 (49) p. 11806

SUMMARY

Tseng and colleagues began their article by saying that the perception of pain is dependent on the individual, but the neurochemical mechanisms involved are common. Little is known or clear about these neural mechanisms that encode the information about painful stimuli so that it can guide future behaviour and allow the individual if possible to avoid dangerous and life-threatening situations. Therefore, Tseng and colleagues looked at the process involved in working memory encoding and non-encoding of somatosensory information in painful or non-painful situations.

In their investigation, Tseng and colleagues used fMRI with a delayed discrimination task. Results were obtained from 20 healthy subjects (male and female, mean age around 27) who had no previous experience of thermal or vibrotactile stimulation experiments. Before the fMRI experiment was carried out, each subject was assessed for their anxiety level and awareness to pain. Painful stimuli were elicited through the application of rapid increases of temperature (30 degrees centigrade in 0.8sec) and non-painful stimuli from vibrotactile frequency discrimination. Each subject was attached to a piezo tactile stimulator by having the left palm on 4 stimulators with the tips of the index and ring finger taped securely on the probes and the bases of both fingers taped on thermal stimulators. Three behavioural sessions were carried out. The first session was to find out the stimulation temperatures of each subject within a range of 1 degree centigrade to 42 degrees and then the highest temperature the subject found tolerable. In the second session, each participant was subjected to vibration frequencies between 5 and 50HZ where flutter sensations are reported. The third session had each participant being subjected to alternating pain (42 degrees centigrade to the highest tolerable temperature) and vibrotactile stimuli. The participants were asked to grade the stimulus intensity directly after each application on a visual scale.  Using this scale four pain stimulus magnitudes and 4 vibration stimulus frequencies were calculated for each subject. In the trial period each participant was subjected to a cue period of 3 secs where they were presented with either a red (encoding trial) square or green (non-encoding trial) square. An 8 sec delay followed and then they were presented with the first stimulus. In the encoding trial participants had to keep the first stimulus in mind when they were presented with the second cue period consisting of the same red square and second stimulus. The participants had to decide if the presented second stimulus was higher or lower in pain intensity or vibration intensity than the first. In the non-encoding trial, the subjects had to judge the second stimulus without reference to the first. Intensity in this case was determined by deciding whether it was higher or lower than the 25^th or 75^th percentile of the visual scale determined for each earlier. Participants were trained to use both right and left fingers to signify response and once this was satisfactorily learnt then in total 32 fMRI were carried out.  The fMRI were to investigate whether the same brain areas were activated in both pain and vibration trials. Small volume corrections were applied for example to the somatosensory cortices, thalamus, insula and anterior cingulate cortices (ACC), amygdala and hippocampal areas. Once completed psychophysiological interaction analyses (PPI) were performed to measure interregional functional connectivity between the bilateral thalamus, the right ACC and left somatosensory cortex (left SI) areas. In order to examine the sensitisation of responses the participants were asked to rate task difficulty for non-encoding trials also using a visual scale. Various statistical analyses were performed on all results to examine whether memory task performance was significantly different from the 50% chance level.

The results obtained by Tseng and colleagues using their experimental techniques showed that the painful stimuli gave moderate pain and this did not change across the trials so no sensitisation had occurred. Error rates were significantly lower than chance so it could be said that subjects performed the tasks correctly and attention remained consistent between the application of the first and second stimulus. The reaction time was found to be slower in the case of the painful condition compared to the vibration condition and this was explained as the skin taking a longer time to return to normal after excessive heat stimulus. Task difficulty was found to be assessed by the participants as significantly higher between the pain encoding vs non-encoding situations.

Regarding brain area activation, Tseng and colleagues studies showed that activation was dissociable between neural encoding of painful versus non-painful stimuli. The activity of the bilateral midline and mediodorsal thalamus and rostral portion of right ACC were enhanced during the encoding of painful thermal stimuli, but not with the non-painful vibrotactile stimuli. Encoding of vibration however, led to an increased response in the left SI which did not show increased activity with pain. Both the left and right amygdala areas were activated in the pain trials, but activity was not significantly different between the encoding and non-encoding tasks. The bilateral hippocampus area was not significantly activated for either pain or vibration trials. The results of the PPI analyses showed that the medial PFC was the only region with enhanced functional coupling with the thalamus and ACC during pain encoding trials compared to non-encoding trials and this was not observed during the vibration trials. There was no significant correlation between medial PFC activity and participant`s perceived view of task difficulty error rates and response latency.

Tseng and colleagues also looked at how anxiety would affect the encoding of pain. During the pain encoding trials participants with a high level of anxiety and those who showed trait anxiety levels showed significantly decreased error rates than those with a lower level. This was not observed with the pain non-encoding trials, or either type of vibration trial and error rates did not differ between the different levels of attention towards pain either. Subjects with high state anxiety scores showed a significantly faster reaction to detect the offset of the first stimulus and significantly reduced the reaction time during high pain stimuli, but not during low pain stimuli. The response latency was not affected by different levels of trait anxiety in any trial and neither was task difficulty. The results suggested that inter-individual differences in state anxiety and trait anxiety related to pain encoding behaviour with subjects having higher levels of anxiety performing better on pain encoding trials and reacting faster in detecting painful stimulus. FMRI analyses showed that the activity of the left amygdala was negatively correlated to the level of state anxiety during the pain encoding trial, but not in the pain non-encoding trial or either vibration trial.  The results were only significant in the high level pain trials and the activity of the left amygdala with anxiety in the pain trial correlated to the extent of coupling between the thalamus and mPFC. In the case of trait anxiety, the results showed a trend towards positive correlation between individual trait anxiety and pain encoding thalamic activity with significance achieved only in the case of high pain levels.

Tseng and colleagues summarised their findings by hypothesising that there were distinct brain areas such as the thalamus, SI and ACC active in the encoding of information in both painful and non-painful situations. However, in the pain situation because of the strong emotional contribution then activity should be present or stronger in pain-encoding areas and emotional related areas such as the amygdala. They found that working memory activity was dissociable and dependent on task ie. whether the stimulus that was to be learnt was painful or not. Differences in area activity were observed. In the case of the encoding of the vibrotactile stimulus SI activity was observed and this was also observed in the encoding of pain with activity in the medial thalamus and rostral anterior CC and with significantly increased connectivity between the medial thalamus and medial prefrontal cortex. The hypothesis given by others that the increased SI activity observed with encoding and working memory involvement was associated with attention was found not to be case in this study since the task reaction times were not significantly different for the different trials. The authors also found differences in activity in the SI areas eg. the bilateral SI responded to vibrotactile stimulation as expected through suggested uncrossed ascending tracts or transcallosal connections, but only the ipsilateral area participated in the encoding process. The role of this area was said to be unclear, but it was considered unlikely that it was involved in sensory perception. The activity of the area was shown to vary across different cognitive states.

The authors went on to explain the roles of the various areas shown to be involved in pain encoding. The rostral ACC, suggested as an area participating in the processing of threat-related stimuli, was also now attributed with a role in acute and chronic pain processing. The medial thalamus, associated with pain encoding, contains nociceptive specific neurons and hence, is involved in mediating the emotional aspects of pain. This view was supported by others suggesting that the area had a range of cognitive functions including attentional modulation of nocicieptive processing and working memory.  The authors also concluded that the connectivity between brain areas was also affected during pain encoding with significantly enhanced connectivity between the mPFC, thalamus and ACC. The mPFC is known to be involved in working memory and in regulating emotion and cognition and the medial thalamus was suggested as acting as an interface between the mPFC and hippocampus during the encoding process. The encoding process was said not to be driven by self-monitoring or attention instead the authors suggested that there was a distinct stream in the brain to sub-serve working memory of pain encoding and the emotional part of pain experience receives preferred processing when pain needs to be transformed into neural construct.

Tseng and colleagues also found in their experiments that the level of anxiety experienced by the subjects enhanced the task performance on pain encoding with the more anxious participants demonstrating significant performance advantages. The modulated brain responses were associated with the increased connectivity between the medial thalamus, mPFC and amygdala especially during trials with high pain. The activity of the amygdala area inversely predicted and negatively correlated to the degree of thalamic-mPFC coupling and this was said to correlate to emotional learning.  This supports the view that amygdala neurons project to the hypobasal forebrain area of the periaqueductal gray (PG) to modulate responses to aversive stimuli and is therefore suggested as a coping strategy that attenuates perceived distress. Therefore, inter-individual differences with exhibiting anxiety show that the emotional state engages distinct neural mechanisms. Anxiety also appeared to elicit a more efficient performance in encoding pain with lower error rates and faster reaction rates.

Therefore, Tseng and colleagues concluded their investigation by saying that working memory neural constructs are different for pain than non-pain encoding situations and that anxiety can affect the process.

COMMENT

What makes this article interesting is that we know that processing of sensory information and sensory memories can be altered by the individual`s emotional system, but this article confirms that the pain system which we think of as a basic biological system, its perception and its ´recording` can also be affected by the individual`s emotional state. (In the article discussed here the emotional state is that of anxiety.) This confirms views that if the emotional state can be controlled then the perception of pain can also be affected and hence, this is another avenue by which modulation can occur.

The discussion here begins with a look at the similarities and differences between information being received in the brain for pain compared to that of the sensory system, its processing and recording and effect on future events plus the effect of anxiety on those systems. Discussion will only be at the fundamental level since the systems are extremely complicated, but it may give areas where perhaps research should focus in the future. We begin by investigating the input and perception of pain and comparing this system to that for other sensory information. It is known that both arise from the input of relevant information: pain from real- time pathway activation from the source at cell level, the nociceptors; and sensory information from receptor and sensory cell activation followed by real-time pathway stimulation or, alternatively sourced from reactivation of appropriate cell memory stores. Hence, the two systems are similar in that they are sourced at specific cell levels, but differ in that the pain system becomes active in using real-time information only.

The evolutionary pathways for sensory information appear to be stable with only the capability having changed with development. Specific pathways lead from the sensory cells to higher brain areas and functionality and activity of those brain areas are linked to informational input plus factors such as attention and emotional state. Activity occurs through the firing of the neuronal cells and involves the release and action of different neurotransmitters such as glutamate, acetylcholine, 5HT, dopamine and GABA. Some areas and certain neurotransmitters lead to inhibitory firing eg. GABA and interneuron function in the hippocampus and some excitatory eg. dopamine and the prefrontal cortex. The group of cells firing together and bound together in time and activity is known as a neuronal cell assembly and this is equivalent to the neural representation of the information from the environment that is being acted on at that time. These initial firing groups are equivalent to the memory sensory stores and exist through sustained firing of the relevant cells. This leads on to the formation of short-term memory stores which are capable of further processing eg. the addition of more information. This is just a simplified version of what is occurring, but we can see that information progresses from the external environment into a neural representation that can be for example manipulated and stored or just decays and disappears. Two important physiological systems affect the neuronal firing, group dynamics and what is acted on or eventually what is stored and these are the attentional system and more relevant to the article discussed here, the emotional system. In a way these two systems are linked because attentional state dictates what brain areas and which cells have increased stimulation. There are likely to be three attentional states: normal – occurs when our minds are flitting from one external event or topic to another; focused both diffuse and concentrated where diffuse focus means attention is on a number of objects within an event rather like gist and concentrated where focus centres on one event; and finally the fear attentional state where attention is appropriate to the fight or flight response. Attentional state is dictated by the activity of certain brain areas such as the ACC, lateralintraparietal cortex (LIP), temporoparietal areas, medial temporal area, PFC (dorsolateral and orbitofrontal areas amongst others). This activity affects the quality and quantity of information being considered in real-time so that it is relevant and maximised for the task at hand. In the case of the fear attentional state, there is an increase in quantity through an increase in volume, but not necessarily and increase in quality, since the level of non-relevant material is higher as well as relevant and gist becomes more featured rather than concentrated focus.

This fear attentional state is linked to the fear emotion experienced at the time and therefore, it can be said that emotional state affects sensory information quality, quantity and processing. This is just one example of emotional state affecting brain functioning and as known, emotional state can also relate to positive emotions such as pleasure.  The two emotional systems can be said to be a balance of two neurotransmitter systems eg. dopamine (pleasure) and noradrenaline (fear) and the action of particular brain areas eg. medial prefrontal cortex for pleasure and amygdala for fear. The emotional state experienced at the time is supposed to be recorded simultaneously with the informational characteristics so that memories have an emotional ´value` to them. This can be called the ´emotional tag` and this value is likely to be recorded in the PFC. It has been suggested that the structure of the PFC relating to emotional value can be considered like a ´sliding switch` with pleasure having graded values appropriate to the different levels of positive emotional response an individual can hold, but fear is allotted to just one grade since it is either present or not.  The ´switching on` of the different grades results in a set of values for all experiences and this type of mechanism is important in cognitive functions such as decision-making as well as behaviour. Just like with informational characteristics, emotional values can be modulated by various factors eg. past experiences. There is support for this view with medial PFC activity required for recording in conditioning and the OFC linked to hedonistic experiences of reward. With relation to the pain system and its modulation, Zhou and colleagues extended this work by looking at reward signals and dorsal raphe nucleus activity and found that the intensity of the response of the OFC was affected by the frequency and duration of dorsal raphe nucleus stimulation.

Tseng and colleagues looked at one emotional factor in relation to the pain system that of anxiety and the sensory system like the pain system is affected by this emotional state. Anxiety is defined as a maladaptive response to threat, stress or fear and it can lead to an unpleasant state of anticipation, apprehension, fear and dread. The response may be real or imaginary and may be disproportionate to the actual stress or threat at that time. Although anxiety disorders appear to stem from past behaviour, there is also some evidence that sufferers may have a hereditary disposition eg. 60 gene regions of chromosome 15 are duplicated in about 90% of family sufferers which may lead to an over-sensitivity of neuronal communication. There are many physiological symptoms, but what are of interest here are the psychological ones, eg. fear, dread, obsession, distress, unease, and difficulty in concentrating. The physiological mechanisms relating to anxiety and these psychological symptoms being experienced include reduced activity in brain areas such as the frontal cortex and prefrontal cortex, but also increased activity in areas such as the amygdala and hippocampus.  Several neurotransmitters appear to be involved, for example: acetylcholine with cholinergic systems being increased in the hippocampus in aversive memories; and GABA binding to the GABA A receptor and acted on by barbiturates and benzodiazepines in association with anxiety directly. Barbiturates potentiate GABA induced increases in chloride conductance due to an increased affinity for GABA at the GABA A R. The reticular formation appears to be an important site of action with the middle area forming the pontine region which activates cortical areas whereas the medulla suppresses. The actions of noradrenaline and 5HT are however more interesting with changes in emotional state relating to increased release or decrease of the relevant neurotransmitter.

Noradrenaline acts at the locus coeruleus and results in anxiety when stimulated and arousal and vigilance when threatened. This area has alpha noradrenergic 2 receptors (alpha-AR2) which when blocked increase the release of noradrenaline and when stimulated decrease its release. In an elevated maze experiment binding of antagonists to the alpha-AR2 appear anxiogenic whereas agonists (eg. clonidine) are anxiolytic. The blockade of post synaptic beta-noradrenergeric receptors (beta-AR) appears to have opposite effects since inhibitors appear to be anxiolytic whereas agonists are anxiogenics. This effect is possibly mediated by peripheral receptors which mediate the peripheral autonomic effects of anxiety such as increased heart rate tremor and perspiration, but do not contribute to the conscious awareness of anxiety. They may contribute indirectly by autonomic activation leading to feedback which could be interpreted negatively eg. the beta-AR antagonist, propranolol, which is used to treat some of anxiety symptoms such as tachycardia, but not psychological symptoms or affects the conscious experience of anxiety.

In the case of 5HT, depression, which can be interpreted as being at the opposite scale of emotional disorders to anxiety, results from a decreased 5HT level in neurons of certain brain areas and decreased numbers of the inhibitory serotonin receptors. Studies showed that reduced numbers of 5HT1A receptors are also involved in the mechanism of anxiety whereas antagonists at the 5HT2 and 5HT3 receptors produce anxiolytic effects. The effect on receptor number mirrors the situation in depression and the effect of SSRIs.

The importance of this balance of neurotransmitters and activity of brain areas can be seen if we consider one cause of anxiety that observed in the case of conditioned aversive stimuli. In this case the behavioural inhibition system (BIS) is responsible for anxiety due to increased sensitivity to the non-reward or punishment stimuli and this may be relevant in the experiments described in this article which rely on the administration of pain. The BIS is a septo-hippocampal system which involves the neurotransmitters GABA, 5HT and NA and a competition between fear and anxiety. The fear response is controlled by the amygdala response to immediate threat whereas anxiety is mediated through a septo-hippocampal system that deals with future threats. Anxiety is therefore created by competition between conflicting goals requiring resolution and leading to uncertainty. The hippocampus, an important relay station for informational input and binding, tries to reduce this by helping to inhibit responses that may put the individual in danger eg.  approaching for food even when there is a chance of threat, and to assess the risk whilst employing reactivation of past experiences to facilitate the resolution of the conflicting goals. However, it has been shown that anxiety causes an exacerbation of pain associated with increased activity in the hippocampus and therefore clinical strategies have been suggested to reduce pain by disengaging the hippocampus during potentially painful clinical procedures. One method used by the BIS to resolve conflict is to increase the negative value of stimuli and to associate this with the emotional state of worry and anxiety. Therefore, the individual is more sensitive to negative stimuli which create activity in BIS which in turn increases the sensitivity to negative stimuli. It is likely that this involves inappropriate emotional tag storage at the time of the initial event or inappropriate processing of the PFC sliding switch scale of the previously stored emotional tag at the time of recall.

From our simple descriptions of the sensory information mechanisms and brain areas and those affected by anxiety we are able to see points where there is overlap with the pain system and where manipulation of the system can occur. The mechanism of pain or nociception also like the sensory system begins with areas on nerves that are sensitive to outside factors and these here in the case of pain are the nociceptors. These respond to damage and transmit neural signals identifying location, intensity and duration of the stimuli. Melzack in 1990 developed a theory of pain which has similarities to that developed for consciousness. He proposed the ´neuromatrix` consisting of neural firing loops between the thalamus and the cortex and leading to a ´neurosignature` of activity. This neurosignature corresponds to all information from the various areas and it includes input from attentional systems, emotional systems and senses. Melzack concludes that neurosignature projects to the ´conscious experience` area and this he termed the ´sentient neural hub`.

The pathways responsible for the input and perception of pain can be divided into two groups: ascending and descending and this too reflects to some extent those that are involved in sensory informational processing. In this case, the ascending neuronal pathways refer to that carrying the sensory input and the descending pathways, the influence on that firing from higher cortical areas such as the PFC as seen in decision-making and other systems such as working memory, attention and emotions. The ascending pathways for pain reflect different uses eg. pain control is the responsibility of the ACC and mPFC, but information about the pain sensation eg. about temperature differences that cause pain is the responsibility of the spinothalamic pathway. This pathway channels signals to the thalamus, then ACC, somatosensory cortex and dorsal cortical areas. Other ascending pathways include: the spinoventricular pathway to the reticular system and then cortex responsible for coordinating the responses to pain eg. turning the head; the spinotectal pathway leading to the superior colliculus; and the trigeminal pathway responsible for signalling between the face and the thalamus before ascending higher. Therefore, brain areas responding to pain perception include the ACC (intensity of pain, self-administered vs administered different areas), cortex, thalamus, somatosensory cortex (exact location) and insular cortex (pain integration). The functional disruption of one system leads to augmentation in the pain-induced activation of one or more other pain relevant brain regions including the PFC.

The neurotransmitters that appear to be involved in pain transmission are glutamate which is a common signalling mechanism in sensory information and dopamine already discussed as important in emotional pleasure expression and firing in the PFC and basal ganglia. Studies of pain processing have shown that there is also dopaminergic activity in the basal ganglia and this is linked to variations in the emotional aspects of the pain stimuli. Nigrostriatal dopamine DA2 receptor activation can be attributed to the sensory aspect of pain, while mesolimbic dopamine DA2/DA3 receptor activity can be related to the negative affect of pain and fear. We have also discussed how anxiety affects GABA firing in the reticular formation and since one ascending pain pathway involves the reticular formation then this is one area of overlap where anxiety could have an effect on the level of pain being experienced by an individual. GABA could also be involved in the pain pathways observed via the action of interneurons which can be either excitatory (eg. activated by glutamate for example in sensory systems) or inhibitory (eg. activated by GABA for example in sensory systems). The activity of the latter plays a role in the Gate Theory of Pain. This explains that certain neurons are excited by large sensory neurons and inhibited by pain axons. For example, there are multifunctional neurons in the substantia gelatinosa dorsal horn (admittedly, not the brain) that are excited by pain and also excited by other input leading to interneuron firing (excited by neurons and inhibited by axons). Therefore, if the neuron is excited by sensory stimulus it is excited and then fires its interneuron and the pain signal suppressed.

Attention just like with sensory information plays a role in the quality and quantity of information transmitted. The level of attention applied is determined in the case of the pain system by the threat value of the event. This can produce conflict when there is a need to disengage from the pain signal in favour of more important signals such as those for the ‘fight or flight’ response and survival. On the other hand, attentional bias towards the pain signal can be modulated by: the nature of the stimulus itself and previous experience (eg. heat is worse than cold), novelty and through anticipation and uncertainty; the individual and his/her own personal characteristics; and the environment in which the pain occurs. For example, attentional bias has been shown by studies that show increased engagement to pain signals and difficulty disengaging from for example by cognitive interference associated with pain-related words and visual-processing bias to the pain location. This prioritisation of pain over other stimuli is an innate response to threat. The threat value of pain may be modulated cognitively by providing information about the pain in advance and this may be the case in the experiments used in the Tseng`s study described above since the individual is aware of intending pain administration. Therefore, the individual has an expectation of pain which can alter activity and patterns of connectivity of relevant brain areas. Negative expectations can also affect activity in the PFC (particularly medial PFC and OFC), ACC, hippocampus, insular cortex, nucleus accumbens, amygdala, thalamus, somatosensory cortex, head of caudate, cerebellum, and the PAG – all areas associated with pain pathways. Hence, pain is anticipated and these expected levels of pain can alter the perceived levels of pain. On the other hand, positive expectations only affect activity in the dorsolateral PFC, ACC, striatum and frontal operculum.

The pain signal can also like the sensory signal be modulated by emotional state and this is relevant to the experiments described in Tseng and colleagues` article where they investigated the change of perception of pain with the emotional state of anxiety. Pain is also an emotional reaction since there is a mental feeling of pain and this feeling is individual and cannot be imagined. It can, however, just like sensory information come under cognitive control and like the case of values, the perception of pain can be manipulated. Pain perception arises from nociception although feelings can be absent even if the pain signal is transmitted. It is an immediate reaction and just like with sensory events and consciousness only one emotional value can be perceived at any one time. The pain system responsible for this emotional recognition is also like the emotional system for sensory information dependent on a descending pathway. Strong emotional stress for example can suppress the feelings of pain through the activation of several brain areas, but one of the most important appears to be the periaqueductal gray region (PAG) which is a zone of neurons in the midbrain which receives input from several brain areas that have a role in transmitting emotional state eg. medial PFC, hypothalamus, amygdala and locus coeruleus. PAG neurons also send descending axons to various midline regions of the medulla and these neurons project axons down to the dorsal horns of the spinal cord which depress noradrenaline activity and also particularly firing to the raphe nuclei. Therefore, the PAG area can be modulated by descending pathways that arise from brain areas responsible for emotional state such as the mPFC and amygdala and this ultimately results in pain signalling effects lower down the neuronal hierarchy. Whereas cognitive modulation may alter both intensity and emotional feeling of the pain being experienced, the emotional modulation of pain is more likely only to change the unpleasantness of it.

   The question is therefore, where pain fits in with the medial PFC sliding switch of pleasure/fear values attributed to information events. We know that pain values exist, are individual and have a threshold only above which can pain be consciously experienced. This threshold can be lowered by certain factors (eg. ill-health, cold, hunger, pain from another source, fear, worry, anxiety, boredom, insomnia, depression and frustration) or raised (eg. by  painkillers, acupuncture, heat application, anaesthetics, alcohol, excitement, concentration, interest, self-confidence and faith). We assume that like emotional values, pain is attributed to medial PFC activity and is part of the grading of ´emotions` experienced portrayed by the ´sliding switch`. It is likely that in addition to the ´sliding switch` having graded values relating to positive pleasure emotions and one grading for fear, it also has one grading for pain. This can be explained by the observations that one experiences fear or pain, but there is no grading ie. there is no little pain or a little fear. The role of the PFC in this function is supported by another pain pathway, the cortico-cortical modulatory pathway which is known to involve the higher areas of the brain and demonstrates connectivity in prefrontal regions such as the dorsolateral PFC and ventrolateral PFC. This pathway is responsible for the cognitive and emotional modulation of pain and does so at these higher brain areas rather than changes in the lower pain relevant regions.

Support for the modulatory role of the cortico-cortical pathway comes from looking at the functions of particular brain areas already known to be associated with the emotional system. The PFC is said to play a role in ´keeping pain out of the mind` and it is thought this is achieved by the modulation of the cortico-subcortical and cortico-cortical pathways, employing both somatosensory (non-emotional) areas and areas that process emotionally salient stimuli. The perceived control over pain activates the dorsolateral PFC during the anticipation of pain and the ventrolateral PFC during painful stimulation. The activation of the latter is negatively correlated to pain intensity and it acts as a controller of attentional engagement and so is linked with amygdala action. The other region of the PFC involved in the emotional system and pain perception is the OFC (synonymous with the ventromedial PFC) which is linked to the attribution of emotional values and reward. In relation to pain, this particular area may play two distinct roles. In the case of distracting tasks carried out at the same time as administration of pain, then the perception of pain is higher and this arises from decreased PAG activation which would normally decrease pain perception, but a more dominant increased activation of the OFC which is linked to increased sensitivity. Zhou extended this view by looking at reward signals and dorsal raphe nucleus activity and found that the intensity of the OFC response was affected by the frequency and duration of dorsal raphe nucleus stimulation and we have already shown that this particular region is linked with arousal and pain. Hence, the OFC modulates in two different ways the perception of pain.

Another area linked to the emotional system and the pain system is the amygdala region. The amygdala is part of the descending pain modulatory pathway and is known demonstrate increased activation in the case of threat to the individual ie. essentially the ´fight or flight` response. This is achieved by a reduction of PFC influence. Anxiety appears to reduce the activation of the PFC and hence, increase amygdala activation under a lower perceptual load and hence, sensitivity to threat is heightened. The amygdala has also plays a role in conditioning involving aversive and emotionally charged events and this is relevant to the experiments described by the authors in the article. In this case, the subjects are conditioned to respond to the pain experienced by pressing the relevant buttons. It is thought that in fear conditioning the amygdala is the place where the unconditioned stimulus (UCS) and conditioned stimulus (CS) is formed and responses have been shown to be linked to synaptic changes in the basolateral amygdala. Recently, distinct neuronal circuits within this area have been identified to differentially mediate fear expression versus inhibition and this has led to suggestions that there might be specific pharmacological target areas for inhibiting fear and enhancing fear extinction.

Therefore, we can see that there are a number of areas where there is overlap, similarities and differences between the systems in play for the input and perception of sensory information and that for pain. The same could be said for the memory mechanisms. In the case of sensory information, it is stored in the form of neuronal cell assemblies where the individual cells represent features of the event and have been physiologically changed to reflect there binding with other cells. The processes that lead to this are complex, but begin with the sustained firing of the cell in response to the appropriate feature and resulting in long-term potentiation (LTP) or long-term depression (LTD). The sensory information is stored with the emotional tag, a record of the emotional worth of the event information to the individual. Recall of the stored information means firing of the same cells that made up the initial input so that a neural representation is formed. This neural representation can be modulated, manipulated, added to and then re-stored if required.

Not much is known about how ´pain memory` is stored. It has been suggested that neuropathic ´pain memory` lies within the peripheral nervous system and evidence suggests that this may be correct to some extent. Plasticity in the pain system can occur at the primary afferent nociceptor just like with primary sensory cells and the involvement of the neurotransmitter receptors such as NMDA R and AMPA R. The mechanism here involves long-term potentiation changes which cause physiological adaptations resulting in long-term changes in cellular functionality responsive to certain stimulus. Changes at the lower levels translates to signal alterations higher up in the neuronal hierarchy and in the case of pain, LTP has been observed in synapses activated by C-fibre afferent activity such as in the dorsal horn. Although the LTP observed shares the same physiological mechanisms as that for sensory information, there is one important difference. Low frequency afferent stimulation causes LTD at most synapses in the brain, but low frequency stimulation of C-fibres, their normal firing frequency in most cases, causes a two-stage LTP at a subset of dorsal horn neurons. It appears that it is the early stage LTP that causes the typical physiological changes eg. gene transcription and translation alterations and it therefore requires the activation of CaMKIIalpha, PKA and PKC leading to the phosphorylation of AMPA receptors. The changes in gene expression cue the transition to late LTP which is less well-researched. However, it is thought that this stage involves an atypical PKC isoform which is involved in the trafficking of the AMPA receptors to the synaptic membrane for the sustained glutamate signalling required for the long-term cellular changes to occur.

Although the dorsal horn may demonstrate physiological changes to pain consistent with sensory information memory, it is unlikely that what we would call ´pain memory` actually resides there. If we think of what we mean by pain memory, we actually think of information that is to us painful or will elicit pain. Therefore, pain memory is likely to be as suggested above a recording of the ´emotional tag` relating to the pain grade associated with the neural cell assembly corresponding to the event we associate that pain with whether in its entirety or just in part. It is likely that the quality and quantity of information recorded at the time corresponds to the same level as that observed in a fear situation. This supports in part the view of pain researchers who suggest that the other possible location for pain memory is at the level of the neurons receiving nociceptive input throughout the brain.

Another approach to looking at the memory mechanism involved in pain is to look at fear conditioning. Tseng and colleagues` experiment carried out here also required memory storage of the pain experienced albeit of a short-term nature and therefore, we can look at the memory mechanisms taking place if we consider it as a case of fear conditioning of an operant nature. Fear conditioning means that the reinforcement is pain/fear rather than reward. The presentation of the pain and the fear conditioning process produces neuronal responses area in particular brain areas. For example: the hippocampus is required for contextual associations, but activity in the raphe nuclei results in no ripple activity in the hippocampus, an area important in the consolidation of memory; the amygdala is important in response to the emotionally charged reinforcement and it was found that the central area directly projects to the PAG inhibitory neurons; medial PFC neurons also project to this area so it is susceptible as shown above to medial PFC activity; and the other area is the ACC which demonstrates theta brain wave activity in fear expression.

We can assume that fear conditioning memory has the same mechanisms as those used to form other informational memories. For example: a reliance on protein phosphorylation and dephosphorylation; increased CREB phosphorylation; changes in spine morphology (observed in amygdala); persistently active protein kinases; microtubules requirement; and calcium/calmodulin-dependent protein kinase II requirement. However, there are one or two differences. Whereas memory formation is associated with NMDA or AMPA R LTP and/or LTD, in the case of fear memories NMDAR LTD is required for their consolidation, but not their acquisition. Fear learning however, does force AMPA R into the amygdala as expected. The presence of NMDA antagonists not AMPA in the amygdala leads to the prevention of the fear memory becoming labile or recalled (Mamou). There is also heightened activity of hippocampal extrasynaptic GABA A receptors in contextual fear conditioning which would normally impair fear and memory, but in this case enabled state dependent encoding and retrieval.  Also sleep which has an important role in normal memory formation has a different structure to that seen in fear conditioning. Here, REM sleep is disrupted and hence, there is higher fragmentation resulting in a disruption of any extinction learning that might follow. This has been observed with PTSD sufferers also. Extinction of fear conditioning is believed not to be an erasure of memory as thought, but a formation of a new memory. This requires the involvement of the hippocampus (ie. the remapping of place cells) and beta-adrenergic receptor and glucocorticoid receptor activity.  Successful fear extinction also requires ventral medial PFC gamma brain wave activity. The extinction of conditioned fear by the glucocorticoid agonist DEX is blocked by NMDA R antagonists which suggest that the conditioned fear mechanism has a different requirement for acquisition than for consolidation in the case of the amygdala and this supports work given above. This also explains the action of the GABA A R agonist, muscimol, whose administration leads to the disruption of extinction whereas the antagonists have no effect. The addition of a partial NMDA R agonist with muscimol reverses the effect.

The experiments carried out here by Tseng and colleagues also involved decision-making and therefore, we should look at how decision-making and working memory capabilities can be affected by pain. The results of the experiment showed that the level of performance errors was reduced with pain administration implying that the level of performance was higher through better accuracy and judgement. Many brain areas are involved in decision-making and working memory eg. left PFC for planning, slower right PFC for execution of those plans, medial PFC for value and upcoming action (dopamine dominant), OFC for the encoding of values (GABA and glutamate dominant), the ACC/CC for mediating response to the visual field and learning the values of outcomes and the amygdala where lesions lead to an increased choice of risky reward and the caudate involved in trade-offs.  Also connectivity between particular areas appears to be important eg. connectivity between the OFC-hippocampus-amygdala whose activity can lead to increased dissatisfaction with results, or the thalamus-medial PFC where a decision based on STN activity can be modulated. Therefore, any factor that affects the activity of any of these areas will have an effect on decision-making and working memory capabilities and this is observed in both the cases of pain and anxiety eg. anxiety causes the reduction in the activation of the PFC, or threat increases the activation of the amygdala.

The above simplified explanations of the mechanisms involved in the sensory information and pain pathways show how both are perceived and ´stored` in order to benefit the individual. The pain mechanism is what it is – a mechanism for the protection of the individual from physical harm, but due to the nature of the pathway it can be modulated by top-down systems that also control how sensory information is inputted and processed. We have seen how the neuromatrix loops for the pain pathway relate to the views of consciousness and how perception of pain can only occur in one area just like conscious awareness. But, we have also seen that unlike consciousness pain can be ´stored` although not as an event itself, but as an emotional value, which is as individual as the emotional values attached to pleasure and this is stored with the sensory information and characteristics of the event to which it applies. There are neuronal differences in the pain mechanism eg. interneurons stimulate firing, GABA neurons lead to excitation, but there are also many similarities. Therefore, bearing pain therapy in mind, advances in neuronal research that can lead to modulation and manipulation of sensory systems can only help advancements in knowledge of this equally important pain system.

Since we`re talking about the topic…….

….bacteria loaded with antibodies and magnetic particles can be guided through the blood to appropriate parts of the body to provide local treatment. Can we assume therefore, it would be possible to do the same sort of thing using neurotransmitter agonists or antagonists to provide localised analgesia so that pain signals are not given out? Capsaicin can cause analgesia by desensitising pain fibres so could this also be used in this way?

….each individual has his/her own pain threshold that can be manipulated by certain factors. Would it be possible through hypnosis to manipulate the threshold to such an extent that the individual becomes desensitised to pain due to injury or disease and not suffer from a general feeling of numbness?

…..it is said that only one site of pain can be perceived at any one time and there can be pain signals without the perception of pain. Could an individual or the pain mechanism itself be manipulated into perceiving pain from a lesser source in preference to a more serious source by increasing the importance and perception of the former and by also giving methods of management so that the more serious source is ignored?

inhibition of visual input by top-down modulation in the case of conscious awareness of information in working memory

Posted comment on ´Attention, working memory and phenomenal experience of WM content: memory levels determined by different types of top-down modulation` by J. Jacob, C. Jacobs and J.Silvanto and published in Frontiers in Psychology volume 6 Article 16033 October 2015

SUMMARY

Jacob, Jacobs and Silvanto explored in their article the role of top-down attentional modulation of the content of working memory and concluded that the representation of the original memory in the centre of the focused attention achieved conscious awareness and this process also requires the suppression of all incoming visual information via inhibition of the early visual cortex.  They began their article by defining their accepted model of working memory (model of Cowan, 1988) where working memory is seen as an activated long-term memory able to retain a number of activated representations in parallel some of which may be re-enacted long-term memory representations. They also quoted the extended model of working memory by Oberauer (2002) which suggests a store of reactivated long-term memory representations plus a capacity limited short term store (zone of direct access) and a store containing a single item linked to focused attention (FOA) which provides the content for goal-directed processing and is the only item to reach conscious awareness. The content of this working memory was said to be experienced as an image with qualia and could be scrutinised and modulated. The authors argue that reaching conscious awareness involves more than just modulation of the actual memory trace involving attention and requires in addition inhibition of visual input.

Jacob, Jacobs and Silvanto continued their article by proposing own hypothetical model relating working memory to attention. They explained that information exists in different states dependent on the level of attention and that this translates to memory. Three memory levels were proposed with relation to attentional control: one level with non-attended, non-conscious memory with no attentional modulation; second level with attended, phenomenally non-conscious memory with an enhanced actual memory trace due to attention; and the third level with attended, phenomenally conscious content with an enhanced memory trace and top-down suppression of visual input. The authors claimed that their model was distinct from previous ones because of distinct, non-conscious memories and conscious, attended memory states.

What followed was evidence indicating a dissociation between attention and phenomenal experience of memory content. Jacob, Jacobs and Silvanto began with the proposal that non-conscious items can be attended to, encoded and maintained in working memory. Research supporting that view was quoted as coming from the work by Soto et al. (2011) where subjects were able to maintain encoded information and use it later even when at the time they were unaware of the cue and distractors were present in the maintenance period. This indicated to the authors that attention was on the information held in the memory store (ie. FOA) in the delay period. Further research from Feredoes et al. (2011) showed that the working memory trace maintained in presence of distractors was brought about by the top-down facilitation of visual cortical regions maintaining the working memory content. Other brain areas were also reported as being involved in maintaining non-conscious items in working memory as changes in the right mid-lateral prefrontal cortex, orbitofrontal cortex and cerebellum were observed with activity in the dorsolateral prefrontal cortex, anterior prefrontal cortex and posterior parietal regions observed in visual working memory. Also, it was found that subliminal shapes in visual working memory guide attention and facilitate working memory performance. The authors therefore concluded that working memory can have non-conscious representations and so information in the FOA may not be necessarily conscious. This supported the view that attention may be allocated to working memory content without the content being conscious and hence, additional processes are required for a phenomenal experience to occur.

The dissociation between attended and conscious representations was reported in the article as being because once memory content has been brought to conscious awareness then it interacts differently with external input than with non-conscious memory content even if both are attended. The encoding of concurrently presented visual information could be changed by for example raising the level of the threshold at which information is detected and this could be independent of the similarity between the mental image and visual input. This was said to prevent the mental image from being weakened, but the reverse could also occur with external input impairing the conscious experience of memory content. The encoding of external stimuli matching working memory content was reported as being enhanced and reaching visual awareness more effectively whereas dissimilar information was said to be suppressed. In this way, working memory was said to act as a ´gate keeper`.

Jacob, Jacobs and Silvanto continued their discussion about their model demonstrating that consciousness and attention are dissociated and that more than just attention is required to bring content to conscious awareness by saying that their 3 state model supports well-known work on consciousness by Dehaene et. al. (2006). Dehaene et al. 2006 described information processing as being subliminal, preconscious (both considered unconscious forms) or conscious. Subliminal was defined as limited bottom-up processing due to attenuated stimulus strength potentially interacting with top-down attention. Preconscious was described as being dependent on stronger signal strength, but was limited, or had no top-down attentional modulation, but did have a potential for conscious access. Conscious processing was described as involving top-down and bottom-up activation beyond a sensory threshold and consistent with working memory models with information assigned to the FOA and activated long-term memory representations as being non-conscious. Jacob, Jacobs and Silvanto stated that Baars classic Global Workspace Theory was not a model of working memory since it described how visual input reaches awareness, but they did quote it in their article as providing evidence of the dissociation between consciousness and attention. In the Global Workspace Theory subliminal, attended information processing is short-lived and there is no attended but non-conscious content whereas in their model, non-conscious, attended representations in the working memory exist and are maintained for longer periods and can survive distractor appearances.

The article then went on to discuss the different requirements for the achievement of conscious awareness with reference to the 3 memory level model put forward by the authors, Jacob, Jacobs and Silvanto. As given above, it was stated that in the working memory model, attentional modulation of the content with conscious awareness requires an additional factor and this was proposed as the suppression of visual input from external sources via early visual cortex inhibition. This view was supported by fMRI studies where subjects carried out visual motion imagery and had were observed to have reduced early visual cortex activity, but enhanced activity in the motion-selective extrastriate region V5/MT. This was explained by the necessity of knowing the source of the content. The authors said that working memory would have to know that the conscious material was internal (the so-called Perky effect where external input is confused as part of mental imagery). They claimed that this was important so that external information is not concurrently consciously experienced as it would have a stronger neural signal than the competing conscious trace from the same item sourced from visual imagery.  Therefore, when the content of the working memory needs to be brought to conscious awareness there should be a bias towards internal, reactivated information and this is achieved by inhibiting the visual input from real-time external events.

The authors concluded their article by re-stating their hypothesis that there is a relationship between phenomenal experience of content in working memory and attentional control and that conscious awareness involves the creation of a second, distinct representation (previous work) generated and by the top-down facilitation of the original memory trace and favoured to incoming external information by suppression of incoming visual input at the cortical level. They concluded their article by suggesting a possible future neuroimaging study to prove their hypothesis.

COMMENT

What makes this article interesting is the continued exploration of the cognitive capability, working memory. This article by Jacob, Jacobs and Silvanto links working memory with attentional modulation and conscious awareness and they propose two things: one, that the working memory is multi-facetted with facets having different attendance and awareness characteristics; and two, that the facet relating to attended, conscious information obtained from the reactivation of long-term memory relies on the suppression of incoming information from the external environment in order to reduce neural competition and this suppression is elicited by top-down attentional system modulation.

Before we can discuss their hypothesis, we have to look at and provide a neural mechanism as to what working memory is and what affect attention can have on it. My view is that working memory is not like Jacob, Jacobs and Silvanto suggest that of a ´melting pot`, a single area or brain ´splodge of activity`, but a state where multiple areas are active at the same time working on multiple ´tasks` most of which are unconscious, but at least one can reach conscious awareness. (I say at least one, because divided attention can mean that conscious awareness flits between at least two cognitive events and may appear virtually simultaneous, but from a neurochemical basis are not). Working memory is not a process, but is a ´condition` where processes can occur and these processes involve common, shared tools such as sensory input, attention and decision-making and involves information which can either come from reactivated long-term memories or from newly inputted information from the external or internal environment. The idea that multiple processes are involved is not new and well-known descriptions of the working memory (eg. that of Baddeley and Hitch, 1974) has it as a group of capabilities such as the central executive (synonymous with attention), episodic buffer, phonological loop and visuospatial pad. Jacob, Jacobs and Silvanto concentrate on only the attentional system`s contribution to function, but other tools participate as well in either the bringing of the information into the working memory state or in the information`s scrutiny or manipulation.

The informational content of the working memory can be from differing sources such as reactivated long-term memories including associated emotions and event value (Jacob, Jacobs and Silvanto`s equivalent of information with quale) as well as newly sourced material from incoming input from the external environment or internally sourced information or created material via manipulation. Whatever the source, we can assume that the time the information spends in the working memory state is dependent on the firing mechanisms in play and to an extent, its relative importance to the individual whether from task performance or personal value. For example, information said to be in the short-term memory store according to Jacob, Jacobs and Silvanto is from the neuronal firing of relevant cells and therefore, the period of activation would be dictated by the time the neuronal cells could fire before chemically being exhausted and they shift into their refractory periods to replenish. The information would then fade. Repetition or manipulation of material would lead to sustained neuronal cell firing and the holding of that information in the working memory state for longer periods just like in the case of formation of long-term memories.

In neurochemical terms, the firing of neuronal cells depends on the source of the material eg. visual working memory activates different brain areas to language and this allows the scope of the working memory state to broaden if multiple skills and senses are involved in the content. The cells themselves are considered to be multi-tasking (Messenger) which allows cells to be representatives of information, but also susceptible and instigators to tools such as attentional modulation and the whole state representing an event depends on connectivity of multiple areas so that items are inputted, maintained and manipulated. Visual working memory is said to involve many areas such as the prefrontal cortex, cingulate cortex, hippocampus and entorhinal cortex (for relaying the signals, synchronicity and binding), fornix and thalamus (for basic sensory information relays), V4 and medial temporal lobe and inferotemporal lobe (for visual pathways and visual attention) as well as the cerebellum (for procedural memory and movements). Manipulation and holding of material is the responsibility of the lateral- and post-parietal cortex.

Whether the informational content of the working memory state has conscious awareness or not depends on several different factors and is independent of the source (eg. internal or external) or type of information (eg. visual, auditory). Jacob, Jacobs and Silvanto hypothesise 3 working memory levels dependent on whether material is attended or not, or conscious or not. What selects information for conscious awareness is essentially the strength of firing and strength of firing is dependent on quantity of information (ie. the more cells active, the greater the chance of reaching conscious awareness) and quality (ie. the more characteristics available including emotional status and value, the greater the chance of reaching conscious awareness) and the task at hand (ie. the more difficult the task or more complex for example the greater the chance of conscious awareness). In the case of, for example, the simple recall of a procedural memory like riding a bike then this is unlikely to evoke conscious awareness especially if other more challenging input is available at the same time, but it will and will enter the working memory state if it is coupled with learning to ride a new bike with a different gearing system. Therefore, conscious awareness of one facet of the working memory will reflect the strength of firing of the information independent of its source. However, it should be remembered that conscious awareness represents only one ´draft` of an experience if the multiple drafts theory for consciousness is to be believed. This can be compared to working memory which can also be considered as one ´draft` of information with manipulation, the addition of supplementary information (associated with ´filling in` of consciousness) providing the other ´drafts`. The link is also supported by looking at the brain areas involved. Consciousness involves the firing of particular brain areas such as prefrontal cortex, cingulate cortex and parietal cortex and these are as shown above the same areas as those said to be involved in working memory. Therefore, the two capabilities can be said to be linked although in most cases dissociated.

So, we have looked at what working memory is and how conscious awareness of some of its content can be brought about, but Jacob, Jacobs and Silvanto expanded this by hypothesising that working memory and consciousness relies in 2 cases of their 3 memory model on the involvement of the attentional system. Only the non-attended, non-conscious form had no attentional modulation according to them and this can be is explained by considering this form of working memory as being associated if at all with recall of memories without the need for any further processing. The only disadvantage of this definition is that it is unlikely that in this case the working memory state at all since the working memory state is usually associated with information manipulation and pure recall does not require any further processing to be totally effective. One of the other forms of memory described by Jacob, Jacobs and Silvanto states that information is attended, but phenomenally unconscious and so attentional modulation can lead to enhancement ie. more processing, but still remains under the threshold for conscious awareness. It is only the final form where conscious awareness is attributed to working memory content and this attended information is in the centre of attentional focus and can be scrutinised and modulated. The link between attention and consciousness is not new with Dehaene and Changeaux`s 2011 Neuronal Global Workspace Theory proposing that attention acts as a selecting mechanism for conscious contents and working memory as a specific store.

Although we think of attention as a discernible force, biochemically it is not. It is a mechanism that instigates the strengthening of certain information and the weakening of other rather like a dial so that some information is attended and other not. This is an important quality if we want a working memory state where some of the information active at that time can be scrutinised and manipulated and the rest ignored or simply carries on. Competitive selection of information can be based on feature strength or even biased because of stimulus colour, movement or emotional value. The level of attention can also vary with a low level described by Marchetti as being with or without consciousness or a high level as associated with selected events, manipulation and decision-making for example. It can also be bottom-up based on the stimulus features and lower level sensory pathways and automatic recall of values, memories for example, or top-down meaning that the allocation of the resources is under the control of the higher cortical areas and dependent on memory, values, associations and decision-making. Independent of which attentional system is exerting its control, the first 270 milliseconds of any event is neurochemically the same and it is only after this time that the allocation of further deciding attentional resources occurs. This early period could be described as the preconscious period for some events with them either reaching conscious awareness later if focused attention is applied (after 300 milliseconds) or if no attentional resources are directed at them being the ´never-conscious`.  Attention may not also be considered as a single focused capability centred on a limited area since Marchetti also described attention as being ´diffuse` ie. like ´gist`, spread over a number of aspects of one event. In Jacob, Jacobs and Silvanto`s model the focus of attention is the conscious event and the diffuse attentional state produces no conscious awareness which differs from Marchetti and others who say that even in this condition, conscious awareness can occur. This is credible if we consider diffuse attention as ´gist` – we may not be exactly aware of all facts, but we have an overall understanding.   Therefore, linking information in working memory and how some of this reaches conscious awareness and some not relative to the amount of attentional resources aimed at it is understandable.

However, Jacob, Jacobs and Silvanto went further with their working memory model by saying that the attentional system inhibited certain informational input into the working memory state if the informational content was of a particular kind ie. was information obtained from reactivated long-term memories for the same event as being observed in real-time. This inhibition was brought about to reduce the competition for working memory capability from the incoming information from the environment which they said would produce naturally stronger neuronal firing and therefore, evoke conscious awareness in preference to the reactivated familiar material. Two copies of the same material would exist with the copy of the newly inputted material being stronger. Therefore, Jacob, Jacobs and Silvanto´s inhibition hypothesis is understandable since: competition would increase the perceptual load and therefore, certain characteristics may be ignored; it provides a reason why material is not processed again if it has already been processed and stored (eg forms capability of object recognition); and the reactivated information may have more recalled associated material with it such as emotional state and personal value than the new input. The hypothesis is supported by the observation that we are often unaware of a change in a person`s appearance for example (attentional blindess). This implies that our recognition of that person relies on recall of stored information in response to cues from the real-time encounter, but close examination of the person`s appearance in real-time does not occur. Although this may be negative in that changes are not observed, using recalled information has the advantage that it is more than just visual characteristics for example and that other information and associations are also part of the reactivated information and hence, the capability of the working memory to process material is strengthened and so is also the chance to reach conscious awareness. However, this inhibition may not be possible as already indicated if the reactivated image is too different from the incoming information and may be detrimental since no updating of the stored information from sensory information will be possible.

The mechanism hypothesised by Jacob, Jacobs and Silvanto as bringing about the inhibition was top-down attentional control at the level of the early visual cortex V1, but herein lies a problem. Jacob, Jacobs and Silvanto compared the informational content in the working memory of the reactivated memory as visual imagery and visual imagery is associated with activation of the V1. Therefore, suppression of activity in this brain area by the attentional system would automatically inhibit the working memory state and suppress the visual information being recalled. Possible explanations to explain this discrepancy if this hypothesis is correct are: that the neurons firing in response to input in the V1 are not the same ones of the neuronal cell assembly group representing the stored image; or that the inhibition occurs at a lower level than the V1 cell hierarchy so that visual features are not discernible. Further research into visual imagery particularly using real-time neuroimaging is required to explore the capability.

Therefore to conclude, Jacob, Jacobs and Silvanto`s model for working memory and the levels of information relating to attendance and conscious awareness has merits in that it provides support for the view that working memory is not a single item capability, but is multi-facetted with each facet having different characteristics regarding informational content, conscious awareness and attentional system involvement. In the case of conscious awareness of one type of working memory content, that of reactivated visual images, then Jacob, Jacobs and Silvanto`s model proposes that top-down attentional modulation occurs that inhibits the input of real-time visual information if the event being observed is the same as that of the reactivated store. This has the advantages that competition for cognitive resources from the stronger real-time image is removed and that associated information such as emotional worth is also recalled and is available for processing and manipulation in addition to the event features. This strengthens the firing and is likely to increases the chance of bringing the content to conscious awareness. However, inhibition of visual input also has the disadvantage that the updating of stored information from real-time external events is prevented. Inhibition of this type could be detrimental for those people that suffer from memory recall problems since the information reactivated may be sufficient enough to trigger inhibition, but not substantial enough to aid working memory performance. Therefore, the topic deserves further investigation.

Since we`re talking about the topic………………..

…..can we assume that the same type of inhibition can be observed with auditory memories under the same conditions and also that the same type of inhibition occurs when the events are multi-sensory?

…..if the experiments are repeated using the same reactivated event as the external event being experienced, at what level of dissimilarity of content is the inhibition of the external input removed? Does this correlate time-wise to when the subject becomes conscious that a change has occurred? Does this occur even if that change refers only in a change in emotional worth?

……the administration of ketamine leads to an increase in irrelevant information in working memory. Therefore, if ketamine is given and the experiments repeated, would conscious awareness be on multiple events akin to divided attention and how would this affect the suppression of real-time visual input? Is it possible that attention is diffuse rather than focused, or is it focused on multiple events (like divided attention) and hence, not reach the threshold value for visual input inhibition?

… what effect could working memory training have on the inhibition of V1 activity?

 

discussion about hypothesised link between the menopause and Alzheimer disease

Posted comment on ´Changing your mind`  by J. Hamzelou and published in New Scientist 3141 2^nd September 2017 p.36.

SUMMARY

Hamzelou began her article by describing some of the symptoms experienced by some women going through menopause. She stated that the cognitive changes observed in menopause, eg. migraines, mood swings, anxiety, short-temper, forgetfulness and insomnia, resemble the presenting symptoms observed with sufferers of Alzheimer disease and may in fact signal the start of that disease. In order to support her view Hamzelou quoted work by Brinton, a Californian scientist who studies the hypothesised link between the menopause and Alzheimer disease. Brinton hopes to develop therapies that artificially boost hormone levels that would lead to protecting the brain from the detrimental changes that could lead to dementia later in life.

Hamzelou continued her article by describing the biological basis of menopause and listed the common non-cognitive symptoms observed, eg. fatigue and weight gain. She said that in comparison to those obvious symptoms, cognitive symptoms are often overlooked since they occur at a time when other reasons can be given to their appearance eg. ageing and also because society demands a level of expectation and endurance when considering mental health problems. However, research given by Hamzelou as being carried out in the last decade, has shown that a decrease in oestrogen level has effects on memory, mood and even what has been termed the  ´brain health` of men and women. Research by Brinton and others has shown that reduced levels of oestrogen are correlated to alterations in the type of energy the brain cell uses and to a reduction in the production of energy. Under normal conditions, oestradiol increases the activity of the mitochondria in brain cells involved in normal cellular energy production and therefore, it helps cells recover from damage associated with normal ageing. Grimm of the University of Queensland, Australia supports Brinton`s view and was quoted in the article as saying that the drop in oestrogen makes the brain more sensitive to damage that could lead to death of neurons. Brinton believes that the fall in oestrogen that occurs in the menopause causes the brain to produce less energy and to change the type of energy it uses. Glucose is the normal energy source of brain and this is reduced by 25% in tissues of menopausal sufferers. To overcome the shortage of glucose the cells, according to Hamzelou and Brinton, begin a ´starvation` response and use fats as their energy source instead. They are also believed to use myelin as well which can be found in the protective shield around the neurons themselves. Although their studies were carried out on mice, Brinton suggested that the results could also apply to humans and some research supports this. A decrease in glucose metabolism, a change in white matter volume and grey matter volume and an increase in beta amyloid production relative to men have been observed. The switch in energy source was also suggested by Brinton to provide an explanation for some of the other symptoms of menopause. For example, the metabolism of fat because it is a less efficient energy source than glucose creates more heat and this excess heat in the brain was suggested in some animal studies as possibly triggering the menopausal non-cognitive symptom of  hot flushes.

Hamzelou continued her article by describing why some researchers link the supposed protective effect of oestrogen on cognitive function and hence why the menopause and its cognitive symptoms can be linked with symptoms observed in Alzheimer sufferers. Brinton investigates why women are more susceptible to Alzheimer`s illness and thinks that the hormonal transition occurring  in the perimenopause stage and full menopause may be the cause and start of Alzheimer illness in some women. Studies have shown that two thirds of people with Alzheimer`s illness are women and even though the disease is diagnosed when they are in their seventies, the disease actually starts around 15-20 years earlier when the natural menopause occurs.  The link to energy production during the hormonal transitions occurring in menopause was supported by work from others. For example brain scans measuring how much glucose is being metabolised across different brain regions were carried out in 2005 by Mosconi and colleagues of the New York University and they observed reduced glucose metabolism with Alzheimer sufferers and women who were in perimenopausal or postmenopausal stages. These observations compared favourably to Brinton`s observations in mice and suggested a link between a decline in glucose metabolism in the menopause, ageing and Alzheimer illness.

Hamzelou then went on to describe the natural progression of such results – if oestrogen has a brain effect when it falls, then what happens when it is replaced? Some studies suggested that hormone replacement therapy (HRT) could prevent dementia, but a trial of 7500 women in 2005 by the Women`s Health Initiative Memory Study found that HRT actually quickened cognitive decline and increased the risk of not only dementia, but also breast cancer and cardiovascular disease.  Hamzelou quotes researchers who believe that the study was flawed and describes the study by Pinkerton at the University of Virginia who looked at women given conjugated equine oestrogens. They stated that the negative link between HRT and cognition was incorrect since the administered oestrogen was obtained from pregnant horses and therefore, not an appropriate hormone source for premenopausal women and that the women taking part in the study were already over 65 and were therefore, too old to be described as suitable menopause subjects. They said that their brains had already adapted to low oestrogen levels and that the number of relevant receptors had already decreased. Pinkerton went on to say that there appeared to be an optimum time for HRT treatment (termed ´window of opportunity`) and that this time period was limited to between the appearance of the menopausal symptoms and the time when the brain was still responsive to treatment. They said that oestrogen can work better on healthy cells and therefore, HRT works better when women take it around the time of the menopause. In response to the increase of detrimental side effects observed with HRT administration, Pinkerton said that in the case of breast cancer, administration of HRT was linked to only an increase in breast cancer of under one case in a thousand. Pinkerton concluded by saying that HRT should be used only if women experience unpleasant symptoms, but the view of ´lowest dose for shortest amount of time` should be replaced by the caveat of ´making sure that the treatment is appropriate`. The determination of what is appropriate has not yet been made. Hamzelou continued by suggesting that the better solution may be to use oestrogens that only work on specific organs eg. one that works on brain, but by-passes breast tissue. She quoted in her article work by Raber of Oregon Health and Science University in Portland who reports that drugs of this nature are already in development. Hamzelou also quotes Brinton who suggests a nutritional approach to protect the brain from the effects of hormone loss. This view is linked to food obtained from the diet and brain function. For example, ketogenic diets appear to benefit epilepsy sufferers. In the case of the menopause, a high fat diet is not advised for people at risk of weight gain and against the view of a healthy diet rich in fruit, vegetables and grains being good for brain health. She also recommended exercise and keeping active, which has been shown to boost mood and cognition and can increase bone mass.

The article concluded with Brinton describing the future with individually tailored hormone therapies given at the right time to treat menopause symptoms and prevent Alzheimer`s illness.

COMMENT

The menopause can be regarded as a ´sensitive` topic at the best of times particularly with women, but when it is linked in scientific research to the appearance of Alzheimer disease then the feelings it evokes are intensified. Therefore, any research into the association between these two topics should be rigorously examined because unlike other factors causing changes in memory and cognitive capability (eg. the administration of certain drugs or a stroke) the natural decline of a hormone due to increasing age is something that transcends effects under the control of the person herself. Experimentation into the menopause in humans is beset with problems. For example, because the onset is variable and the occurrence of relevant symptoms is individual. We know that natural occurring menopause is clearly defined as existing one year after the last menstrual period, but definition of the ´last menstrual period` is difficult to define itself since women experience differing forms of menstrual periods in the perimenopausal phase. The definition of the beginning of menopause is therefore easier to establish when it occurs through surgical intervention eg. hysterectomy or also through disease such as polycystic ovarian syndrome. Even if the beginning of menopause can be determined accurately time-wise the variation in symptoms whether physiological, cognitive or emotional makes interpretation of results difficult in humans. Physiological symptoms such as hot flushes or loss of sleep are probably easier to see and measure, but the cognitive symptoms (eg. irritability, loss of spatial memory) on which this Blog is focussed are more difficult since they are in part ascertained through self-reporting which can be unreliable and are subjective with daily variations and differences depending on personal situations. However, we can say that the menopause is a physiological condition or state brought about by decreased levels of circulating oestrogen/oestradiol and therefore, we can assume that whatever symptoms are observed then they occur as a result of this decrease in circulating hormone.

Oestrogen is produced from progesterone by the ovaries and instigates a wide variety of effects in the whole body. However, it is also produced in the brain, blood vessels and bone synthesised from cholesterol to various intermediate compounds eventually to pregnenolone which then is converted to 17alpha-hydroxyprogesterone then to androstendione (to estrone),  to testosterone and eventually to oestradiol. Since this Blog focusses on the brain and neurochemical processes we shall concentrate here in this post on effects of oestrogen in the brain and on neurons. It can be said that the presence of oestrogen in this organ and on these types of cells has a general effect on neuronal firing and is said to elicit intracellular effects associated with changes in the DNA, membranes and from the article reviewed here on cellular energy production. This general positive synaptic effect translates into an influence on firing and is said to provide a protective effect on neurons and their functions. Exposure to oestrogen or oestradiol can mean that cells are more likely to survive hypoxia, oxidative stress and exposure to neurotoxins for example and hence, also elicit a protective effect against the development of certain mental illnesses such as multiple sclerosis, Parkinson´s disease and dementia.

When considering the effect of oestrogen on brain cell firing we should assume that the effect is not major since for example there are other systems in play which have far more wide-ranging effects (eg. NMDA concentration, glial cell functioning) and that there is a natural variation in oestrogen level anyway with the menstrual cycle with no major signal transmission shut down when oestrogen is at a low level. Therefore, we should probably consider oestrogen more as an instrument of ´fine tuning` of the neurobiological system in the same vein as the emotional system (eg. a positive influence from dopamine on the emotional system and neuromodulation of prefrontal cortex firing) or like the effect of tiredness and sleep deprivation. In order that such an influence can occur the cells in question must have oestrogen ´acceptor` capability and this will be described in more detail later on. The possession or absence of such a capability could explain why some brain areas are affected by oestrogen and why some are not and hence, why some cognitive functions are affected and others independent from oestrogen influence.

For now in the context of a positive effect on synaptic firing, oestrogen has been shown to increase neuronal firing due to the growth of neurites (increases cell viability) and an increased number of dendritic spines. For example in the case of the hippocampus, the number of spines varies with the level of oestradiol in vivo with both peaking together. The presence of oestradiol also shows that the area grows more excitatory synapses and the new spines have more NMDA receptors on them. Hence, the long-term plasticity of the hippocampus is increased in the presence of oestrogen. Also oestrogen can initiate its effect directly in the hippocampus by depressing the synaptic inhibition mechanism. Oestrogen receptors have been found on the inhibitory interneurons in the area which do not grow more spines on exposure.  The oestradiol causes the inhibitory cells to produce less GABA so there is less inhibition of firing and hence greater general neural activity which somehow triggers an increase in spine growth in the area and increases the number of excitatory synapses on the pyramidal cells. In the presence of low oestrogen then decreased spine density and a decreased number of NMDA receptors is observed as expected, but also increased acetylcholinesterase activity is seen. This implies that an effect on the cholinergic firing mechanism in the area is also influenced. These effects on the hippocampus give an explanation in part as to why certain memory systems are said to be affected in menopause since the hippocampus is believed to be responsible for the relay of information in the brain and with the neighbouring entorhinal cortex area responsible for the binding of information together. Hence, effects on object recognition and verbal memory in menopause where there is a reduced level of circulating oestrogen are seen.

Another brain area said to be affected by oestrogen is the prefrontal cortex. It has been found that dopamine activity in this area is enhanced by oestradiol and in its presence then bigger synapses are observed. The effect is associated with the presence of oestrogen receptors of the alpha type. Therefore, in this case oestrogen could influence the neuromodulatory control associated with this area and dopamine, thus explaining in part the observed cognitive symptoms in menopause linked to the emotional pathway eg. irritability, and lower decision-making capability eg. assessment of values of events.

The synaptic and firing effects observed in the presence of oestrogen are brought about by intracellular processes involving the hormone. These are believed to be associated with DNA binding and/or cellular membrane effects and also as suggested by the authors in the article reviewed in this blog, by changes in the energy producing mechanisms taking place in the cell`s mitochondria. The DNA effect is well documented and begins with the transfer of the hormone through the cell`s membrane – a process that is simple due to its non-polar molecular structure. Once inside the cell it binds to a highly specific soluble receptor protein in the cell`s cytosol. These oestrogen receptors are of the alpha or beta type and are known as nuclear oestrogen receptors (ERalpha, ERbeta). It is thought that it is the alpha type in the hippocampal CA1 area that is linked to the increased synaptic plasticity described above. The hormone/receptor complex then interacts directly with specific binding sites on the DNA called oestrogen response elements (EREs) and ultimately, this binding modulates gene transcription. The DNA binding domain is highly conserved with 9 cysteine residues, 8 of which bind zinc ions which stabilise the structure of the domain (called zinc finger domains). The ligand binding site exists at the carboxyl end and is comprised of alpha helices. Ligand binding in a hydrophobic pocket in the centre leads to conformational changes that allow the recruitment of a coactivator protein such as SRC-1, GRIP -1 or NcoA-1. These have a common modular structure and bind to the ligand binding domain of the receptor dimer. Binding to the DNA ultimately changes gene transcription so that certain proteins are either down- or up-regulated so that the oestrogen influence on the cell is realised.

The other known cellular effect of oestrogen is its binding to membrane-bound receptors (mERs) eg. GPER (GPR30), ER-X and Gg-mER. These receptors can be rapidly activated on exposure to oestrogen and their effects are believed to be associated through the attachment of caveolin-1. Complexes are formed mostly with G protein coupled receptors, striatin, receptor tyrosine kinases (eg. EGFR, IGF-1) or non-receptor kinases (eg. Src) and each causes different effects. Although binding through G protein coupled receptors eg. GPR30 has an unknown role, binding to other structures cause cellular effects eg. through striatin – some of the membrane bound oestrogen receptor complex may lead to increased levels of calcium ions and nitric oxide; through receptor kinases – signals sent to the nucleus via the mitogen activated protein kinase MAPK/ERK pathway and the phosphoinositide 3 kinase (PI3K/AKT) pathway; and finally through glycogen synthase kinase 3 (GSK- 3beta) which inhibits transcription by the nuclear oestrogen receptor by inhibiting phosphorylation of serine 118 of the nuclear oestrogen alpha receptor. Phosphorylation of the GSK-3beta removes its inhibitory effect and this is achieved by PI3K/AKT pathway and MAPK/ERK pathway via rsk.

Another possible mechanism involving the membrane is the oestrogen receptor complex`s effect on the lipid domain as a whole and the subsequent increased or decreased action of other neurotransmitter complexes existing in that same lipid domain. It is known that oestrogen elicits an effect on NMDA receptors in the hippocampus, but it also affects acetylcholine binding to the M2 acetylcholine receptor. In general, however it is likely that the overall effect of oestrogen by binding to membrane-bound receptors is increased firing activity as described above.

The third action of oestrogen at the intracellular level is that suggested by Hamzelou and researchers such as Brinton who hypothesise that the presence of oestrogen supports the use of glucose as fuel source in the cell in its energy production mechanisms, but its absence causes a change in fuel source to fats and even myelin and a decrease in mitochondrial function. What does this actually mean? In the brain, the sole source of fuel for cells is glucose under normal circumstances and we have to assume that there are normal circumstances even in the low levels of oestrogen in parts of the menstrual cycle because of diet and that the glucose transport into the cells is below maximum capacity and hence, an increase in brain cell activity will still keep glucose transport into the cell within its limits. As already described in another Blog post, cell energy production mechanisms can change according to certain conditions eg. conditions of low oxygen/high altitude. In this case, the lack of oxygen means cellular adaptation of the biochemical processes supplying energy to the cell occurs.  In low oxygen conditions, the normal mechanism of energy production means that glucose is still being metabolised by a chain of enzymatic reactions (called glycolysis) to produce pyruvate just as that occurring in aerobic respiration (ie. in the presence of oxygen),  but the second stage of the process is altered. This stage is where the pyruvate is converted by another chain of reactions into the energy molecules, ATP.  If oxygen is not present at the level required for this aerobic mechanism, then a process called lactic acid fermentation is initiated (anaerobic respiration). Lactic acid fermentation means that pyruvate is then converted to lactate by the enzyme lactate dehydrogenase (LDH). However, this anaerobic process does not produce the same number of ATP molecules as normal aerobic mechanisms, but it does provide some. The other potential problem of this scenario is the build-up of lactate which is observed in muscle cells. However, it is likely that in the brain which is dependent on a constant supply of glucose and energy that a safeguarding mechanism is in place called the Cori cycle which transports the lactate out of the cell, back to the liver where it is converted into glucose by a process known as gluconeogenesis. Again the LDH enzyme is involved and this conversion could explain the lack of appetite experienced by some when undergoing rising altitude.

In the case of the menopause, Hamzelou and researchers such as Brinton suggest that the source of fuel in the brain cell changes from glucose to fats when oestrogen levels are low. Normally, fatty acids are bound to albumin in the blood and cannot cross the blood brain barrier, but under conditions such as starvation for example,  ketone bodies are generated by the liver and transported in the blood across the blood brain barrier to partly replace the glucose as fuel in the brain cells. Therefore, Hamzelou and Brinton suggest that ketone bodies are used as fuel source. The acetyl coA formed in fatty acid oxidation enters the citric acid cycle only if levels of fat and carbohydrate degradation are balanced. This is because of the availability of the substrate oxalocitrate which forms citrate, the next substrate in the cycle. Oxalocitrate concentration is lower if carbohydrates are not available.  In fasting or diabetes, oxalocitrate is used to form glucose by the gluconeogenic pathway and therefore the substrate is not available for acetyl coA production. Therefore, acetyl coA is converted to acetoacetate (by a 3 step mechanism) and D-3-hydroxybutyrate (formed by reduction of acetoacetate in mitochondrial matrix) which with acetone (formed from slow spontaneous decarboxylation of acetoacetate) forms compounds known as ketone bodies. The major site of production of ketone bodies is in liver mitochondria and these are transported via the blood to other tissues. They are used as fuel sources in the muscle, renal cortex and brain in cases of starvation (75% of fuel in prolonged starvation) and insulin-dependent diabetes mellitus. In the latter, the absence of insulin means that the liver cannot absorb glucose and as a result cannot provide oxaloacetate for the fatty acid derived acetyl coA process and cannot prevent fatty acid mobilisation by the adipose tissue. Therefore, the liver produces large amounts of ketone bodies which are strong acids and the presence of such high levels causes severe acidosis. This results in a decrease in intracellular pH which impairs tissue function – a condition already described in a previous Blog post when considering cell function in high altitude conditions. The brain begins to use acetoacetate after 3 days of starvation (a third of energy needs met), but after several weeks it is a major source. The advantage is that ketone bodies are built from released fat and this preferable to breaking down muscle instead.

Although the hypothesis by Hamzelou, Brinton and supporters about the switch from glucose to fat and even myelin may be true and that glucose metabolism is reduced in the brain in low oestrogen, then if this hypothesis is correct, then we must assume that in menopause, the brain cells are not getting their normal fuel source because of the lack of oestrogen. Therefore, under normal conditions oestrogen would then aid the transport of glucose into the cell by affecting the insulin signal on the glucose transporters, or by directly effecting the glucose transporters themselves. Is there any proof of this? There are no reports of significant effects on insulin sensitivity or levels or glucose levels in the menopause. However, there is a report of the change in insulin metabolism. Therefore, the cause of effect could be indirect through reported changes in diet in menopausal women where diet is altered to counteract the increased weight gain and fat deposits observed around the middle. A strict diet could translate into starvation conditions and hence, changes in fuel sources as indicated above could be observed. It is likely that if a normal diet is maintained then such an effect on fuel source would not be seen.

Therefore, the overall conclusion about the action of oestrogen in the brain is that it is a molecular compound that affects cell functioning of susceptible cells by either binding to the cell membrane or by internally binding to receptors which bind directly to the DNA and affect gene transcription. This can result in either a negative or positive effect on cell functioning. If the cell has oestrogen acceptor capability then oestrogen can affect that cell, that area and ultimately have an effect on cognitive function of some sort linked to that brain area eg. oestrogen influences the activity of the hippocampus by inhibiting the interneurons and hence, increasing synaptic firing and increased plasticity of area in question. An absence of the hormone will lead to observed changes in verbal memory, object recognition, spatial memory (only rats), short term memory, learning new associations, long term memory, working memory plus depression. However, the action of oestrogen should be considered more in terms of ´fine tuning` systems and mechanisms already in place rather like the effects of tiredness. This would in part explain why there appears to be a neuroprotective effect with oestrogen ie. cells are more likely to survive hypoxia, oxidative stress, exposure to neurotoxins for example or protection against diseases such as multiple sclerosis, Parkinson`s disease and dementia if exposed to oestrogen or oestradiol. The positive oestrogen effect on gene transcription and synaptic firing would counter-balance the negative effects caused by the cellular stresses.

This leads on to the hypothesis proposed by Brinton and others and explained by Hamzelou in her article about a link between the menopause and Alzheimer`s disease. It is said that there are several similarities between the two conditions eg. the start of menopause is considered to be linked to the same time as the start of Alzheimer`s disease; women are far more susceptible than men; and the presenting symptoms relating to cognition appear to be the same or similar. Therefore, we must question whether this is just circumstantial or whether there is a real link. With regards to timing, the menopause or reduction in oestrogen as described above could initiate some minor temporary changes in physiology which could lead on to changes in sleep patterns, depression and anxiety and small changes in performance of some cognitive functions. Although the physiological changes seen with Alzheimer`s disease are known for times later on in the disease progression, the physiological changes associated with the early stages are to date not clearly defined. It could be that these are actually the same changes as those observed in menopause ie. changes in sleep patterns, susceptibility to depression and anxiety, reduced levels of interest and hence, lower levels of mental stimulation etc. and therefore, the timing of the menopause and onset of Alzheimer disease would appear to be the same. Of course, it should be remembered that not all women who experience the menopause go on to develop Alzheimer disease and menopause and Alzheimer disease  are associated with more elderly people and hence, timing could be a reflection of the normal ageing process and the changes in life style, aspirations, emotional stability that could accompany this particular life period.

The second association between the menopause and Alzheimer disease according to Hamzelou, Brinton and others is the observation that Alzheimer disease is more prevalent in women and understandably, the menopause is a female condition. Since brain neurochemical mechanisms are independent of gender then we must assume that the difference is due to either physiological differences between the female and male brain, or possibly could the reflect the way in which men and women mentally approach and carry out events. The latter is probably a product of the former and therefore, the observation that oestrogen level has an effect on the performance of the hippocampus (described above) could explain why there is a gender difference in the appearance of Alzheimer disease. The hippocampus is an important brain area with multiple roles in cognitive functions such as information intake and binding, memory mechanisms, working memory and decision-making and is known to be progressively and extensively negatively affected as Alzheimer disease progresses. Women appear to have naturally larger hippocampal areas and therefore, this could provide a possible reason why women appear to suffer from Alzheimer disease more than their male counterparts.

The third similarity proposed by Hamzelou, Brinton and others linking menopause to Alzheimer disease is that the cognitive symptoms of the menopause are similar to those seen with sufferers of Alzheimer disease. Both appear to be a collection of cognitive symptoms linked to relaying information taken in, binding of information together, value assessment for example and hence, the similarity of symptoms of for example lack of memory, decision-making problems and emotional status changes are understandable. As stated above, circulating oestrogen appears to affect synaptic functioning and as with the timing association physiological changes would instigate observable performance changes. Since both produce to some extent permanent changes in physiology eg. menopause causes minor changes due to its ´fine tuning` role and Alzheimer disease massive changes because of amyloid deposits and abnormally high apoptosis of neurons then symptoms would be appear to be the same.

There is however a difference between menopause and Alzheimer disease in relation to whether cognitive performance can be restored by treatment of oestrogen replacement therapies. With Alzheimer disease in the later stages of the disease, administration of oestrogen replacement therapies appears to have no beneficial effect. In the former however, treatment can reverse some of the symptoms eg. verbal memory, short term memory are improved and there are positive effects on sleep and emotional state disturbances, eg. depression is reduced. This is understandable since falling levels of circulating oestrogen are being boosted by the administered oestrogen compounds and hence, the positive effects on DNA transcription and synaptic firing are being restored. Increased expression of the oestrogen alpha receptor in hippocampal CA1 area and increased NMDAR synaptic transmission have been observed with the administration of oestrogen compounds to mice menopausal models. However, most research appears to suggest that the positive effect of this oestrogen administration appears to be limited to only a short period when falling levels are minimal (called the ´window of opportunity`) which implies that in the long-term other changes are occurring in the synapse and brain areas that are not associated with the fine tuning mechanism brought about by the presence of the oestrogen hormone. These physiological changes could be those linked with natural ageing for example or instigated through life-style changes brought about by a variety of reasons. This may be important because other things appear to be beneficial for reduction of menopausal cognitive symptoms eg. exercise, proper diet, social contact, mental stimulation, appropriate sleep patterns. These can possibly restore the balance or counteract the loss of oestrogen experienced in the menopause. One factor that should be considered relating to this is the importance of zinc in brain cell functioning. Zinc deficiency is known to cause anorexia, lethargy, diarrhoea, impaired immune system, growth restriction, intellectual disability, depression, loss of appetite and disorders of fear conditioning. There is a range of effects because zinc ions have important functions in general in nerve conduction in the brain, roles in correct enzyme functioning such as carbonic anhydrase, aspartate transcarboamylase, aminoacyl –tRNA synthase, metalloproteases and in neurons in particular an important role in the phospholipid cell membrane signal and in relation to menopause in  steroid binding to the receptor as seen in the case of oestrogen. It is possible that menopausal women could suffer from zinc deficiency due to dieting and/or poor diet. Food stuffs containing zinc are bread, eggs, oysters, liver, meat, dairy products and pulses and weight gain associated with falling oestrogen levels may mean that the diet is restricted of these zinc containing foods. This deficiency could lead to the wide range of effects attributed to the multiple cellular roles of zinc.

Therefore, can we definitively say that oestrogen reduction in menopause is linked with Alzheimer disease? It is likely that oestrogen is not a major player in neuron function rather it provides a ´fine tuning` mechanism for synaptic physiology and function in the same way as tiredness or emotional state changes can. It appears that its effect in the brain is limited to particular areas such as hippocampus and prefrontal cortex which play important roles in cognitive functions such as memory and decision-making. Therefore, the presence of oestrogen may provide a neuroprotective effect on certain neurons which allows these cells to more likely survive extreme negative conditions such as those seen with  hypoxia, oxidative stress and exposure to neurotoxins. This of course naturally translates then into positive changes on cognitive functions so that oestrogen is said to have a protective effect against certain mental illnesses such as multiple sclerosis, Parkinson`s disease and dementia. It is then understandable that conditions where there is an absence of oestrogen or where levels are low such as the menopause lead to minor effects on cognitive performance. However, the effect could be also attributed to normal ageing processes being experienced at that time. The association between menopause and Alzheimer illness, although symptoms appear similar, is likely to be indirect with general ageing, certain conditions such as stroke and lifestyle changes being the main causes of the appearance of the disease. Therefore, it is understandable that boosting the level of oestrogen when it is naturally falling can provide some positive effect on certain cognitive functions, but only temporarily. Probably of more benefit to women experiencing the menopause is the continuation and maintenance of good life style practices.

Since we`re talking about the topic………..

…..if synaptic firing is enhanced by the presence of oestrogen because of the inhibition of hippocampal interneurons can we assume that the administration of a GABA antagonist preferably targeted to the hippocampal area simultaneously with the administration of oestrogen to ovariectomised mice will block this positive effect? Would the expected behavioural changes relating to restored spatial memory also be absent?

…..using real-time functional MRI would it be possible to chart connectivity between certain brain areas eg. hippocampus, amygdala and prefrontal cortex during the course of a problem-solving type task using menopausal subjects and to monitor the effects that the administration of either oestrogen or progesterone pre-testing would make on those connectivity patterns?

…..performance of place recognition tasks was found to be reduced in female rats who were in the proestrus (high oestrogen) phase of their oestrous cycle. An excessive consumption of sugar sweetened drinks daily beginning 14 days before testing was found to protect the rats from this negative change. This was attributed to the sugar consumption causing functional changes in the hippocampus. Object recognition appeared not to be effected. Can we assume that the same pattern of results would be unlikely to be observed with human females because of the effect of insulin, but may produce a problem in those that suffer from diabetes?

bioelectric signals and membrane ion channels

Posted comment on ´Bioelectrical Signals and Ion Channels in the Modelling of Multicellular Patterns and Cancer Biophysics` by J. Cervera, A. Alcaraz and S. Mafe published online in Nature Science Reports February 4^th 2016 6:20403 doi 10.1038/srep20403

SUMMARY

Cervera, Alcaraz and Mafe state in their article that bioelectrical signals and ion channels are central to spatial patterning in cell assemblies (ensembles) and are therefore, important in determining cell positioning and the organisation of multicellular groups. Defects or deficiencies of the bioelectric signal are also implicated in cancer. The research group proposed a model or an approach to a bioelectrical network of non-neural cells where the biochemical coupling of the assembly is brought about by the action of an ion channel blocker. Their approach was based on three concepts: that a cell`s electrical state was characterised by the membrane potential of that cell which was regulated by voltage gated channels that had depolarising and hyperpolarising capabilities; that the long-range electrical coupling between neighbouring cells of multicellular assemblies was brought about by gap junctions; and that the electrical state of the whole multicellular assembly could be changed by the administration of a biochemical agent locally in the cell`s microenvironment and this could alter the conductivity potential of the membrane`s ion channels. Using their model, Cervera, Alcaraz and Mafe investigated the electrical effects of small neuronal assemblies in spatial patterning, the role of ion channels in cancer biophysics and the distribution of charged nanoparticles over neuronal assemblies. They found that spatial patterns arising from their model are characterised by a map of cell potentials which are ultimately regulated by the voltage gated channels on each cell. The spatiotemporal patterns could normalise areas where there were abnormal cell electrical states ie. from the administration of charged nanoparticles and these findings indicated that bioelectric signals of multicellular groupings provide a new insight into the biophysics of cancer.

Cervera, Alcaraz and Mafe began their article by describing the connectivity between cells of multicellular groupings as not just biochemical in nature, but also bioelectrical, a fact they said is often disregarded. They cited examples of where tissue morphology and morphostatic fields are important in building and maintaining cell group architecture and where cancer results from the destruction of this architecture. Cancer cells have been observed to have an overexpression of specific ion channels (ie. an up-regulation of sodium ion channels and a down-regulation of potassium ion channels) and so tend to exhibit sustained depolarisation (ie. have a low membrane potential in absolute value). According to Cervera, Alcaraz and Mafe, depolarisation of the cell leads to a significant level of spatial distribution of negatively charged lipids and a significant effect on their interactions with positively charged proteins which may lead to the activation of intracellular biochemical pathways that result in cell proliferation.

The authors continued in their article with a description of the cell electrical state. They said that the electrical state of the cell is described by the membrane potential (Vmem) being less than zero and this is defined as the potential difference between the cell cytoplasm and the extracellular microenvironment under zero current conditions. This potential difference regulates the entry of sodium, potassium, calcium ions and other biologically-relevant molecules into the cell. It is a dynamic system transitioning between low (depolarized, abnormal) and high (hyperpolarized, normal) electrical states. Cervera, Alcaraz and Mafe said that the cells of their model exhibit bioelectric bistability (observed with for example neural cells) and others have demonstrated it also in their experiments. Hence, there are 3 values for Vmem: one for the hyperpolarised state, one for the depolarised state and one for the intermediate unstable transition state. Transitions are induced by modifying the equilibrium potential (Ein) which is dependent on ionic concentrations within the cell. It may also be associated with changes in biological parameters such as the pH and the ionic concentrations of the salt solution regulating conductance ratio (Gout/Gin). Other ion channels aside from those of sodium and potassium are also likely to be involved.

Cervera, Alcaraz and Mafe went on in their article to describe the case of cancer cells with relation to membrane potential and conductance ratios. In the case of cancer, anomalous inward-rectifying potassium channels were found in tumour cell lines giving values of Vmem different to those found in normal cells. It was also hypothesised that voltage-gated sodium channels are associated with depolarized values for Vmem in cancer cells. Abnormally low absolute values of Vmem correspond to plastic (not yet differentiated) cells while high absolute values of Vmem are found in terminally differentiated cells. Therefore, it was concluded that there is in the case of cancer cells simultaneous up-regulation of sodium ion channels (and hence, increased sodium ion inward currents) and down-regulation of potassium ion channels (leading to a decreased potassium ion outward current) giving a conductance ratio for Gout to Gin greater than 1. This implies that the membrane potential Vmem decouples from the normal hyperpolarized value Ein, bistability and cell depolarization regime. Therefore, depolarization is seen as a characteristic of abnormal cells that promotes the initiation of biochemical signal cascades. The addition to the external microenvironment of an ion channel blocker which would act on the outward rectifying channel in these circumstances could then decrease the channel conductance ratio Gout to Gin to less than 1 and have an effect on the existing bioelectrical state of the cell.

The authors then went on to extend their model (using long-range electrical coupling simulated by effective conductance ratios (G) and capacitances (C) arranged in parallel) to describe electrical states within multicellular groupings. This topic was of interest to the authors since abnormal tissues appear to have defective intercellular communication. Using their model, Cervera, Alcaraz and Mafe described the bioelectrical states of the cells in terms of protein channels acting as gap junctions between neighbouring cells of the cellular assembly. External ionic concentrations were ignored in the model with potential changes by the cell only occurring because of the gap junctions present.

Having described the role of non-functional junctions and defective intercellular communication in uncontrolled growth regulation, Cervera, Alcaraz and Mafe went on to report on the anticipated normalisation of small regions of the cell`s membrane of abnormal, depolarised cells brought about by modulation of the ion channels present. This would have the potential for restoring long-range gap junctions occurring in cell patterning and result in the removal of the conditions associated with tumorigenesis. The authors hypothesised that it would be possible to do this if the conductance ratios of coupling cells were high enough. The non-uniform distribution of gap junctions was suggested as allowing the coexistence of spatial regions of cells having increased intercellular communication (ie. having high G values – strong coupling and hyperpolarised state) acting as an electrical buffer with other cellular regions having decreased communication (low G values – depolarised state and abnormal). However, this was suggested as not being possible if there were a high number of abnormal cells within the multicellular assembly. Another reason given for the unlikelihood of this scenario was that low values of G produce cell isolation with inhibitory electrical signals characteristic of hyperpolarised potential neighbouring cells. This could be modulated however and reversed by the administration of particular agents in the external cell microenvironment. The authors did suggest that this was unlikely in this scenario because of the slow speed of diffusion of the agent and the biochemical reactions that may result.

A second scenario discussed in the article was the effect of the upregulation of the ion channels which leads to abnormal cell functioning and stimulates uncontrolled proliferation. In some cases, this causes persistent depolarisation and modifies the spatial distribution of negatively charged lipids in the cell membrane leading to the clustering of signalling proteins with positive residues around them. This can result in the initiation of biochemical pathways promoting cell proliferation. Cervera, Alcaraz and Mafe gave the example of upregulation of the outward rectifying channel by increasing conductance Gout to Gin thus promoting depolarisation. They said that this condition could not be normalised, but instead the region could expand and invade the normal cell region. Blocking of the outward rectifying channels by the administration of an external ion channel blocker would have the opposite effect with the conductance ratio Gout to Gin decreasing locally. The authors described this effect as immediate and coupling between the cells means that the effect on a single cell would spread to the multicellular assembly as a whole. Therefore, there would be a gradual transition from the depolarised abnormal state to the normal hyperpolarised cell state. This scenario would, according to the authors, require specific blockers for specific ion channels. If instead channels associated with membrane hyperpolarisation were blocked then the depolarised area would be extended and the authors gave as an example the situation in CHO and HeLa cells where the blocking of the potassium ion channels that are responsible for maintaining the membrane potential by cation nanoparticles would lead to significant cell depolarization.

Cervera, Alcaraz and Mafe then discussed in their article the association between bioelectric cell status and cellular patterning. They said that the coordinated action of external effects along defined spatial directions could produce cellular patterning providing it was a fast channel blocking action and the spatial map of the electric potentials closely followed the concentration of the blocking agent. In the example they gave in their article, the patterning required no anisotropic electrical coupling between the neighbouring cells and so they stated that the positional information from the individual cell could be determined by its electrical potential value plus individual cell properties such as its ion channels and the charged lipids and proteins in the cell membrane. The multicellular assembly was regarded as a bioelectrically coupled network of cells supported by intercellular gap junctions allowing the transmission of bioelectric signals. Interactions among the cells were crucial for positional information in addition to the presence of specific morphogens.

This led the authors to discuss the distribution of charged nanoparticles on multicellular assemblies. With the number of particles scaled to an exponential function of local electrical potential, they found that positive nanoparticles concentrated around negatively charged cells. Therefore, the spatial map of potentials would in their opinion influence the local uptake of charged nanoparticles over the tissue as a whole. They also discovered that the binding of the nanoparticles could also disrupt the cell membrane potential leading to modified intercellular communication. This indicated to them that the administration of nanoparticles would be a method for altering maps of potentials for multicellular groups by external means.

Cervera, Alcaraz and Mafe concluded their article by summarising the importance of bioelectrical signals in cancer. They suggested that the cancer cell`s microenvironment may show long-range bioelectrical signals and gap junctional cell insulation which may be involved in tumorigenesis and that the membrane depolarization caused may trigger gene transcriptional changes that regulate morphogen transport. Changes in single cell membrane potential and polarization states can occur when specific ion channels are up-regulated, down-regulated, or physically blocked. These modulations change the activity of those particular ion channels and possibly lead on to a promotion of depolarisation and normalisation. However, modification of the bioelectric state can also be brought about by modifying the gap junction conductance ratio by shifting the balance between the outward- and inward-rectifying channels. This can be achieved by blocking specific ion channels. Although the role of ion channels in cancer biophysics is already known, the authors reminded their readers that studying them in vivo in humans with ion channel blocking agents has problems since for example, these agents can cause cardiac arrhythmias. The authors also concluded that the bioelectrical characteristics associated with the spatial distribution of external morphogens over the multicellular assembly can be of significance for cellular patterning. The map of bioelectric potentials resulting locally from cell coupling regulates the experimental uptake of charged nanoparticles over the tissues themselves. These nanoparticles can disrupt the cell membrane potential and modify intercellular communication which is suspected of having a role in the biophysics of cancer.

COMMENT

What makes this article interesting is that it links together cell outer membrane potential (transmembrane potential, or Vmem), bioelectrical cell status and cell connectivity within multicellular groupings and describes dysfunctioning of these mechanisms in terms of ´limiting causes` for problems of cell proliferation, cell organisation and patterning and possibly to the disease, cancer.  Cervera, Alcaraz and Mafe stated that what they called the bioelectric status and bioelectric signaling of the cell are often disregarded in research on diseases with emphasis usually on physiology and biochemical systems reactions. This is probably not completely correct since the source of the topic being considered, that of the change in electric potential of cells through membrane channel functioning, is an enormously important topic in neurochemistry and forms the basis of neuronal firing mechanisms, neuronal firing patterns and cell connectivity and these are associated with cognitive functioning and mental health. Although this type of research may be centered on brain tissue, bioelectric signals or the movement of charged particles whether simple ions, charged molecules, protons or electrons underpin a huge number of biochemical reactions in the whole body and are always in consideration when looking at cell functioning.

That said we must consider what the authors are saying in their article. They concluded from their simplified model of bioelectric signals relating to outer cell membranes that the bioelectric status of the cell in question comes about by the presence and functioning of cell membrane ion channels. Through an imbalance of concentration of charge across a membrane due to their action then a voltage difference across the membrane would exist (just like that seen in neurons) called the transmembrane potential (TMP). The passage of ions through these ion channels, according to Cervera, Alcaraz and Mafe, could lead to cellular depolarisation (inward flow of sodium ions and up-regulation of channels leading to abnormal cell functioning) or hyperpolarisation (outward flow of potassium ions and down-regulation of channels leading to normalisation of function). The authors also concluded that connectivity between multiple cells within a group could be achieved by shared bioelectric signals through gap junctions that require functioning ion channels between neighbouring cells and this connectivity could lead to cell positioning and organisation within the cell`s grouping. In addition, another conclusion made was a logical extension of the two and that was that modulation of ion channels by a number of different measures (eg. administration of ion channel blockers, surface distribution of externally administered nanoparticles) could lead to changes in individual cell functioning and multicellular group functioning some of which could be detrimental to the organism, for example in the case of cancer.

Cervera and colleagues first conclusion ie. that the bioelectric status of the cell relies on cell membrane ion channels and the passage of ions through them results in the electrical potential of the cell (designated Vmem) or the voltage of the cell, is supported by neurochemical studies and is well-known for neurons and neuronal firing. It is also known in the case of non-neural cells in the brain, eg. glial cells where neurotransmitter binding can cause the opening and closing of associated ion channels. The authors link their ion channel functioning to the electrical status of the cell in non-neural cells and formulated a two-channel model. One type of channel was the voltage gated sodium ion channel which was associated with the inward flow of the ion and subsequent depolarisation of the cell. An upregulation of this type of ion channel and resulting persistent depolarisation was proposed by the authors and others to be linked to cell dysfunction observed in cancer. The other type of channel was a voltage gated potassium ion channel which was associated with the outward flow of potassium ions and hyperpolarisation leading to the ´normalisation` of cell functioning in the case of, according to the authors, cancer cells. If we look at these conclusions, we can say that there is support for the ´limiting cause` of cancer being at the level of the membrane since essentially if there is going to be a root cause then the cell outer membrane is the logical first step, the first encounter, the protector of cell functioning from external influences. The ion channels also span from outside to inside (or vice versa) of the cell and therefore, provide a route between external causal event and internal effect (or vice versa). Describing the effect as a signal is also supported since the mechanism relies on the transfer of charged particles eg. electrons from one side of the cell to another thus promoting a change in the voltage across the membrane and this acts as the message/signal being transmitted from external sources to internal mechanisms (or vice versa). It should be noted however, that according to Levin and colleagues the Vmem of non neural cells is itself important and not how it is achieved, ie. ion transfer must occur but not linked to any type of channel in particular. This is not the case of neural cells where specific cell surface receptors and channels are involved in signal transfer. However, the idea that the voltage difference across a membrane is a signal is further supported by evolutionary evidence linking change in bioelectrical status with cell movement as with amoeba, cell functioning as with paramecium, regeneration as with frogs and salamanders, and voltage differences observed between organs as in the so-called life fields in humans, plus on a smaller scale in correct muscle functioning and bone repair.

However, a change in bioelectric status is only a signal. It may lead to a change in proteins or molecules at the membrane level possibly leading to conformational changes that result in a change in reactions or function of the cell internally. To keep this comment relating to cancer, we should look at what effects this bioelectric signal could have on cell differentiation and proliferation, both systems appearing to be defective/abnormal in cancer. If we take a look at the observations made in life fields research we see that the bioelectric signal is believed to be associated with the health and well-being of an organism and is thought to be associated with growth and repair of an organism. In the case of new bone growth an application of an electric current is reported to lead to new growth around the negative electrode implying that the bioelectric signal was negative on the outside of the cell and positive on the inside. (This is opposite to what is known about the resting potential of the neuron which has a negative charge inside the cell relative to the positive charge on the outside.) The observation was explained by looking at bone stress and the so-called pizoelectric effect where the stressed bone acts as a matrix biphasic semiconductor with positive apatite outside (P) and collagen inside (N negative). With the PN junction and P to N current flow then the current rectifies the charge on the compressed internal side leaving it with a negative signal that stimulates cells to grow to form new bone.

Continual cell proliferation relating to the bioelectric signal and life fields was described in the case of the frog and its ´current of injury` reported with leg amputation and limb regeneration. Early ideas of ´electric currents coming from a wound` (Galvani) developed into ideas about changes in transmembrane potentials across the cell membrane dictating repair. In the frog it was reported that a stream of electrons (negative current) came from the wound from an amputated limb as a result of cell damage. With the transmembrane potential ions leaking out this was said to lead to the reversal of the polarity of the cell to positive. This only occurred on the wound surface and the current of injury was found to be proportional to the amount of nerve present. The change in current was associated with regrowth. In the case of the salamander a similar experiment was reported where a limb was amputated and the wound washed to remove blood clots. An immediate fall of membrane potential from minus 10 to plus 22 was observed. However, after about 3 days there was no membrane potential and no blastema (a mass of undifferentiated cells capable of proliferation and differentiation). After 6-10 days the Vmem rose to negative 30 and a blastema formed. This indicates that an external negative charge promoted cell proliferation. However, the potential was recorded as changing relative to regrowth since the external negative charge dropped to around minus 10 up to 22 days and the limb regenerated over that time. The view that cell proliferation is linked to bioelectric signals was further supported by studies where an external application of an electric current (AC current) or field was applied to tissues or tissue samples and this caused them to divide in a manner similar to cancer.

The implication from the observations of life fields researchers and cancer is that cell differentiation and cell proliferation mechanisms involve in some form bioelectric signals relating to the external membrane (ie. via the transmembrane potential). They may have an indirect effect through influencing gene transcription and the release of factors in response to signal changes. We know that differentiation is guided by transcription factors and morphogens and that differentiation is changed by a bioelectric signal. Therefore, we should ask whether the bioelectric signal could act as a ´transcription factor`. This is unlikely since the conditions arising from the signal are more like a ´state` rather than a specific factor. Transcription factors bring about DNA methylation and modification of the histone structure, but the bioelectric signal instigates changes in molecules to bring about these reactions and therefore, cannot itself be described as a transcription factor, but as a factor that modulates indirectly transcription via another party.  More likely is the consideration of it as a ´morphogen` with the signal arising from its own cell and/or from others and beginning at the level of the outer membrane (ie. differentiation`s  ´limiting cause`). Since a morphogen usually acts by a general mechanism of ligand binding to a receptor (possible bioelectric signal involvement here through conformational changes of the receptor induced by the presence of ions) leading to active ligand-receptor complexes which result in the catalysation of reactions through phosphorylation type reactions (processes requiring proton exchanges and conformational changes of molecules involving ionic charge changes also indicating bioelectric signal involvement). These can lead to the activation of dormant transcription factors (the direct intervention) or cytoskeletal protein changes (indirect intervention via the action of actin and myosin in microfilament and microtubule functioning – also involving the bioelectric signal in their mechanism and direction of movement) that result in changes to the differentiation process. Alternatively, cell differentiation can be affected by matrix elasticity which is also dependent on an actin-based intracellular microstructure reacting to the tugging motion of the cell`s contents during the cell`s division. This acts as a signal and hence, the bioelectric signal is again involved through molecular motor molecules and their reliance on the bioelectric signal for their correct functioning.

Therefore, Cervera, Alcaraz and Mafe`s model that bioelectric signals relating to outer cell membranes may play a role in cell differentiation and dysfunctioning of that system in the form of cancer may be valid. However, although there is support, there are also factors that count against such an easy and plausible cause. We must consider that in the case of neuronal depolarisation, persistent depolarisation arising from only a change in membrane function is unlikely to occur since under normal circumstances there are feedback mechanisms that switch off the mechanism, eg. the refractory period for neuronal cell recovery after sustained firing. Also, nothing is ever simple regarding cell functioning. In neuronal cells there are multiple receptors in play and multiple stages in any mechanism with multiple areas for feedback mechanisms to possibly right any wrong.  For example, it is unlikely that any cell has just two types of membrane bound ion channel and their actions are often linked to the performance of a third (eg. calcium channels with depolarisation or GABA channels with hyperpolarisation) or changes in intracellular conditions eg. pH changes counteract enzymatic mechanisms. Therefore, the bioelectric status of cells is likely to be highly controlled and to override this control there must be continual gene transcription changes and possibly involving many cells. This is seen in the case of cancer cells where transcription changes induce continual division of the cells against normal instructions.

Cervera, Alcaraz and Mafe also made a hypothesis in their article about the bioelectric signal and physical connectivity between multiple cells in a functional grouping. Their view is that connectivity occurs through one type of cell signaling mechanism, that of gap junctions and that disruptions of that connectivity can have catastrophic effects on the cell, the grouping or even the organism itself. Support for connectivity between cells of a group is clear, eg. from neuronal cell assemblies in memory, consciousness (connectivity supported by the observations of  brain wave synchronicity between participating cells), and cancer with connectivity between tumour cells. There is also support for connectivity between long-range parts of a total organism, for example: Planarium and organism polarity; cell positioning and growth alignment (eg. matrix arrangements of Planarium, hydra, salamander and frog); ´current of injury` in regeneration (frogs and salamanders); and more controversially humans and the so-called chi meridians. Although support is there the situation is more complicated than just having coupling via gap junctions since some examples exhibit long-range connectivity and the gap junction is a close-quarter communication mechanism with connexon proteins spanning physical connections between neighbouring cells. Membrane ion channels effects may explain the bioelectric state of the cell at the level of a single cell, but it must apply to a whole number of cells to get to the large scale connectivity required to explain positioning, polarity etc. A possible way of approaching this is to view connectivity more like Jenga or dominos ie. a multitude of single pieces making up the shape of the whole or the shape is made by the hole that is left (template vs stencil). In this way, maybe only certain cells in close quarters need to have gap junctions as the signaling mechanism and these can produce other signals that act long-range by alternative transmission mechanisms, eg. by exuding neurotransmitters and/or hormones. However, either way assumes that each single cell has a property that makes it special from one that does not experience the ´switched on/active` bioelectric state and the whole takes on that ´quality`. (Salt describes this as the ´E` quality meaning ´energised state`.) This property must be apparent for other cells within the group and guesses could be made as to what the cell with the ´switched on` bioelectric state  does eg. does it vibrate, shine, hum etc whereas the non-active cell remain indistinguishable from other non-active cells. This question still remains to be answered. However, continuing with the view that the multitude of cells within a grouping (Cervera and colleagues` ensemble or in the case of memory, the neuronal cell assembly) has a particular electrical status then the authors` view that a quantity of abnormal cells in the multitude grouping determines the overall status of the group is also plausible. When the number of abnormal (or non-switched on) cells is low then the ´quality` of the grouping is likely to be minimally effected, but the effect would increase in some manner (a threshold and then total dysfunction or a sliding scale effect for example) as the number of abnormal cells rises.

Observations on life fields lend support to bioelectric signals and electric fields having a role in connectivity between cells. The overall view is that they act as a signal for new growth as described above and then as a mould or matrix for that cell growth and in the organisation and maintenance of positioning. For example, life field studies have shown that specific voltage potentials reflect the arrangement of the nervous system of worms and fish. One area of positive potential in the brain is reported to match the one major nerve ganglion and in the case of the salamander there is a report of strong positive charges in three areas (the brain, brachial plexus and lumbar nerve ganglions) and negative charges (8-10millivolts) towards the extremities and tail. Organisation based on voltage potentials was also proposed for humans with voltage potential differences shown to be just like with the salamander (head and spinal cord, brachial plexus between the shoulder blades, lumbar arrangement at the base of spine dictated as positive and hands and feet as negative). There are also reports of the human brain having direct current coming from the reticular activating system and flowing from the back to the front of the brain through the middle with the olfactory lobes (positioned at the front) several millivolts more negative than the occipital lobe located at the rear. Current was assumed to flow up from the brain stem. Naturally, the automatic arrangement of the neurons with its polarised positive end at the input dendrite end and negative at the axon output end lent support to the idea of the bioelectric signal being used to organise cells and connect them.

As indicated by Cervera, Alcaraz and Mafe, the bioelectric status of a multicellular grouping associated with connectivity achieved through gap junctions dictates the functioning capability of that grouping. Supporters of the life fields and bioelectric signal views suggest that the bioelectric signal is linked to correct functioning and conversely abnormal changes in fields or bioelectric status can be an indicator of dysfunction and possibly disease eg. voltage differences have been observed between certain organs with cancer. The bioelectric signal and field exhibits normal changes according to the function assigned to that individual cell or grouping of cells, eg. changes of voltage potential in salamanders is associated with sleep status. In this case, it was reported that negative potentials are linked to frontal regions of the salamander brain and the periphery of the nervous system and these potentials are associated with wakefulness, sensory stimuli and muscle movements. The greater the shift to the negative, the greater the activity recorded whereas a shift to positive charge was found to be associated with rest and sleep. Studies on anaesthesia revealed that the negative charge vanished from the extremities and a reversing of the voltage occurred with the limbs and tails becoming positive and the brain and spinal cord negative. The administration of minute currents from the front of the salamander`s brain to the back was found to render the animal unconscious. An examination of brain waves during the process showed the presence of delta brain waves with the unconscious salamander and these increased in size as the current grew bigger. This was plausible since delta brain waves are believed to coordinate brain area connectivity during slow wave sleep. As the anaesthesia wore off, it was found that the normal potential differences returned to normal. A similar pattern was observed in humans with the brain becoming more negative during physical activity, declining during sleep and reversing to positive under general anaesthesia. Therefore, the association between bioelectric status of individual cells linked to others within the grouping via gap junctions (or as suggested by other means) and overall function appears plausible and may give an alternative approach to explaining how certain cognitive functions that rely on connectivity of large areas, eg. consciousness and memory, may occur. Applying knowledge obtained from studies on cell growth (eg. proliferation, organisation and positioning) of non-neural cells and cancer relating to the bioelectric status of a cell achieved by its ´limiting cause` the ion channels present at the cell membrane, the effect of this on neighbouring cells and the corresponding connectivity of those cells of the groupings may give an indication of how these cognitive functions could occur.

The third area explored by Cervera, Alcaraz and Mafe leads on from the other two points that they made, and that is that deliberate modulation of the outer membrane ion channels and hence, modification of the bioelectric signal, leads to changes in bioelectric status and changes in cell functioning. This can be positive for the organism as in for example the life field`s observed ´current of injury` and regeneration of a missing limb, or they can be negative as in cancer where there is a upregulation of sodium ion channels resulting in unwanted changes in cell proliferation, differentiation and organisation for example. We can assume that modulation is achieved in the same way as that for neurons at the first level (´limiting cause`) at the outer membrane stage, eg. by changing the number of  cell membrane receptors or ion channels, or by changing the secondary messenger systems (eg. by a change in conformation of molecules). In their article, Cervera, Alcaraz and Mafe describe direct methods for changing the bioelectric signal at the outer membrane level. This was achieved by the administration of blockers that act directly at the ion channels or receptors or nanoparticles that produce positive or negative changes in the microenvironment of the membrane where the relevant ion channels/receptor molecules are located. Other methods observed to have effects at this level and known to influence the bioelectric status of a cell is the administration of silver (eg. silver electrodes are shown to bring about the destruction of bacteria in close vicinity) and bleach or a weak acid. It is thought that these methods produce a change in the phospholipid basis of the cell`s membrane physiology and hence, affect the functioning of the ion channels and other membrane proteins by changing the conformation of the proteins within their phospholipid microdomains. With all methods the likely result of such a modulation is a detrimental end-effect on gene transcription, which could have positive or negative consequences for the cell. For example, stripping the outer cell membrane of its bioelectric charge using diluted bleach leads to changes in cell growth and proliferation and as reported here a change in transcription rate and cancer. Similarly, leaving cells bathed in diluted bleach takes electrons off a molecule that activates NF- Kb transcription factor resulting in an inhibition of the inflammatory pathway and faster healing of burns and increased skin cell production. Studies have also shown that a weak acid bath can lead to the reprogramming (de-differentiation) of lymphocytes to STAP cells.

In the context of voltage, bioelectric signal and transmission of charged molecules other researchers report the effect of external electromagnetic fields whether electric, magnetic, geomagnetic, or EMF on cell functioning. Cell alignment and cell positioning changes have been reported for applied EMF as well as changes in cell-to-cell communication, membrane physiology and change in cell division for magnetic fields. If such influences on gene transcription rates should occur as the authors suggest through the starting point of the electrical voltage differences at the inner and outer membrane surfaces of the cell, then administration of electric currents or magnetic fields can have probably effects at the single cell level. A problem may arise in explaining how this can influence the whole organism since there are multiple cells present. Again the hypothesis that the bioelectric status of one cell can translate to an effect on a multitude of cells through the connectivity mechanism of gap junctions may provide an answer, but the situation is made more complex by an organism being made up of many different materials within close vicinity to each other all having different physical characteristics eg. fluids, gases, bone.

To summarise, the bioelectric status of a cell through voltage differences across a the outer membrane dependent on two ion channels acting in opposite polarising directions as proposed by Cervera, Alcaraz and Mafe is likely to be an over-simplification of what is actually occurring, but the knowledge obtained from the studies can be applied to the areas of cognitive function like consciousness and memory which have yet to be fully explained. From what is known it can be said that bioelectric signaling is likely to be the first step (the ´limiting cause`) in the action of many cell types. It can also be said that this type of mechanism can provide a means of signaling from the external environment to the inner workings of the cell and hence, is a mechanism of cell-to-cell communication providing connectivity between a multitude of cells. It is unlikely that gap junctions are the sole mechanism of such connectivity as suggested by Cervera and colleagues since this method in neural cells is intended only for close neighbouring cells. Instead a mixed system may be more plausible with gap-junctions and a longer ranging signaling system eg. exudation of another transmitter or signaling molecule such as oestrogen. Cervera, Alacaraz and Mafe suggest that the bioelectric status of a cell from the membrane voltage differences attributed to ion channels present may provide a link to cancer and since it has been reported that bioelectric signals attributed to life field observations are linked with cell proliferation, cell organisation and cell positioning from other species such as the frog and salamander this may be plausible. It definitely needs further investigation, but in the case of humans, the mechanism may not be so applicable. It may be the case early on in development and later on in some areas where pluripotent differentiation and growth occurs normally eg. hippocampus and neurogenesis, skin cells and skin repair, but it may not be applicable in all organs and in all situations. The area needs more research especially in its application to explain the mechanisms behind memory and consciousness and also to the fields of specific targeting of electrical ´charge-busting` molecules on to cell membranes in the hope of normalising cell dysfunction, but maybe more emphasis should be placed on looking at whole organisms or larger groupings of cells rather than single cells in culture.

Since we`re talking about the topic……………………………

…..calcium ion channels have been implicated in the growth of some fish cells. What effect on the bioelectric status of the cell is there if calcium blockers are used? Would the use of specific calcium blockers at each stage of the intracellular calcium signaling mechanism show a dependency for growth on the calcium ion channel at the outer membrane and hence a link to bioelectric status?

……does stripping cells of their external electrical charge with diluted bleach cause a change in structure and performance of the sodium ion channel or potassium ion channel? Could this be observed with fluorescent imaging techniques?

…..would the cooling of cells or the use of specific blockers of phospholipids definitively show that microdomains of the cell outer membrane and particular phospholipids are important in ion channel functioning and hence, bioelectric status of the cell?

….reversine is said to de-differentiate cells by inhibiting the phosphorylation of a histone and preventing the activation of a specific cell differentiation kinase. Can we assume that the dual administration of a targeting cell membrane sodium ion channel blocker and reversine would have no greater effect on cell regeneration than the former alone because the signal for cell growth would have been prevented at the level of the cell membrane and not at the level of gene transcription?

…would the use of voltage sensitive fluorescent dyes to track non-invasively ionic gradients allow the changes in Vmem to be measured for each cell type and a comparison to cell function and cell division and proliferation be made?

 

effect of high altitude on cognitive functions

Posted comment on ´Acute and Chronic Altitude-Induced Cognitive Dysfunction in Children and Adolescents` by S.F. Rimoldi, E. Rexhaj, H. Duplain, S. Urben, J. Billieux, Y. Allemann, C. Romero, A. Ayaviri, C. Salinas, M. Villena, U. Scherrer and C. Sartori and published on website Researchgate.net/publication/282245481

SUMMARY

The investigation carried out by Rimoldi and colleagues was to see whether short-term and long-term exposure to high altitude (around 3450m) induces cognitive dysfunction in children and adolescents. They found that short-term hypoxia had a significant negative effect on executive functions (inhibition, shifting and working memory), some memory functions (verbal short-term and verbal episodic memory), but not visuospatial memory and processing speed. The impairments observed were found to be even more severe for those individuals living at high altitudes permanently. A return to lower altitude for one group of study participants gave performance values back in the control range.

Test subjects consisted of 3 groups of healthy European children and adolescents. The control group consisted of 14 children and adolescents (aged between 10 and 17) living at low altitude (less than 800m).The acute, short-term high-altitude group consisted of 48 children and adolescents who were tested 24 hours after arrival at high altitude (Switzerland, 3450m) and 3 months after their return to low altitude (46 living at an altitude less than 800m, 2 at 1100m). The chronic, long-term high altitude group consisted of 21 matched subjects who had lived for longer than 3 years at high altitude (La Paz, Bolivia 3500m). The general cognitive abilities of each subject was assessed before testing using Raven`s Progressive Matrices (a non-verbal reasoning task) and it was found that subjects of all groups performed similarly. The cognitive tests given assessed each subject`s executive function, memory and processing capabilities. Testing of executive functions was carried out using the Attentional Network Task (tested inhibition),   the Trial Making Test Part B (TMT- tested shifting) and the backward Digit Span Task (tested working memory). Memory functions were tested using the  Forward Digit Span Task (tested verbal short term memory), the California Verbal Learning Test (tested verbal episodic memory) and the Corsi Block Tapping Test (tested short-term visuospatial memory).  Speed processing ability was tested using the TMT Part A. Paired student t tests were carried out for all results to indicate statistical significance.

According to Rimoldi and colleagues their study showed that short-term exposure to high altitude produced significant negative effects on all executive and memory performances except for visuospatial memory (tested by the Corsi Block Tapping Test) and processing speed (TMT part A). The value for inhibition was found to be 30% higher (a rise in value from 92 to 129) indicating an increase in reaction time and a decrease in attentional performance. The test of shifting capability (Trial Making Test Part B) showed that the subjects required on average 20% longer (63 to 74) to complete the task demonstrating that cognitive flexibility was reduced and the value of the working memory test (Backward Digit Span Task) was 10% smaller (4.9 to 4.5)indicating that working memory performance had decreased. On return to low altitude the same group of subjects was said to show performances in all of these tests similar to control values, ie. inhibition (Attentional Network Task) demonstrated a shift from 129 to 93 indicating a quicker reaction time and increased performance;  shifting (Trial Making Test Part B) with a shift from 74 to 66 also indicating a quicker reaction time and increased performance;  and working memory (Backward Digit Span Task) with a shift from 4.5 to 4.8 indicating according to the authors an improvement in working memory performance.

In the case of long-term exposure to high altitude, Rimoldi and colleagues said that the detrimental effect on these executive functions were the same or even more severe as those observed for acute exposure compared to the control, ie.  inhibition (Attentional Network Task) had a significantly longer reaction time (92 to 148);  shifting (Trial Making Test Part B) an approx. 20% longer reaction time (63 to 71); and working memory (Backward Digit Span Task) demonstrating an approx. 10% lower capability (4.9 to 4.6).

Rimoldi and colleagues found for the tests of memory capability that both verbal short-term memory and verbal episodic memory were impaired by acute exposure to high altitude. Verbal short term memory tested by the Forward Digit Span Task produced a decrease in length of word series remembered compared to the control from an average of 6.1 to 5.6. In the verbal episodic memory test (California Verbal Learning Test) the number of correct words recalled fell from an average of 12.4 to 11.9 compared to the control. However, in the case of short-term visuospatial memory measured by the Corsi Block Tapping Test, acute short-term exposure to high altitude was said to produce no effect (values recorded of average number of blocks remembered was 6.1 for the control and 6.6 for exposure). The authors concluded that the recall of both numbers and words were significantly impaired with short-term exposure to high altitude, but visuospatial memory capability was not. After return to low altitude the performance of the acute exposure group was said to improve back to the control values (verbal short term memory by a recorded value shift from 5.6 to 5.9 and verbal episodic memory by a recorded value shift from 11.9 to 13.5 and short term visuospatial memory by a recorded shift from 6.6 to 6.8).

In their investigation of the effects of long-term exposure to high altitude, Rimoldi and colleagues  stated that verbal short term memory determined by the Forward Digit Span Task and the verbal episodic memory test determined by the California Verbal Learning Test were no different to those experienced with only short-term exposure. Values recorded were shifts from 6.1to 6.3 in the former and 12.4 to 10.8 for the latter. However, the results of the visuospatial memory test (Corsi Block Tapping Test) was said to show more severe impairment in capability with long-term exposure in comparison to short-term (a recorded negative shift in performance from an average of 6.1 blocks remembered to 5.6) . Therefore, the authors concluded that the recall of words and numbers was not affected by long-term exposure to high altitude compared to short-term, but visuospatial processing impairment was even more severe.

The authors also tested their subjects for speed processing capability using the TMT Part A test. They said that on short-term exposure to high altitude there was no detectable effect on speed of processing, but recorded a shift in average performance time for joining the dots or letters from 25.3 to 27.6. On returning to low altitude, this value was decreased to 26.0. On long-term exposure to high altitude speed processing ability was said to be reduced by more than 25% since the subjects needed longer to complete the task (average recorded values of 25.3 for the control and 32.5 for subjects exposed long-term to high altitude).

Therefore, the authors concluded that changes in some cognitive areas were observed with exposure to high altitude. These changes it was said could not be attributed to acute mountain sickness (AMS) since the neuropsychological changes observed with hypoxia were different to those seen with this condition. In the case of hypoxia, acute short-term exposure to high altitude was found to induce marked cognitive deficiencies in healthy children in the areas of verbal short term memory, episodic memory and executive functions such as inhibition, shifting and working memory. Impairments were said not to be observed in visuospatial memory capability and speed processing for acute exposure, but longer exposure would lead to deficiencies not only in these areas, but also to a more severe level. A return to lower altitude after acute exposure led, according to the authors, to impairments being no longer discernible. The causes of the impairments observed for the executive functions were attributed by the authors to dysfunction of white cerebral matter, perhaps at the level of the prefrontal cortex and anterior cingulate cortex and this dysfunction had been induced by hypoxia. The differences observed between acute- and long-term exposure in the cases of visuospatial memory and processing speed indicated to them that children and adolescents may have a greater resistance to a hypoxia effect at neuronal level in these areas than adults. Since semantic cues were of no help in recall in the verbal episodic memory task the authors hypothesised that the defect in the memory process was at the level of information encoding. This observation was found to contradict the results observed for adults who do not present with episodic deficits with high altitude. Therefore, the authors suggested that the hippocampus which is involved in episodic memory is more sensitive to effects of hypoxia in children than adults. With reference to speed processing problems seen with long-term exposure in children, the authors reported that other research shows that this processing deficit has been reported even at lower altitudes (2500m) for adults and with short-term hypoxia exposure. Hence, they surmised that speed processing capability is better preserved in children than adults.

The authors concluded their article by saying that the results of their investigation into high altitude and executive, memory and processing capabilities were important not only because major tourist destinations are located at high altitudes that would expose tourists to hypoxic conditions that could have effects on their cognitive capabilities, but also that more than 15 million people live permanently at high altitudes and appear to have little or no neurophysiological functional adaptation to living in these conditions.

COMMENT

What makes Rimoldi and colleagues` article interesting is that it indicates an element of influence on cognitive processes that is frequently ignored or forgotten:  that is, the influence of the body`s hormonal system.  Emphasis is often placed on changes to cognitive performance through personal thinking, our attitudes, effects of our experiences and other mental reasons, but this is not the sole explanation for differences in capabilities observed.  The article commented on in this Blog post describes the effect of one example of hormonal influence on cognitive capability. It describes how hormonal changes brought about by low environmental oxygen affects the attentional and memory systems. In this case, low environmental oxygen is achieved by the individuals enduring either a short-term or long-term stay at high altitude (above 3500m). Low oxygen causes the body to experience what is termed ´environmental stress` and researchers have shown that this induces physiological changes that lead to cognitive and emotional effects and they can be different for altitude, duration of stay and age of individual.

Before discussing the cognitive effects observed by Rimoldi and team, we must answer the question as to what influence does high altitude and low environmental oxygen have on cell physiology. One of the most important and obvious is the change in energy production mechanisms. We are all familiar with the processes involved in muscles during intense and prolonged exercise – a situation where cells cannot get enough oxygen. Hence, the normal biochemical mechanisms of energy production cannot continue and other pathways are used instead which leads to the formation of lactate. Continued production means a build-up of lactate in the muscles and a fall in intracellular pH until this inhibits even this energy producing pathway. A similar problem occurs with the energy production in brain cells exposed to high altitude. High altitude is characterised biochemically as ´environmental stress` since the fall in external oxygen leads to a decrease in inhaled oxygen pressure (50% at 3000m of that at sea level) and this results in reduced driving pressure for gas exchange in the lungs. Therefore, the individual has less oxygen intake for each breath and so blood is not fully oxygenated at the alveolar level and ultimately, circulating blood level. Hence, brain cells are unable to obtain enough oxygen for their normal biochemical processes relying on it and as a result cellular adaptation has to occur.

In the case of the energy-producing systems in low oxygen conditions, the normal mechanism of glucose being metabolised by a chain of enzymatic reactions (called glycolysis) to produce pyruvate that occurs in aerobic respiration (ie. in the presence of oxygen) still takes place, but the second stage of the process is altered. This stage is where the pyruvate is converted by another chain of reactions into the energy molecules ATP.  If oxygen is not present at the level required for this aerobic mechanism, then a process called lactic acid fermentation is initiated (anaerobic respiration). Lactic acid fermentation means that pyruvate is then converted to lactate by the enzyme lactate dehydrogenase (LDH). However, this anaerobic process does not produce the same number of ATP molecules as normal aerobic mechanisms, but it does provide some.

The other potential problem is the build-up of lactate which is observed in muscle cells. However, it is likely that in the brain which is dependent on a constant supply of glucose and energy that a safeguarding mechanism is in place called the Cori cycle which transports the lactate out of the cell, back to the liver where it is converted into glucose by a process known as gluconeogenesis. Again the LDH enzyme is involved and this conversion could explain the lack of appetite experienced by some when undergoing rising altitude.

The processes of this energy switch due to the rise in altitude plus the short fall of ATP production are made easier by increasing the production of a cellular factor that influences gene expression. The production of hypoxia-inducible transcription factor (HIF-1) is induced as the oxygen concentration of the blood falls. This transcription factor leads to increased gene expression of many glycolytic enzymes eg. hexokinase, aldolase, phosphoglycerate kinase (ie. those taking part in the first stage of the energy producing cycle), plus LDH (which we have seen converts lactate to ATP) plus the glucose transporter molecules  GLUT1 and GLUT3, which transport glucose across cell membranes. Therefore, the energy producing mechanisms that are required for any metabolic energy changes due to lower oxygen availability have been optimised.

Changes to the blood and circulatory system can also occur to alleviate the absence of oxygen.  This is understandable since oxygen is transported via the blood from the lungs to the brain and the brain has a high need for oxygen and for glucose. Therefore, increased ventilation has been observed from altitudes of 3000m and this response varies with individuals and is not related particularly to performance. There is also an increased level of cardiac output initially leading to normal values with time. However, there is increased heart rate and decreased stroke volume since hypoxia acts as a vasodilator in systemic circulation and a vasoconstrictor in pulmonary areas which can lead to a high risk of pulmonary hypertension and pulmonary oedema. There is also increased viscosity of the blood and increased coagulability which can result in an increased risk of stroke and venous thromboembolism. Blood components can also adapt in travelling to high altitude leading to increased haemoglobin concentrations by the decrease in blood volume due to dehydration. Later on, hypoxia leads to an increased production of erythropoetin and increased haemoglobin production to compensate.

Although clearly the production of energy molecules for the cell is very important, the presence of oxygen in the blood is also required for a number of other biochemical processes that could also influence cognitive function, eg. the degradation of aromatic amino-acids by oxygenases, the formation of steroid hormones by hydroxylation, oxidative phosphorylation, the elongation of unsaturated fatty acids in endoplasmic reticulum of cells, the formation of nitric oxide, the synthesis of plasmologens and the degradation of nucleotides. Systems influenced by low oxygen availability affecting in particular brain cells and cognitive functions are not specifically known, but certain paths could be possible. For example, the degradation of aromatic amino-acids by oxygenases could in the case of the amino acid phenylalanine mean reduced tyrosine levels and tyrosine is used in the brain to form dopamine, a major brain neurotransmitter and instrumental in inducing hypoxic changes as we will describe later. Another example is the elongation of unsaturated fatty acids in the endoplasmic reticulum of cells could influence cell membrane structure ie. affect the formation of membrane lipid rafts and affect the cell surface receptors and other membrane proteins and molecular functioning. Also, an example of an effect on the synthesis of plasmologens could mean that the plasmologen phosphatidal choline which corresponds to the phospholipid, phosphatidyl choline, which is important for cell membrane structure, is not formed.

So we have seen how environmental stress from low oxygen availability causes physical adaptations and these can as with all other influences include effects on brain physiology and because of that, on the functioning of cognitive and emotional systems. For example, increased vigilance and arousal is observed with high altitude exposure and these are associated with increased activity in the ventral tegmentum area (effects linked with increased dopamine availability), raphe nuclei (increased 5HT), locus coeruleus (increased noradrenaline) and other brain stem and basal forebrain areas. The increased neurotransmitter production and increased functioning of these areas in response to high altitude exposure all lead to increased firing of the thalamus and cortical areas which result in changes to the input, processing and storage of information.  High altitude exposure also causes the activation of the autonomic nervous system (ANS) which leads to the production by the adrenal cortex of adrenaline (associated with the ´fight or flight` response) and noradrenaline (associated with the activation of the locus coeruleus and the ´fight or flight` response). Also, there is an observed release of cortisol from the adrenal glands and this has an important role in promoting changes to cognitive capabilities since it leads to the activation of the hypothalamus-pituitary-adrenal axis (HPA). HPA activation from cortisol release leads to increased amygdala responses and decreased hippocampal activity (inhibits corticotropin-releasing hormone CRH release and hippocampal cells can wither and die during chronic stress).  Cortisol can bind to receptors in the cytoplasm of neurons and travel to the nucleus where it stimulates gene transcription. It can also promote calcium ion entry into neuronal cells by either changing ion channel function or by changing energy metabolism in the cell, but independent of method both cause cell depolarisation. The effects on cellular firing and neuronal firing connectivity between brain areas can be linked to performance of attention and memory systems and hence, high altitude exposure can have multiple effects on these dependent cognitive systems.

Rimoldi and colleagues`  investigation looked at certain cognitive functions in relation to exposure to high altitude in European children. We must say at this point that our interpretations of their results were not always in exact agreement with the interpretations by the authors, but in this comment we attempt to understand what is being observed and provide an explanation of the neurochemical mechanisms involved. The first area we will discuss is that of attention. Rimoldi and colleagues say that there is decreased attention capability with exposure to high altitude and with duration of that exposure. We disagree about the interpretation of the level by which this occurs since the authors say acute exposure produces a 30% decrease with chronic exposure greater, but our interpretation gives a greater negative influence at 40% on acute exposure and more severe at chronic (60% decrease in capability). The neurochemical explanation for such a large decrease in capability (whether at the authors´ level or ours) could be that the brain areas associated with attention (ie. of the fear system, the association between the cingulate cortex and ventral tegmentum and the dopamine activation observed) are affected by the physiological changes brought about by hypoxia. Lower levels of firing seen in low-oxygen conditions could result in the level of connectivity between brain areas not being achieved and hence, decreased performance occurs. For example, neuronal cell group connectivity is required as shown by alpha brain waves between that group`s  members to maintain informational items and therefore, if connectivity is disrupted to an extent that the items cannot be held in the working memory, performance on the task (in this case the assessment of arrow direction in relation to a given example) would be reduced.

However, this hypothesis conflicts with the view that increased dopamine would lead to increased attention and processing and increased hippocampal functioning. Therefore, it is possible that the decrease in attention seen with high altitude exposure can be explained by the mechanisms involved being more akin to those observed with increased dopamine seen in individuals with attention-deficit disorder (ADHD). In this case, there is a fear state observed with increased prefrontal cortical and cingulate cortical activities which would lead to an increase in task relevant information and task irrelevant material. The result of this is that the tasks given in Rimoldi and colleagues` study are more difficult to perform since a decision has to be made between whether the arrow points in the same direction as the example or not. If there is an increase in task irrelevant material then it is more likely that errors would increase and reaction time decrease and hence, the overall performance level observed would drop. In Rimoldi and colleagues` attention experiment only correct congruent and incongruent results were measured and the level of errors was not considered. It should be noted however, that decreased blood flow is seen in the frontal lobes of ADHD patients akin to the symptoms observed with high-altitude exposure, but there is also decreased glucose availability and no prefrontal cortex activation of the amygdala, both of which are not observed under hypoxic conditions.

Under chronic environmental stress conditions decreased attention is still measured since there is continued increased dopamine release from the production of cortisol and there is decreased hippocampus functioning via feedback inhibition of the HPA. There is also an increase in amygdala activity because of this continued HPA activation. This could explain why the decrease in attentional capability is more severe with longer duration of high altitude exposure. Long term changes occur at the neuronal level due to over-excitability of the amygdala. Apoptosis of neuronal cells can occur as a result of over-excitability of certain areas and decreased functioning and reduced area size is observed. However, there must be in the areas the capability of readjusting once the stressor is removed since the subject`s return to low altitude is met with a reversion back to control values for certain cognitive functions. Therefore, long-term changes such as the overly large thalamus and decreased size of the hippocampus as seen with full-blown ADHD are not observed in the case of exposure to high altitude. Readjustment of an area`s capability occurs in response to a reduction in HPA activation and cortisol production.

Another cognitive capability investigated by Rimoldi and team was working memory. The authors saw a lower decrease in capability after acute high altitude exposure compared to attention (10% as seen with increased reaction time with our interpretation slightly less at 8%). A greater change was measured (20%) as expected with the Trail Making Test Part B (shifting task) since this task measures a combination of attention and working memory capabilities. Although an association between dopamine and working memory is not definitive (working memory is thought more of a GABA process), an explanation for the negative effect is likely to be decreased hippocampal function (glucocorticoid receptor effects) and increased amygdala function as a result of increased HPA activation and cortisol production. The changes in firing of these areas lead just like with attention to alterations in connectivity of the brain areas involved in the working memory function.  Working memory requires cell group connectivity with theta brain waves (gives temporal order) and alpha (maintains relevant items and keeps non relevant information away) for correct performance and hence, changes in individual brain area functioning could lead to the working memory conditions of binding and presentation of information for the correct completion of the task not possible. Chronic exposure to high altitude appears to bring, according to Rimoldi and team, no change to the level of impairment observed from the acute (although our interpretation is that there is slight improvement with chronic exposure) and therefore, negative influences on the physiology occur on immediate exposure and there is no long-term adaptation to the systems. This is supported by the observation that once the stressor is removed (ie. there is a return to low altitude) the mechanism returns to normal and therefore, any element effected is only temporary and only in the presence of the stressor.

Another capability investigated by Rimoldi and team is the cognitive capability of visuospatial memory. They found that acute exposure to high altitude had no effect on visuospatial memory, but long-term exposure did. Our interpretation is slightly different in that a slight increase (8%) in capability was observed with acute exposure, but chronic exposure led to a slight decrease (8%). An explanation for the observations could be that there is a ´fight or flight` response to acute exposure due to increased amydala activity induced by the activation of the HPA and production of cortisol. The change is amygdala function would lead to both increased levels of task-relevant and task irrelevant material, but the low effect could be due to the task (the Corsi Block Tapping Test) not being complicated enough.  Therefore, what would be observed would be the increased performance from the initial high dopamine concentration which induces ventral tegmentum activity and greater cortex and thalamus activities. Since there is greater activity and greater connectivity, better decision making and higher end-performance would be observed. The effect still remains to some extent (our interpretation 3% increase above control) once the stressor is removed (ie. by returning to low altitude) which implies that some physiological changes are not readily reversed.

However, chronic exposure to high altitude decreases visuospatial capability and this is consistent with the idea that long-term stress causes permanent physiological changes. Other researchers have already shown that there is an age-related decrease in spatial working memory that is exacerbated by increasing HPA activity therefore, it is likely that the chronic effect on the HPA,  downregulation of hippocampal activity, upregulation of amygdala activity and long-term cortisol production leads to decreased capability observed through permanent changes in brain area functioning and connectivity.  In the case of spatial memory, other researchers have said that the dorsal lateral prefrontal cortex is essential (although this is disputed) and this area and the ventral hippocampus and medial prefrontal cortex form a pattern of area connectivity that sets up a theta brain wave for correct functioning. A parieto-temporal circuit is also involved which integrates valuable sensory information. Dopamine receptor stimulation appears to demonstrate an inverted dose activation plot. Therefore, it is assumed that if visuospatial memory capability is decreased with chronic exposure to high altitude then all of these essential areas are affected by long-term administration of the stressor. This also applies to the workings of the visual working memory system where connectivity between the prefrontal cortex, cingulate cortex and hippocampus is involved. Therefore, it is thought that increased dopamine production leads to decreased connectivity patterns resulting in poor visuospatial performance.

Rimoldi and colleagues also looked at the cognitive capability of verbal memory.  They said that exposure to high altitude caused a decrease in capability independent of duration for both verbal working memory and episodic verbal memory. Our interpretation is slightly different with short-term verbal memory demonstrating an 8% decrease in capability, but only 3% after long-term exposure and episodic memory with decreased capability with long-term exposure even more severe (8% to 3% acute).  Researchers have already stated that hypoxic conditions lead to problems with memory encoding, retrieval and retention. In this case, it is likely that there is a decrease in encoding in the acute exposure situation as expected because of decreased working memory and attentional capabilities described above, but there is readjustment with time (long-term exposure leads to a decrease and return to values observed at low altitude albeit that the recovery is not at the same level as control). The decrease in episodic memory capability can be explained by decreased prefrontal cortex functioning as observed and decreased hippocampal activity (through the sustained HPA activation) causing problems with encoding (input and binding), retention and retrieval of information. It has also been reported that glucocorticoid receptors activity associated with hypoxic exposure eliminates newly formed cortical spines and disrupts previously acquired memories. Hence, episodic memory performance would be diminished. Again, hypotheses that cortisol production and HPA activation due to exposure to high altitude would lead to disruption of the brain area connectivity patterns required for memory mechanisms to function normally (ie. adequate and correct cellular connectivity within the groups with theta brain wave patterns required for encoding and gamma brain waves for learning) would apply. This is supported by other research that memory mechanisms under stress when the HPA axis is stimulated are different to those when not.

Although Rimoldi and colleagues provide adequate evidence that physiological changes are induced on exposure to high altitude and these can change according to duration, one factor not investigated by them, but which provides support for their conclusions, is the effect of high altitude exposure on the emotional system and mood. Mood changes are associated with high altitude exposure and duration. Initial environmental stress causes euphoria and arousal, a system which is dopamine dominated and hence, provides support to the observation that there is a rise in dopamine levels on stress exposure.  Corticotrophic releasing factor (CRF) is produced in response to hypoxia and this leads to cortisol production that is known to act on the brain area ventral tegmentum to cause increased dopamine function. As given above, the increased activity of this area leads to firing of axons connected to the frontal cortex and to parts of limbic system such as the nucleus accumbens (known to fire with unpredicted rewards plus known for its role in encoding anticipated rewards) and striatum (increased activity also seen with increased environmental stress). Both systems are thought to be associated to the pleasure system and reward and hence, this supports changes in mood observed. There is also release of other neurotransmitters eg. 5HT and NA recognised as being linked with cortisol and HPA axis activation and both of these can lead to increased dopamine release in the prefrontal cortex by the action of the raphe nuclei.

Continued exposure to stress (ie. high altitude) leads to a change in mood from euphoria to depression. This condition is normally associated with decreased serotonin levels and has symptoms such as a lack of concentration, disinterest, insomnia and loss of appetite. There are two ways of looking at this. The first is that there is decreased serotonin because there is physical acclimatisation to the environmental stress as physiological systems such as the energy production mechanism adjust and therefore, the lack of oxygen is seen as a ´controllable stressor`. This leads to inhibition of the raphe nuclei and decreased ventral medial prefrontal cortical activity which is linked to the System 1 automatic, fast decision-making system and has a role in assessing value. Or, secondly, that there is a down-regulation of the dopamine receptor system in response to its high exposure to its own ligand neurotransmitter as seen in drug addiction for example. Excessive dopamine levels leads to decreased prefrontal cortex activity, decreased numbers of D1, D3 and D4 receptors, decreased glutamate receptors and decreased working memory capability. 5HT follows suit with the down- regulation of its receptors and down- regulation of prefrontal cortical activity leading to the symptoms of depression.  Again, the neurochemical systems readjust to this state and with time will lead to another mood change that of anxiety, irritability and belligerence which are all symptoms normally attributed to increased amygdala activity. This type of mood response supports the documented increase in amygdala activity that HPA axis activation is said to lead to.

Therefore, what can we conclude about the effect of exposure to high altitude on brain functioning? What we have seen is that there are physiological effects at the cellular level due to the low oxygen availability and at the area level because of the activation of the HPA and cortisol production and these translate into effects on cognitive functioning and performance and mood. These effects change in response to duration of exposure and level and naturally, to individual`s own physiological levels.  What makes this topic interesting is that here is another element that has to be considered when we are talking about cognitive capability and bringing about change to cognitive capability. We know about sleep and oestrogen and now we have the HPA system and the action of cortisol and glucocorticoids. It also reinforces the view that changes to physiology and biochemical functioning can have repercussions on how and what we think and even things like environmental stress should not to be underestimated. The results recorded in this investigation with exposure to high altitude shows that individuals should consider it with concern and perhaps for some preventative measures, eg. cognitive training could be advantageous.

Since we`re talking about the topic………

…..can changes in pH in brain cells be observed with exposure to high altitude and do these change with time?

…..would the use of serotonin receptor knock-out mice show definitively that the serotonin system is involved in the effects on working memory and attentional performance caused by exposure to high altitude?

…would administration of serotonin selective uptake inhibitors and other antidepressants lead to a decreased level of anxiety and counteract the depressive mood change observed with longer term exposure to altitude? Would the administration of ketamine also lead to protection from depression as observed with stressed mice?

….what would be the effect of the administration of the ADHD treatment, ritalin? Would there be a shift to normal prefrontal activity as measured by neuroimaging techniques that could accompany the return to normal performance levels for working memory, attention and memory capabilities?

….would there be an increased number of glucocorticoid receptors observed in the hippocampus on acute and chronic exposure to high altitude?

lithium in drinking water and dementia incidence

Posted comment on ´Association of Lithium in Drinking Water with the Incidence of Dementia` by L.V. Kessing, T.A. Gerds, N.N. Knudsen, L.F. Jorgensen, S.M. Kristiansen, D. Voutchkova, V. Ernsten, J. Schulllehner, B. Hansen, P.K. Andersen and A.K. Ersboll and published in JAMA Psychiatry 2017 74(10) 1005-1010 doi 10.1001/jamapsychiatry.2017.2362

SUMMARY

Kessing and colleagues` article addresses the question of whether the incidence of dementia in the general population of Denmark co-varies with long-term exposure to micro-levels of lithium in the drinking water. Their study suggested that higher levels of lithium could be associated with a decreased incidence of dementia.

A nationwide, population-based nested case control study with 733,653 control individuals and 73,731 dementia sufferers was performed in Denmark. Dementia sufferers were aged between 50 and 90 years of age and had received a diagnosis of dementia from 1^st January 1970 to 31^st December 2013 and had hospital contact either as an in- or  out-patient. Each dementia sufferer was matched by both age (median age 80.3 yrs old) and sex (approx. 60% female and 40% male) to 10 controls. All control subjects had to be alive and have no diagnosis of dementia when their matched subject had been diagnosed. The residence of each study subject was recorded from 1986 and each location was cross-referenced to the level of lithium in the drinking water at that time measured according to municipality between 2000 and 2010. The lithium level was assumed to be stable within the study period.  Data was analysed from 1^st January 1995 to 31^st December and Kessing and colleagues looked at 4 levels of lithium exposure (2.0uG/L to 5.0; 5.01 to 10; 10.1 to 15; and greater than 15) and recorded the incidence of dementia in their test groups.

Kessing and colleagues found in their study that the level of lithium exposure was lower for patients with a diagnosis of dementia than for the controls (dementia – median 11.5uG/L whereas controls -median 12.2 uG/L). Also, this association between exposure and dementia was found to be non-linear. A comparison of incidence rate ratio (IRR) for dementia for lithium exposure of 2.0 to 5.0uG/L to exposure greater than 15.0uG/L showed a decreased value for the higher exposure (IRR 0.83, 95% CI, 0.81-0.85 P>0.001). However, the ratio value was the same for a comparison of exposures between 10.1 to 15.0 uG/L (IRR 0.98, 95% CI, 0.96 – 1.01; P= 0.17), but the value increased when it was compared to 5.1 to 10.0uG/L (IRR 1.22, CI 95%, 1.19-1.25 P>.001. Similar patterns were obtained for both Alzheimer and vascular dementia sufferers.

Therefore, Kessing and colleagues summarised that their results suggested that increased lithium exposure in drinking water may be associated with a lower incidence of dementia in a non-linear manner. However, they described the association as not being definitive since the nature of their study set-up meant that patterns and links could be identified between factors, ie. lithium exposure and incidence of dementia, but no definite conclusion could be made because other factors may have influenced  their results.

COMMENT

What makes this article interesting is that it looks at the topic of dementia not from cause or treatment, but from protection. This article hints that naturally occurring high levels of lithium in drinking water can have a protective effect from dementia, ie. there is a slightly lower risk of dementia occurring with exposure to high levels of naturally occurring lithium in this case sourced in the drinking water. Others have jumped to suggesting that adding lithium to drinking water may provide some form of protection from developing this disease, but it should be said that Kessing and colleagues indicate that their results are not definitive and the incidence of dementia could be influenced by other unknown factors. Unfortunately, because of the nature of the disease and the fear that it induces, the field of dementia research and non-academic thinking is awash with different hypotheses about causes, treatment and protection and also unfortunately, although we may be gradually explaining its biochemical basis, we are still not far enough forward to finding a ´one-tablet` cure. The problem with dementia is it`s elusiveness – many causes and triggers, many possible culminations of causes and its wide-ranging physiological effects that may be individual in both degree and nature. The value of this article is that it provides another element to studying this disease in the hope that every fragment of information is another piece of the puzzle.

The article commented on here in this blog is about the effect of lithium exposure on dementia. The beneficial health effects of lithium comes about from studies on bipolar disorder which is where sufferers experience periods of mania and periods of depression.  Sufferers of this particular disorder exhibit associated negative cognitive effects with both of the two phases and it often develops later into dementia. Treatment with chronic administration (but not acute) lithium helps the sufferers by stabilising moods so that the periods of mania and depression are reduced and has also been found to delay the onset of dementia if occurring.

The first point about lithium exposure and dementia is that cognitive disorders, especially those not genetically predetermined and where the emotional status and system are involved, seem to appear over a period of time and require treatments that are also needed over a period of time before reduction of symptoms is observed. This delay between administration and effect means that the long-term ´fix` is not reliant on instantaneous changes of biological processes. There may be observable influences on processes after single administrations, but these may not be the same as those occurring after long-term administration, or even be related to symptoms.  The exploration of the effects of lithium exposure on dementia hence requires two approaches – the instantaneous effects observed perhaps in the ´test-tube` although more advisable on the whole body after single exposure and secondly, what is happening at the biological and physiological levels after a longer period with or without further administration of the ligand or sustained administration. This type of exploration mirrors the approaches to research on depression where we know that the beneficial effects of antidepressant administration require 3 weeks to occur whereas certain biochemical changes are instantaneous eg. the immediate effect on neuronal adenyl cyclase activity compared to long-term effects on neuronal connectivity and neurotransmitter receptor number . The therapeutic effect of any drug (or therapy) has to either reverse these changes in order that treatment is deemed successful, or provide alternative means by which correct neuronal functioning may occur. In the case of some treatments this reversal is also temporary and administration must continue. Without sustained administration the biological processes and systems can fall back into ´dysfunctioning status` resulting in the reappearance of symptoms and an example of this is lithium itself and its role in the treatment of bipolar disorder.

If we are to understand what is happening in dementia we have to look at the conditions that cause it and the mechanisms that are subsequently put into play. Kessing and colleagues` article prompts an investigation of the metal, lithium and its supposed effect on the incidence of dementia. We have to assume that the biological mechanisms involved in dementia and associated with cell death and destruction of neuronal pathways are independent of the cause of the disorder, ie. the biological mechanisms with abnormal functioning are the same whether the dementia observed is a result of long-term bipolar disorder or injury for example. In the same vein, as given above treatments can either normalise the dysfunctioning processes or provide alternatives that compensate for those dysfunctions caused by the disease or injury. If we apply this hypothesis to lithium and dementia, the biochemical effects of lithium which lead to the proposed neuroprotective effect can then be grouped according to whether they affect neuronal firing (eg. reducing levels of over-excitation in neuronal firing in certain brain areas as observed in dementia) or by reducing cell apoptosis (ie. reducing the mechanisms employed in cell degradation and cell death, also perceived as important in dementia). Therefore, lithium could be said to promote a reduction in susceptibility to dementia by affecting one or both of these groups of functions.

If we look at the first group of functions relating to lithium exposure that of effects on neuronal cell firing we can see that the action of short term administered lithium acts at many different neuronal sites and cellular functions. Biochemically, neuronal over-excitation results in excessive firing in individual cells or in groups of cells within brain areas at a level which is outside normal expectations for that cell or area. For lithium to have an effect on this mechanism then it must interact with normal firing mechanisms. (Here in this comment we concentrate just on the brain and neuronal systems since we are looking in general at possible mechanisms for cognitive failure, emotional system upset and dementia.) Lithium ions due to their size and electronic charge can act where two other common ions in the brain act. These are sodium ions (a cation like lithium with a single charge) and magnesium ions (also a cation, but with an electronic charge of two, however having the same ionic and hydrated radius as the lithium ion). Since a lithium compound is medically administered then the lithium is already in ionic form and so is already able to accept an electron. Therefore, electronically lithium ions can replace sodium ions in firing mechanisms.  One way in which it can do this is to transfer through the sodium channels of the neuronal cell membrane. The possible result of this action is cell depolarisation and this is observed in brain areas where hyperpolarisation occurs. Researchers have also found that lithium can regulate the expression of different isoforms of sodium channel and therefore, effect on firing through increased presence of sodium channels is possible. Cellular firing levels can also be affected by lithium causing the release of the neurotransmitters, serotonin (5HT) and noradrenaline (NA) which can result in cells that are activated by these molecules actually depolarising. This is important since in some brain areas, 5HT and NA have an inhibitory effect on cell activity, or have an inhibitory effect on cells further down the pathway.  In the absence of these neurotransmitters for whatever reason, the administration of lithium ions could substitute for their loss and firing whether excitatory or inhibitory results. It should be noted however, that under normal firing circumstances, lithium ions do not cause excessive firing and cellular depolarisation.

Whereas lithium ions can directly replace sodium ions in neuronal firing mechanisms, their action relating to magnesium ions is a little more complicated since they disrupt processes important in cellular firing that rely on the involvement of magnesium ions for correct molecular conformational structure.  For example, lithium ions can have an effect on cell firing via its action on sodium potassium ATPase ( Na+K+ATPase) which is magnesium ion sensitive and responsible for ionic gradients across the neuronal cell membrane during firing and another transport system, the mitochondrial sodium-calcium exchanger. The sodium-calcium exchanger is important in the removal of calcium ions from the mitochondrial matrix. The activity of this depends on previous action of the Na+K+ATPase which pumps sodium ions out of the cell and potassium ions in during firing. This action allows the entry of calcium ions into the cell in the presence of high concentrations of sodium ions (ie. at depolarisation). Hence, failure of Na+K+ATPase action (as observed as impaired in bipolar disorder) leads to potassium ion depletion inside the cell and sodium ion accumulation. Therefore, the sodium calcium exchanger begins to pump calcium ions in leading to an increase in the cells hyper-excitability (also observed in bipolar disorder). Lithium ions in this case activate Na+K+ATPase, hence normalising cellular ion concentrations and depolarising the cells where hyper-excitability was previously observed.  It was found that lithium ions at high doses actually replace the magnesium ions in the complex. The presence of the metal ion is important because of its role in the nucleotide phosphorylation stage of the process eg. in the breakdown of ATP (the transport of ions requires energy) in this case. Nucleoside triphosphates require magnesium ions or a manganese complex to be active since the magnesium ions neutralise some of the negative charges present on the physical polyphosphate chains of the molecules, hence reducing non-specific ionic interactions between the enzymes and the polyphosphate groups of the nucleotide.  This link with nucleotides, magnesium function and lithium action as a substitute ion can also be observed with cellular cyclic nucleotides, eg. in the case of adenyl cyclase and the production of cAMP. Here the enzyme interacts indirectly with the magnesium ion through hydrogen bonds to coordinated water molecules. The inhibitory effects or activation effects of lithium ions on adenyl cyclase (cAMP levels are reduced in depression and increased in mania) relates to G protein binding (likely to be inhibited) and hence, this is another example where lithium has an effect dependent on what the normal functioning of that area or system is.

Therefore, we have seen how lithium ions can work at the level of cell firing, but how does it reduce levels of neuronal firing over-excitation and ultimately, levels of excitotoxicity where cells begin to die? Both over-excitation and increased excitotoxicity have already been reported as involved in dementia and occur in areas such as the entorhinal cortex and hippocampus. The end effect can be local cell death and in dementia there appears to be a natural progression of cellular degradation as inactivity and death in one area leads to underactivity and cellular death in another and so on. It should be remembered that only the hippocampus appears to be capable of high levels of neurogenesis (new cell formation functionally linked to memory) and hence, any loss of cells in other areas will have serious effects on firing of the system as a whole.

So, how can lithium ions reduce over-excitation where needed? The different mechanisms influenced by lithium ions will lead to different effects on neuronal cell functioning restoring normality. The administration of lithium ions can lead to neuronal firing in areas which would normally have an inhibitory effect or could force depolarisation in areas which are normally hyperpolarised. Neuronal firing forced by lithium ion presence could be by substitution with sodium ions in membrane-bound sodium channels leading to cellular depolarisation directly or through its action on neurotransmitters 5HT and NA release. Normalisation of the other ion transfer systems required such as Na+K+ATPase and sodium-calcium exchangers can also be induced. The overall effect is that lithium ions substitute for dysfunctional firing systems and cause neuronal activation which leads to inhibition of cells further down the pathway. This knock-on effect on others could remove the higher levels of excitation seen in some conditions that lead to excitotoxicity and cell death. The overall inhibition of hyper-excitable cells may also explain the activation effects of lithium on adenyl cyclase (cAMP levels are reduced in depression and increased in mania) where G protein binding is inhibited and also reports where lithium administration causes an increase in calcium ions through likely activation of the PI3 / Akt pathway as observed with action against alpha-bungarotoxin and muscarinic receptor binding. Therefore, a reduction in firing occurs and since areas work together and firing is interconnected then functioning patterns of firing will also change and long term alterations ensue. Since in the case of lithium ions and bipolar disorder, mood is stabilised on long-term administration the physiological changes that occur after the sustained period of administration of the metal ion result in a likely ´normalising` action of individual cell firing and ultimately, connectivity patterns seen in the disorder.

The second area of functioning where lithium ions may have their effect is on reducing cell apoptosis or death.  Cell death methods can be intrinsic (eg. requires transcription factor effects and subsequent DNA transcription changes) or extrinsic (eg. requires extracellular receptor binding and caspase cascades), but independent of cause (eg. injury, cell membrane signalling detrimental changes, neurotrophic signal changes) they appear essentially to have the same mechanisms. A change in apoptosis because of lithium treatment leads to a change in mitochondrial/ER dysfunction, reduction of negative epigenetic effects, reduction of glial dysfunction, reduction of oxidative stress and inhibition of the enzyme, glycogen synthase kinase- 3 (GSK-3). For example it has been reported that there is a change in IP3 in bipolar disorder which results in a change in calcium ion signalling in offending cells. The rise in intracellular calcium (also reported in bipolar disorder) results in dysfunction of the mitochondria (observed by increased Bcl-2 levels and decreased Bax levels) and increased oxidative stress of the cell (an effect that can be decreased by glutathione administration also observed in bipolar disorder). The increased level of oxidative stress results in cellular apoptosis. Lithium is reported to inhibit GSK-3 which is one of the factors controlling cellular apoptosis and it can either increase activity or decrease it depending on circumstances. This again could be interpreted as lithium ions ´normalising` function in dysfunctioning cells, or having no effect if the cells are functioning normally.

Increasing apoptosis occurs by the disruption of correct mitochondrial functioning and affects the regulation of the expression of the mitochondrial transcription factor, Bcl-2. Decreased apoptosis occurs in the case of lithium administration when GSK-3 inhibits the early phase of the caspase cascade (caspase 8) or by having an active PI3-AKT pathway which leads to a rise in calcium ion release or an increased beta-catenin/wnt pathway. The inhibition of the GSK3 enzyme has wide ranging effects since the enzyme has multiple functions such as phosphorylation of glycogen synthase and ultimately regulation of glucose metabolism, effects on transcription factors such as cJun and cell cycle mediators such as cyclin D. There are also observations that GSK-3 affects proteins bound to microtubules and this could relate to an observed build-up of beta-amyloid protein in the cells – a process which is linked to the observation of dementia.

Therefore, the administration of lithium ions can result in decreased apoptosis or a protective effect  against apoptosis. The latter may be the case if the cell senses that the normal firing function is defective and another unnatural ion has taken the system over as suspected in the case of lithium ions actions on cellular functioning described above. The result is that there is a normalisation of function in the relevant areas which in bipolar disorder may exhibit extensive induced apoptosis. The increase in cell number and functioning cells is supported also by the observation that lithium administration leads to an increase in volume of the hippocampus, an area whose function is observed to be reduced in dementia. Renewing cells is essential particularly for the hippocampus as stated above since neurogenesis here is linked to the cognitive input of new experiences, binding of information and memory. An increase in growth of the hippocampus has been observed with lithium ion administration and this counteracts the area`s shrinkage which has been reported in depression. An increase in volume implies the production of new cells.

Therefore, lithium ions exhibit a wide range of cellular effects, but their action appears to be linked to only those brain areas whose neuronal functioning is abnormal, ie. areas which exhibit over-excitation or are subjected to over-inhibition. Exposure to the lithium ions can ´normalise` firing and also ´switch off` the apoptosis mechanism so that cells can return to normal functioning levels even if not by natural mechanisms. Cells that are functioning normally appear not to be affected and this is supported for example by the action of lithium ions on guanylyl cyclase which is part of the photo-reduction mechanism in humans and important in visual processing.  Guanylyl cyclase activity leads to an increase in cGMP keeping sodium ion channels open whereas light reduces the level of cGMP causing the sodium channels to close and the cell to hyperpolarise. Sufferers of bipolar disorder report reduced visual motion perception, but lithium has no effect on photo-reduction or on ocular functioning even though it is a potent inhibitor of guanylyl cyclase activity. Therefore, just like with neuronal firing, this brings about a discrepancy since the biochemical effects described for lithium ions are independent of cell status and therefore should occur whether the cell is in either an over-excited/over-inhibited state or not and the action should be on all cells and not just those exhibiting excitotoxicity. The action of lithium ions therefore, implies that those cells exhibiting excitotoxicity or lack of inhibition have already different functioning or different physiological characteristics that favour lithium ion action and binding in preference to its action on normal cells. It could mean that the propensity of binding of lithium ions is lower on these abnormally functioning cells than the cells own natural substrates and hence, it only works when these natural substrates are not available. Since the first port of call of the lithium ion is the neuronal cell membrane then it is possible that the over-excited or over-inhibited cell already has a different cell membrane structure or functioning and it is here where the natural substrates are normally favoured, but when absent, then the presence of lithium ions after administration will have an effect. This hypothesis is supported by the biochemical property of magnesium ions which is that they coordinate groups of ions or molecules so that the correct arrangement and correct conformation of the molecule is attained. This could be of importance when looking at lipid rafts and protein conformations as part of the cell membrane physiological structure and functioning. Lithium ions could replace magnesium ions if absent to re-stabilise the neuronal cell membrane lipid raft structure so that normal firing mechanisms can be induced.

Therefore, a look at the biochemical basis of lithium ion action in bipolar disorder and the mechanisms behind its mood stabilising effect gives an indication to the complexity of dementia and to the problem of defining biochemically what causes it and what can be done about it. Lithium ion administration can be described as a ´blanket` treatment affecting many different systems even if the overall affect is a reduction of emotional upsets and a stabilisation of mood. Its possible mode of action is likely to be through normalising cellular firing in areas experiencing over-excitation or lack of firing and this could be caused by imbalances of sodium ions and magnesium ions. It could also act by affecting the cellular apoptosis processes that may be called into play if cell death is ordered due to cell dysfunction or to prevent cell death ordered because of the abnormal lithium ion effects on the neuronal firing mechanisms. Lithium ion exposure also does provide some insight into a possible initial ´limiting` cause for dysfunctional cell firing and this appears to be the cell`s exterior membrane.

Since we`re talking about the topic….

…would it be possible to use radioactive lithium to map areas of neuronal cell action with time using mouse or rat models of disorders such as dementia? Would such studies give an idea of where the ´limiting` brain area for dementia is?

……lithium is said to be an important inhibitor of glycogen synthase kinase 3 which is known to phosphorylate the voltage gated potassium channel type, KCNQ2. Phosphorylation of the channel decreases its activity and ultimately affects the transport of potassium ions important in depolarisation. Therefore, can we assume that lithium ion action ultimately has an effect on KCNQ2  channels` number or function and this can be observed by measuring potassium ion transport and concentration in the neuronal cells using inhibitors or blockers of KCNQ2?

……prolonged lithium ion treatment is said to lead to probably indirect inhibition of PARP-1 (poly-(ADP-ribose) polymerase) by inhibiting 3´5´-phosphoadenosine phosphatase (pAp-phosphotase) function. This causes a progressive accumulation of pAp in the cell that binds to the PARP-1 inhibiting it. However, the hypothesis is under dispute. Can we assume that deletion of pAp phosphatase gene could clarify the observation since in its absence poly -ADP-ribosylation activity inside the cell would be normal and PARP-1 inhibition would not be observed?