Posted comment on ´Neuronal chloride regulation via KCC2 is modulated through a GABA B receptor protein complex` by R. Wright, S.E. Newey, A. Ilie, W. Wefelmeyer, J.V. Raimondo, R. Ginham, R.A. J. Mcllhinney and C.J. Akerman and published in Journal of Neuroscience 31st May 2017 37(22) p. 5447, doi.org/10.1523/JNEUROSCI.2164-16.2017
It is known that synaptic inhibition can occur through the activity of ionotropic gamma aminobutyric acid A receptors (GABA A Rs) which produce fast inhibitory synaptic currents involving transmembrane chloride gradients and also through gamma aminobutyric acid B receptors (GABA B Rs) which produce slower inhibitory actions and are G-protein related (metabotropic receptors). Wright and colleagues investigated the GABA B Rs of the rat hippocampus which are physically associated with the potassium chloride cotransporter protein, KCC2 (solute carrier family 12, member 5 protein – SLC12A5).
In their experiments, Wright and colleagues used cortical membrane samples from 5 Sprague Dawley rats. The peptide mixtures were analyzed by liquid chromatography tandem mass spectrometry (MS/MS) and reference was made to a GlaxoSmithKline non-redundant protein database. Coimmunoprecipitation, biotinylation of cell surface receptors, immunofluorescence and electrophysiological experiments were conducted on organotypic hippocampal brain slices from P7 male Wistar rats (cultured for 7–14 DIV before experimentation), or on CHO cell cultures (or transfected CHO cells cultures) which expressed the rat GABA B R1b and GABA B R2 (termed CHO GABABR1b/R2) . The advantages of using the organotypic hippocampal brain slices were that they allowed tests to be performed on the same sample and also that the method was shown to produce mature and stable chloride homeostatic mechanisms as required. Intracellular chloride concentrations were measured using cyan and yellow fluorescent protein (CFP-YFP) based chloride sensor proteins with the organotypic hippocampal CA3 cells excited at 850nm and 510nm respectively for the two separate ionic channels. The ratio of the two was calculated and used in the results.
Wright and colleagues found in their coimmunoprecipitation and mass spectrometry experiments that the GABA B R complexes of the cortical preparations used contained multiple peptide components, eg. G protein subunits, KCC2, potassium channel tetramerization proteins, NEM sensitive fusion proteins, and 14-3-3 signaling proteins. KCC2 was found in three isolates of the neuronal cell membranes associated with GABA B R1 in samples of the cortex and hippocampus. Western Blot analysis gave two distinct bands at about 130 and 270 kd representing the receptor proteins existing in monomeric and dimeric forms. This occurred with both GABA B R1a and GABA B R1b forms. The protein complexes were found to be associated with the somatic and dendritic plasma membranes of the organotypic hippocampal pyramidal cells. Cortical samples also showed KCC2 co-localised with both the GABA B R 1a and 1b forms and this association was additionally confirmed using the CHO cell line. KCC2 was predicted to consist of a cytoplasmic amino acid domain and a cytoplasmic carboxyl domain existing either side of the transmembrane domain and consisting of twelve transmembrane helices. Biotynlation experiments showed that the fusion products of the KCC2 that contained the transmembrane domain were trafficked to the cell surface. The authors also found that GABA B R can form a complex with KCC2 that does not contain intracellular terminal domains, but it cannot form a complex that does not contain the transmembrane domain, which indicated that KCC2 associates with GABA B R via the transmembrane domain.
In investigating the effect of GABA B R on transmembrane chloride gradients, Wright and colleagues looked at the reversal potential of ionotropic GABA A R (EGABAA) and CA3 pyramidal cells using the GABA A receptor agonist, muscimol. They found that CA3 pyramidal cells demonstrated a hyperpolarizing EGABAA state of -82.8 mV with a resting potential of – 71.5mV with a shift to -70mV on application of furosemide (a GABA A R antagonist). This was said to demonstrate an active KCC2. When the GABA B R agonist, SKF97541, was used there was a depolarizing shift from -82mV to – 78mV demonstrating increased intracellular chloride. This change was prevented by using a GABA B R antagonist. The GABA B R effect was shown to be related to G protein signaling since it was disrupted by using the Gi/Go protein antagonist, PTX. This also blocked the SKF97541 effect. Using SCH23390 which blocks downstream GIRK channels (G protein coupled inwardly rectifying potassium channels), the SKF97541 change was found not to be prevented showing that GIRK channels are not involved in the mechanism. The EGABAA was also not affected by activation of post-synaptic adenosine receptors, which are also G protein coupled and linked to potassium channel activity. This confirmed the hypothesis that activation of GABA B R with SKF97451 involved an intracellular concentration increase of chloride ions.
The authors also investigated if GABA B R activation could regulate KCC2 at the plasma membrane. They showed that the shift in EGABAA occurred because of reduced KCC2 function. Furosemide was used that blocks KCC2 activity and cells were found to have higher depolarizing resting EGABAA (-70mV compared to -83mV of the controls). This occurred within 5 minutes which demonstrated that KCC2 functions continuously to maintain the effect. The effect of the sodium-potassium-chloride cotransporter protein NKCC1, which can also regulate chloride concentrations in hippocampal pyramidal cells, was discounted by an experiment using its selective blocker, bumetanide. The results were the same as the controls and the effect of the GABA B R agonist was not changed. The effects of other manipulations that alter GABA B R activation were also tested. The authors used zero Mg2+ ACSF that reduces KCC2 levels. In this case, they found a depolarizing shift in EGABAA (to -64mV).
Biotinylation experiments were also used to quantify changes in the plasmalemmal level of chloride transporter proteins in neuronal tissue. Here, the authors found that after SKF97451 activation, there was a reduction of KCC2 at the cell surface with both monomeric and dimeric forms reduced (80, 83%). The level of GABA B R1 was also found to be reduced at the cell membrane with a concomitant decrease in electrophysiological recordings (-88mV went to -79mV). The reduced level of KCC2 was found not to be linked to degradation changes, but to the amount of surface protein trafficking (ie. endocytosis and recycling) taking place. Application of SKF97451 to the CHO cells produced the same results as the organotypic slices. Therefore, the authors suggested that the KCC2 chloride transport mechanism is sensitive to KCC2 expression levels, post-translational modifications or that intermediate proteins are involved in regulating the surface expression in neurons.
In order to investigate whether GABAB R regulation of KCC2 involved clathrin mediated endocytosis, the authors performed pretreatment with the blocker dansylcadaverine (DC) and found no change in EGABAA in CA3 pyramidal cells in the presence or absence of SKF97451 ie. the normal EGABAA shift was prevented by the pretreatment. The reduction of surface levels of KCC2 following GABA B R activation reduced with pretreatment with DC indicating that disruption of clathrin-mediated endocytosis prevented the GABA B R mediated change in surface KCC2. Surface KCC2 levels were not altered in the presence of DC. Treating cells with a combination of calcium ion channel blockers, selective protein kinase C inhibitors, general kinase blockers, tyrosine phosphatase inhibitors, or protein phosphatase 1 and 2 inhibitors had no effect on the SKF97451 induced shift in EGABAA, nor on the levels of surface KCC2. This indicated that calcium signaling was not involved. Therefore, it was concluded that GABA B R regulation of KCC2 involves clathrin-mediated endocytosis and is not linked to calcium signaling.
In order to investigate whether synaptically-driven GABA B R activity affects intracellular chloride regulation (ie. that the GABA B mediated effect occurred at the inhibitory synaptic connections at presynaptic GABAergic interneurons) or not, the authors evoked monosynaptic GABA B R responses and measured synaptic EGABAA of -76mV similar to the muscimol evoked responses. GABA B R are thought to be located predominantly extra-synaptically in hippocampal pyramidal cells and are thought to be activated under robust GABA release occurring during periods of high frequency presynaptic firing. Wright and colleagues found a single presynaptic stimulus generated a pure GABA B R response in CA3 pyramidal neurons which was blocked by SR95531 (a selective GABA A R antagonist). A high frequency train of stimuli (6 at 20HZ) produced a postsynaptic response comprising of a large GABA A R conductance (found over a range of frequencies) and a smaller GABA B R conductance that could be blocked by CGP55845 (a selective GABA B R antagonist). They concluded that the optimal presynaptic frequency for activating a GABA B R response was close to 20HZ. Blocking KCC2 with the selective antagonist VU0240551 led to a change in the EGABAA reduced shift due to GABA B R stimulation. Therefore, Wright and colleagues experiments showed that the GABA B R mediated effect occurred via KCC2 and was evoked by the agonist effect and by synaptically evoked GABA release.
Therefore, Wright and colleagues concluded that they had identified an association between GABA B R activity and KCC2 at the cell surface. Agonist binding leads to GABA B R activation and G protein activation and chloride entry (increase in intracellular chloride of about 1.2mm) as part of the signaling mechanism. KCC2 reduced function was involved, ie. agonist activation modulates proteins with which KCC2 is physically associated with the GABA B R. The authors concluded that the GABA B R effect on KCC2 was different to other activity dependent mechanisms that can regulate KCC2. Post-translational regulation linked to calcium signaling events and associated enzymatic modifications (KCC2 function is associated with its phosphorylation state since its turnover is rapid as a function of phosphorylation) was not shown since there were no effects from calcium ion signaling blockers, phosphatases and phosphokinases etc. The reduced function of KCC2 was also not caused by increased degradation since the total level of KCC2 present was not altered. Instead the authors concluded that the effect occurred at the cell surface level. Relating to this there were conflicting reports about the GABA B R level at surface. Some researchers claim that the receptor is stable whether active or not whilst others state that the receptor is mobile and rapidly internalized in a clathrin-dependent manner relating to activation. Wright and colleagues` experiments showed that GABA B R activation led to down-regulation of the receptor and KCC2 surface expression. The discrepancy between the reports was explained on the dimerization state of the receptor complex or the experimental system used. The authors claim that the effect is observed only due to a subset of proteins since protein proportion is less than 25% for both surface proteins. The effect occurred over a similar, but not identical timescale of down-regulation of NCC2 function which could reflect the sensitivity of the experimental method or functional changes in the KCC2 resulting from recycling to the membrane, or changes in membrane domain, cellular location or molecular interactions. Since the authors found that blocking clathrin-mediated endocytosis prevented the GABA B R down-regulation of KCC2 function and expression at membrane their findings supported the observations from other researchers that internalized GABA B R are associated with the clathrin binding adaptor protein 2 complex and that KCC2 also undergoes fast clathrin-mediated endocytosis.
Therefore, Wright and colleagues concluded in their article that GABA B R modulates its effect on KCC2 function via a mechanism involving clathrin-mediated endocytosis. Since their experiments produced a reduction of only 20% in the level of surface KCC2 and a smaller GABA B R mediated shift in EGABAA with furosemide they concluded that different pools of KCC2 must exist in the membrane. This supports observations from others that KCC2 is also localised at glutamergic postsynaptic structures (perhaps NMDAR), functionally associated with kainite receptors and implicated in glutamatergic transmission. This supports the evidence that GABA B R activation could affect NMDA R activation. Therefore, Wright and colleagues` experiments were said to demonstrate an interaction of GABA R systems within the hippocampus and shows how GABA B R can regulate one type of inhibitory synaptic transmission.
What makes this article interesting is that it discusses the topic of synaptic inhibition instead of the more popular topic of neuronal stimulation. The type of synaptic inhibition discussed here is not the normal shutting off of stimulation of a firing cell by hyperpolarization through potassium ion channel opening and potassium ions flooding in and the subsequent readjustment of the cellular electric signal and induction of mechanisms in place regarding the endocytosis, enzyme and protein phosphorylation and dephosphorylation mechanisms for example that naturally end the firing of the cell in question, but instead this article describes the action of another mechanism which actually prevents cellular firing. GABA binding and GABA receptor action take the membrane potential away from its firing threshold and prevents the next cell in the neuronal pathway from initiating an action potential and depolarizing. This means that firing of cells further down in the neuronal pathway is prevented and earns the GABA synapse the name of inhibitory synapses. Inhibitory synapses normally involve post-synaptic receptors that are mainly transmitter gated ion channels and this paper describes one such case, that of the amino acid neurotransmitter, GABA, the GABA receptor and its functional mechanisms. GABA is known to have an inhibiting effect on firing and physiological conditions can arise from extremes in this mechanism, ie. too much inhibition leads to coma; too little leads to seizures and epilepsy for example.
It is known that GABA released from presynaptic neurons in the course of firing of that neuron binds to post-synaptic GABA receptors and synaptic inhibition occurs through either the ionotropic GABA A R (relies on transmembrane chloride channels and chloride ion gradients to generate fast inhibitory synaptic currents), and/or the metabotropic GABA B R which produces a slower inhibitory effect. Two mechanisms are linked to GABA B R activation: G protein signaling which generates cAMP and initiates a cascade mechanism involving protein kinase activation and phosphorylation of proteins; and inhibition via the opening of chloride channels so that the firing threshold of the cell is not reached. There is normally low chloride permeability in cells since chloride ions are linked to several cell functions such as the regulation of cell pH which is tightly regulated.
In the case of the GABA A R this receptor is linked directly to the opening of the ionic chloride channel and causes fast synaptic inhibitory action (80% IPSP from this type). Its agonist is muscimol (used in the experiments described above to generate the EGABAA) and its antagonist is bicuculline. This receptor is particularly known for its binding to certain common substances: it binds benzodiazepines (released on anxiety) leading to increased frequency of opening of the chloride channel; it binds barbiturates which increase the length of time the channel is open; and it binds ethanol, although for this it needs a specific subunit structure. Binding of GABA leads to synaptic inhibition, but it is not always associated with big responses, eg. in CA1 there could be a shunting inhibition. This is where the synapse acts as an electrical shunt preventing the current from flowing from one side to another because the membrane potential at the site of the inhibitory synapse is at the time equal to the chloride equilibrium potential (ECl – about -65mV). Opening of the chloride channel allows chloride ions to cross the membrane in a direction that brings the membrane potential towards this chloride equilibrium potential. If the membrane potential is less negative than -65mV when the neurotransmitter is released then activation of these channels would cause a hyperpolarizing IPSP, but if the membrane potential is -65mV then no IPSP is visible after chloride channel activation because the value of the membrane potential is the same as the ECl. This is called the reversal potential. In this case the positive current therefore flows outwards across the membrane at this site to bring the membrane potential to -65mV and there is formally an equivalent inward movement of negatively charged chloride ions. Tominaga found that theta burst brain wave stimulation could induce spike firing and LTP in CA1 cells. When theta burst stimulation was paired with a NMDA R blocker then enhanced GABA A R spike firing was observed. This enhanced excitatory postsynaptic potential was blocked with a GABA A R antagonist. They suggested therefore that pulsed burst stimulation activated the GABA A R system to cause short term spike firing increases without increasing postsynaptic excitability, thus establishing a link between post synaptic firing in CA1 with GABA shunting inhibition. However, in Wright and colleagues experiments only chloride ion channels and the inhibitory effect of chloride ion movement by GABA was investigated.
The work by Wright and colleagues looked at the other form of GABA receptor that of the GABA B R which exhibits different properties to its companion GABA A receptor. GABA B R has auxiliary subunit proteins that modulate agonist response plus the kinetics of the G protein signaling. G protein activation leads to the formation of cAMP via the conversion of ATP to cAMP free in the cytosol. Free cAMP activates protein kinases which catalyse phosphorylation (ie. the transfer from ATP of a phosphoryl group to serine or threonine amino acid residues of proteins). In some neurons one protein phosphorylated when cAMP rises is a type of potassium channel causing it to close and hence reducing the membrane conductance of potassium so that the cell becomes more excitable. It is also reported in some cells that the rise in cAMP concentration is linked to changes in cellular processes such as the degradation of storage fuels and as in this case the induction of opening of chloride channels.
The GABA B R also has proteins for the control of dimerization or desensitization of the receptor and also has molecular partners for associations that enable the GABA B subunits to regulate gene transcription and intracellular trafficking of other membrane proteins. The recycling of the GABA B R at the cell surface is dynamic and modulated through receptor activation, composition, phosphorylation, or degradation. GABA B R are also associated with chloride channel functioning as seen above with GABA A R and also linked to synaptic inhibition. However, in this case the receptor is associated with KCC2, a potassium-chloride cotransporter protein, where receptor activation leads to down-functioning of the KCC2 function. Rapid changes in KCC2 function have been shown to be elicited in an activity-dependent fashion and involve different post-translational regulation mechanisms of the transporter protein, including its phosphorylation state and regulation at the cell surface. The mechanism could be that cAMP is formed from G-protein activation on GABA binding to the post-synaptic receptor. The cAMP leads to activation of protein kinase which phosphorylates the KCC2`s serine/threonine residues causing a conformational change that reduces the KCC2 activity, but opens the chloride channel. Wright and colleagues found that the level of KCC2 at the membrane surface reduced 20 minutes after stimulus. This indicates that the phosphorylated form of KCC2 is inactive and clathrin induced endocytosis of KCC2 and the receptor complex occurs. In this case, KCC2 in its normal form is associated with the receptor inhibiting the opening of the chloride channel and in its phosphorylated form causes the chloride channel to open. Opening of channels on amino acid phosphorylation is seen with the opening of non-specific cation channels in olfactory epithelial cells. Here, phosphorylation with cAMP protein kinase action allows calcium ions and other cations into the cell with the flow of cations causing depolarization of the neuronal membrane and initiating the action potential. In the case of KCC2 and the GABA B R then either the binding of the agonist on the postsynaptic receptor will cause a conformational change that results in opening of chloride channel directly or the binding of the agonist to the GABA B R itself will cause activation of protein kinase by the cAMP formed which will then phosphorylate the serine or threonine residues of the KCC2. This GABA binding will result in down-functioning of the KCC2 protein and cause conformational changes in the chloride ion channel resulting in it opening and chloride ions to flow inward. The difference to GABA A R binding is that GABA B R binding occurs when there are repetitive stimuli ie. firing is more sustained hence, long term stimuli. Therefore, the KCC2 association with the GABA receptor changes the way in which it is associated with the chloride channel.
Therefore, there are two types of chloride inhibition associated with GABA receptor binding: one fast through the GABA A R and a slower inhibition through GABA B R. We cans ask why there is a need for two systems of inhibition brought about by the same GABA neurotransmitter. It should be pointed out first of all that just because there are two types of receptor this does not mean that one area has one type of receptor and another one has the other. It is known that an area can have both, for example the globus pallidus. In this area, GABA B R are intracellular and presynaptic and GABA A R are on plasma membranes. Therefore, their localization is then probably linked with the two types of inhibition that the receptors are associated with. Fast inhibition is likely to be associated with a strong stimulus with multiple neuronal cells firing and connectivity between neurons and neuronal pathways meaning that the signal is transmitted quickly and efficiently and that neuronal cell assemblies symbolizing the electrical representation of the stimulus are formed. At the cellular level this means that the firing of neurons of a particular pathway occur from sensory level upwards to the higher cortical levels with action potentials, depolarization and release of neurotransmitter into the synaptic cleft occurring at each level. When the neurotransmitter GABA is released it binds to the postsynaptic neuronal membrane and results in the connecting cell being unlikely to reach its firing threshold since it is effectively hyperpolarized by causing a change in the ionic balance of the cell. Therefore, the transmitting signal stops at that cell. However, if the signal is transmitted via other neurotransmitters being released or firing of other cells not containing GABA synthesizing enzymes and substrates then the transmission of the signal continues. The overall pattern of firing results in the electrical representation of the stimuli being established.
The result of GABA B R action is the same as GABA A R apart from it being slower due the associated KCC2 involvement. Why then does any cell or brain area require a mechanism of slower inhibition? The difference between GABA A R inhibition and GABA B R inhibition is that GABA B R inhibition occurs in the case of repeated or sustained stimuli. Repeated or sustained stimuli are required for long-term memory and the appropriate physiological changes of the neuron. If an external stimuli is repeated but then stops then there is no problem. The signal stops as described above and no inhibition of cellular firing is required. However, repetition of cellular firing can be elicited by internal stimulation only and cells have been shown in the hippocampus and PFC to exhibit these characteristics. We also know that many long-term memories are formed where there is no deliberate repetition of stimuli so the conditions for long-term physiological changes associated with long-term memory have to be achieved by internal means. For example in the case of the hippocampus, this area plays a critical role in long-term memory, spatial memory, object and location (timing and order) for example. Activation of the cells occurs along the sensory pathways from the entorhinal cortex to the hippocampal dentate gyrus to the CA3 region and then to the CA1 which can then activate the deep entorhinal cortex again. The firing signal goes from the hippocampus then to the PFC and other areas. After a particular time the cells are then inhibited from firing by activation of the GABAergic cells that release GABA and bind to the postsynaptic membrane receptors, opening the postsynaptic chloride channels to prevent the threshold of firing being reached in the next cell of the pathway. This stops the repeated firing condition of these reverberating firing cells so that the cells are free to experience other stimuli. It is also known that that repetition of firing by internal stimulation can cause hyperexcitability of some cells, which can lead to epilepsy. Therefore, the internal method of stopping the transmitting signal is beneficial if the firing is carried on for too long. Another reason is to support desensitization of receptors through multiple stimuli. Desensitisation is a natural method for preventing the hyperexcitability described above. Repeated stimuli already cause the electrical stimulation for long term physical changes to be put in play, but desensitization of the receptors allow neuron firing to be switched off without exhausting the cell ie. before the absolute refractory period. Hence, the cells are more capable of responding to other stimuli within a short period of recovery time. The activation of chloride channels and the failure of the post-synaptic cell to reach firing threshold complements the decrease in sensitivity of the receptor to the agonist.
Therefore, what does inhibition mean to the overall pattern of firing and the electrical signal? Inhibitory synapses contribute to the overall synaptic integration of the cellular system in which they exist. IPSPs can be subtracted from ESPS making the postsynaptic neuron less likely to fire and elicit an action potential. Also, shunting inhibition acts to drastically reduce membrane resistance and consequently dendritic length constant (depolarization is 37% of at the origin) thus allowing positive current to flow out across the membrane instead of internally down the dendrite toward the spike initiation zone. The reaction of GABA R functioning is that the firing signal is stopped and since there is phase locking of firing and non-firing cells and neuronal cell assembly formation then maybe it is better to think of neuronal cell assemblies not just in terms of firing cells, but also of those non-firing at the same time. For example, cortical firing described by Deneve says that the area tightly balances excitation. Inhibitory currents not only match the excitatory currents on average, but track them on a millisecond time scale, whether they are caused by external stimuli or spontaneous fluctuations. This suggested that a tight excitatory/inhibitory balance may be a signature of a highly cooperative code with the precise, tight balance providing a template that allows cortical neurons to construct high-dimensional population codes and learn complex functions of their inputs. This tight balance of excitatory/inhibitory balance may be critical for correct functioning and this supports the views of others where it is known that neural dynamics are poised at criticality (Zhigelor) and that neural avalanches and long range temporal correlations are hallmarks of critical dynamics in neuronal activity and occur at fast and slow timescales. If there is synaptic integration then it is likely that inhibition occurs at the rich nodes (Nigam) where any excitation or inhibition is likely to have its greatest effect. Also, it is recognized that key to correct functioning of the brain is its capability to reconfigure its network structure to respond to its demands (Cohen). This could mean local, within-network communication (ie. critical for motor execution) or integrative, between-network communication (ie. critical for working memory) and involve excitation or inhibition of cells. Therefore, this balance of inhibitory/excitatory firing is essential for correct brain functioning.
Such a balance has been shown to be important in the development of neurons and reinforces why GABAergic neurons play an important role in connectivity during this time. Restivo showed that new neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits throughout adulthood. Although the addition of these new neurons may facilitate the formation of new memories, as they integrate, they provide additional excitatory drive to CA3 pyramidal neurons. During development, to maintain homeostasis, new neurons form preferential contacts with local inhibitory circuits. During adulthood, new neurons form connections with inhibitory cells in the dentate gyrus and CA3 regions as they integrate into hippocampal circuits. In particular, en passant bouton and filopodia connections with CA3 interneurons peak when adult-generated dentate granule cells are approx. 4 weeks of age, a time point when these cells are at their most excitable. Restivo found that CA3 interneurons were activated robustly during learning and that their activity was strongly coupled with activity of 4-week-old (but not older) adult-generated DGCs. Hence, this indicated that as adult-generated neurons integrate into hippocampal circuits, they transiently form strong anatomical, effective, and functional connections with local inhibitory circuits in the CA3.
The balance of excitatory and inhibitory synapses and synaptic integration may also be important for the brain at rest. We know that that brain commonly exhibits spontaneous (ie. in the absence of a task) fluctuations in neural activity that are correlated across brain regions (van der Brink). The topography of these intrinsic correlations is in part determined by the fixed anatomical connectivity between regions, but it is not clear which factors dynamically sculpt this topography. Potential candidates are given as the subcortical catecholaminergic neuromodulatory systems, such as the locus coeruleus-norepinephrine system which sends diffuse projections to most parts of the forebrain. Here, it was found that catecholamines reduce the strength of the functional interactions during rest and this decrease showed an anterior–posterior gradient in the cortex, with strongest connections between regions belonging to distinct resting-state networks. In this case, the firing noradrenalinergic neurons have NE receptors that are G-protein linked and could provide an increased potassium channel phosphorylation and activity. Therefore, the system is dampened by inhibition of firing. Therefore, it is possible that the GABAergic system could instigate the same effect on the excitatory/inhibitory system via its chloride ion channel opening when the brain is at rest.
Therefore, the study of neuronal firing inhibition is important to the workings of the brain as a whole. This is clear when we see that imbalances of these inhibitory systems produce marked physiological effects eg. hyperexcitability or coma and development problems. Hence, it could be that not only should we be looking at firing for explanations of cognitive defects, but also be looking at the contribution that non-firing cells makes to the overall picture of synaptic integration and investigate why certain cells and systems are not firing. For example, although Alzheimer disease is linked to amyloid deficiency and endocytosis disruption, chloride channel function may provide another reason why hyperexcitability of the hippocampal areas exist and may give another mechanism by which manipulation could have a beneficial effect. Therefore, cellular firing inhibition may be as important as its complementary stimulation action.
Since we`re talking about the topic……………
….the first direct evidence of synaptic connections between interneurons came from paired recording of experiments combined with biocytin labeling and anatomical reconstruction of recorded neurons. Neuronal firing in artificial conditions may not be representative of what is actually going on in the brain with long distance inhibition and excitation coming into play. Therefore, should organotypic slices always be used to confirm results achieved in patch clamp experiments on synaptic integration?
….when the GABA A agonist is tested with the calcium ion dependent protein kinase inhibitor then there is no change in EGABAA effect. Since GABA B R causes phosphorylation of chloride channels, should the experiments be repeated with pyruvate kinase not linked to calcium ions? Would a change in results be observed and if no change is seen does this rule out a phosphorylation effect?
…..it is known that kainite receptors depress GABA mediated inhibition and increase the firing rate of interneurons. This is shown to also require KCC2. What happens in this mechanism? Are kainite receptors presynaptic so GABA is not released and therefore the post-synaptic effect of GABA does not occur? If kainite receptors are blocked will the same effect on EGABAA be observed?
……what would be the effect on signaling of influencing the lipid raft of the GABA B R G-protein complex? Would a ´hardening` of the lipid raft prevent the chloride channel functioning and hyperpolarizing effect?