action of anaesthetic agents at the neuronal cell membrane

Posted comment on ´When the lights go out` by Philip Ball and published in New Scientist no. 3115 4th March 2017 p. 36

SUMMARY

As Ball states in the beginning of his article, after 177 years of using anaesthetic agents for surgery we are still not exactly sure on the biochemical mechanisms behind them apart from that their administration affects particular molecules in the brain and this results in the subject losing consciousness. What is known, with knowledge coming from the fields of biochemistry and biophysics, is that the initial stages of the agents` effects involve them binding to specific ´receptor` molecules in a traditional ´lock and key` binding type mechanism. However, there is a wide range of agents of various molecular sizes that can do this, a concept not completely in keeping with the ´lock and key` mechanism. Ball gives as an example, xenon, which is a fairly common anaesthetic agent. Xenon gas exists as lone unreactive atoms, not compatible with water molecules and preferring negative and positively charged particles, but favouring a non-polar environment which of course is part of the basic structure of neuronal cell membranes. This preference, discovered over a century ago, is a common feature of many anaesthetic agents and it was hypothesized early on that they might bind to lipids in the cell membranes, accumulate and make the cell swell or distort by disrupting the adjacent channels which allow ions to pass through the membrane during normal nerve transmission. As expected, the result is that the cell`s capability to transmit a neuronal signal is disrupted.

This neuronal model was expanded by Cantor in 1997 who hypothesized that binding of the anaesthetic agents to the neuronal membrane molecules was not ´indiscriminate`, but that they bound to molecules associated around the ion channels. This would influence how they clustered together and could cause changes to the curvature of the membrane itself. The hypothesis at the time lacked detail and it was believed that the reported changes would be too small to make a difference to the nerve signal. However, later research has shown that this may be an incorrect assumption since indications are that small changes in membrane structure may have more significant effects on membrane function than expected. Ball reports in his article the views of Machta and Veatch who worked to the physics concept that at a critical point or critical temperature a system can undergo an abrupt change in state. Machta, Veatch and also Sethna believe that binding of the anaesthetic agents may affect the ´critical temperature` of the cell membrane making the cell more sensitive to slight changes (the so-called Meyer-Overton rule). The researchers suggested that close to a critical temperature the neuronal cell membrane molecules are constantly rearranging themselves and that in the membrane there are ´rafts` of regularly packed molecules (mostly cholesterol and saturated fats) that drift within a more disorderly matrix of unsaturated fats. Channels and receptors function when surrounded by appropriate molecules and within appropriate membrane ´rafts`. Machta, Veatch and also Sethna suggested that the anaesthetic agents bind to the membrane molecules and consequently alter the temperature at which the membrane critical state is achieved. This results in the channels not attaining their functioning status. Machta and Veatch`s hypothesis was supported by their own experiments using alcohols such as ethanol that act as anaesthetic agents. These substances were shown to lower the critical temperature of the membrane, ie. it would have to be colder before the appropriate rafts form for correct channel functioning. They also used as evidence the case of two lipid-loving drugs that should act as anaesthetics, but do not and found that these failed to alter the membrane`s critical temperature. The reverse was also found. Compounds that raised the critical temperature counteracted the effects of the anaesthetic agents eg. hexadecanol suppressed the effect of ethanol in tadpoles.  The theory appears to apply to both intravenously delivered anaesthetics as well as inhaled, small molecule anaesthetics according to Forman.

However, there are other hypotheses on how anaesthetic agents work and Ball in his article discusses the quantum-physics based view of Luca Turin. Turin suggests that some general anaesthetics cause electrons of membrane bound molecules to jump from one to another, thus altering the molecule`s functioning. His hypothesis was based on ideas about how the sense of smell works. He proposed that scent is perceived not from the shapes and binding of specific molecules (the biochemical view), but by the vibrations the molecules cause and these influence electrons jumping across gaps in the olfactory receptors. In the case of the anaesthetic agent, xenon, Turin proposed that xenon inserts itself directly into the membrane molecules and influences signaling by providing new, energetically favourable pathways within the molecule along which individual electrons could jump. Such electron currents would produce changes in the spin of the molecules, a property which can be measured. Turin and colleagues produced evidence for the hypothesis in the form of fruit flies which exhibit increases in electron spin when exposed to anaesthetic agents such as xenon, nitrous oxide and chloroform. The researchers also expanded the hypothesis by suggesting that the sites of the anaesthetic agents were molecules of the mitochondrial membrane. Forman, however, warned against the acceptance of what he called ´zombie theories` (ie. where experiments cannot show definitive evidence, but neither can the idea be definitively dismissed) and therefore to date, common acceptance of the action of anaesthetic agents is through binding of the agents to cellular membrane molecules related to ion channel functioning.

Ball concludes his article by expounding the importance of knowing the mechanism behind anaesthetic agents linking it to the design of better agents with fewer side effects and higher efficiency so that surgical doses can be lowered.

COMMENT

What makes this article interesting is that it describes something that gives measurable results, but involves mechanisms that range from the common and provable (e.g. blood concentration, lock and key binding, changes in intracellular ion concentrations) to the more esoteric, unprovable (eg. lipid rafts, electron transfer, consciousness.) Even the question ´Can we use the knowledge about natural sleep, which also involves loss of consciousness to explain the action of anaesthetic agents and vice versa?` is interesting because although sleep is widely researched, it too is not definitively explained in terms of neurochemistry. Hence, it is shown again that we need new ideas, new experimental techniques, and actually probably new physical theories. We cannot assume that the physics of biological materials is the same as the physics of metals, air, stone, and fluids for example since even if we take the example of the brain neuron, we have multiple physical states existing within millimetres of each other, eg we have solid objects, fluids, gaps, all within close quarters and these can experience microchanges in milliseconds within molecules or affecting the outside of molecules. To solve the mechanisms of anaesthetic agents we need to grasp what is going on at this level and because the brain consists of more than one cell, we also need to understand how what happens to one cell relates to a group as the neurochemical changes here relate to consciousness. By studying both sleep and anaesthesia perhaps the biochemical mechanisms of neurons and consciousness may be elucidated.

Ball`s article describes the knowledge associated with anaesthetic agents at the membrane microscale. However, to compare the two, we should probably look first at their effect at the macroscale and compare it to what happens in sleep. It is clear that anaesthesia and sleep differ in their instigation. Surgical anaesthesia normally involves the subject being given a cocktail of drugs applied over different times eg. a sedative (or other anaesthetic agent), a compound to paralyse the muscles and a pain perception blocker. The drugs can be given internally or locally, but this article because of this blog`s emphasis on the brain will consider only those anaesthetic substances given internally and having an effect on the brain. The anaesthetic agents are transported within the blood system and cross the blood-brain barrier to act at specific sites in the brain. However, the instigators of sleep, are molecules internally produced eg. melatonin increases for sleep, increased cortisol for wakefulness, and there are links to adenosine levels (eg. a drop leads to sleep), although there may be circadian rhythms of the levels of these molecules and reactions to external environmental factors such as light and darkness.

Whether sleep or anaesthetic agent the ultimate result is the loss of consciousness and this follows after certain brain areas are affected. In the case of sleep, the brain areas believed to be affected were thought previously to be the hypothalamus and reticular activating system in the brainstem, but now a network of structures is thought to be involved. Recent research has shown that the ventrolateral preoptic nucleus (VLPN) of the hypothalamus appears to be a switch between wakefulness and sleep and output from here during sleep inhibits activity in the brain stem, but maintains stimulation of the cerebral cortex either directly or indirectly. Circadian rhythms appear to be the work of activity of the suprachiasmatic nucleus (internal clock) and pineal gland (melatonin production). Studies on the action of anaesthetics have indicated that the VLPN is also stimulated through the activity of alpha2 adrenergic receptors (although mainly GABA receptors are present in this area) as well as activity in the thalamus, cerebral cortex and brain stem. Therefore, there is similarity in the areas affected by both sleep and anaesthesia.

In general the areas affected by anaesthesia, ie. the brain stem, cerebral cortex and thalamus show functional and effective connectivity which varies depending on the agent used, its dose, and network affected. A study on the anaesthetic, propofol (work by Chennu) showed that increasing the dosage administered to a group of 20 individuals meant that some subjects were still conscious at the maximum dose and were still able to do the given task as demonstrated by EEG readings of alpha brain waves. The experimenters found that even before the test started that some subjects were more susceptible than others to the propofol given and exhibited higher brain activity at the baseline than those less susceptible. This was correlated to exhibited delta-alpha brain wave coupling. Studies by Hudatz on decreases in the global cerebral metabolic rate and blood flow found that the thalamus was a common site of modulation by several anaesthetic agents, but the effect may be secondary to effects on the cerebral cortex. Using fMRI, Bukhari hypothesized that anaesthetic agents demonstrated specific signatures of brain functional networks and interactions, eg. medetomidine exhibited different functional connectivity to isoflurane, propofol and urethane (perhaps a sign of different levels of sodium ion channels or GABA receptors in these areas?). The Default Mode network-thalamic network and lateral cortical network-thalamic network was affected by medetomidine (influences alpha 2 adrenergic receptors) exhibiting a sedative function and vasoconstriction whereas these areas were not affected by isoflurane (demonstrates GABA activity, is an anaesthetic and vasodilator). Cortical-thalamic interaction was found to be modulated by the type and depth of anaesthesia and therefore, it was concluded that it is important to study anaesthesia function in networks rather than in single brain areas.

This is a reasonable assumption especially when we consider the Global Workplace Theory of consciousness where conscious awareness is achieved when there is global connectivity of participating neurons in particular brain areas. Consciousness is linked to firing in the areas of the cingulate cortex, parietal areas, prefrontal cortex, and temporal areas such as amygdala, hypothalamus and insular cortex. The application of anaesthetic agents results in a loss of neuronal firing and action potentials which manifests as dampened stimulation, a disruption of higher order cortical information integration and connectivity and loss of consciousness. This change in activity and connectivity can be observed with monitoring brain waves.

In the case of natural sleep, there is distinctive brain wave pattern activity (amplitudes and frequencies) in the 4 stages of NREM sleep and the REM stage. It is a not clear cut with bursts of particular kinds of activity eg sleep spindle occurring in certain stages. The progress of anaesthetic agent administration can also be observed through brain wave changes with more of the brain going to slow wave oscillations (SWS – slow frequency, high amplitude) typical of NREM stages 1-3 of sleep as loss of awareness proceeds. The SWS oscillations are regulated by the thalamus and the action of thalamic type (T-type) calcium ion channels. Studies on brain waves after the administration of the anaesthetic agent, etomidate, shows decreased 1-4HZ brain waves (theta) observed in wakefulness and increased alpha brain wave oscillations (8-12HZ) and beta (12-30HZ) (in fact, paradoxical since beta waves are actually linked to excitation) and increased sleep spindles (NREM stage 2 of normal sleep). The brain wave activity is said not to be linked to GABA R binding in the thalamus (Mesbata).

Studies on another anaesthetic agent inducing loss of consciousness, the substance profolol, shows that brain wave activity is not simple. After the induction phase, the surgical phase is actually maintained by a combination of different drugs and this produces different brain wave patterns in the different phases. In Phase 1, where there is a light state of general anesthesia, a decrease in beta brain wave activity (13 to 30 Hz) is observed as well as an increase in alpha activity (8 to 12 Hz) and delta activity (0 to 4 Hz). In Phase 2 the beta activity decreases and alpha and delta activity increases and brain wave activity resembles that seen in NREM stage 3 sleep. Phase 3 is defined as a deeper state of anaesthesia and the EEG activity exhibits flat periods interspersed with periods of alpha and beta activity (burst suppression) with time between the alpha bursts lengthening as the anaesthetic state deepens.  Surgery is actually carried out in Phase 2 and 3. In the final phase, Phase 4, EEG is completely flat (isoelectric). The REM stage of normal sleep involves acetylcholine firing and a highly active cortex. However, it is believed that in the case of anaesthesia, activation occurs through the GABA mediated inhibition of striato-thalamic pathways and in fact, direct injection of acetylcholine in thalamus has been shown to overcome anaesthesia.

Therefore, we have shown that anaesthesia and sleep although both cause loss of consciousness, do not share exactly the same brain wave activity patterns. These differences may be due to dissimilarity in the physiological structure of the brain areas involved and may indicate the different molecular mechanisms in play between sleep and anaesthesia. The action of neurotransmitters is one such factor. In the case of sleep, many different neurotransmitters play a role. For example: histamine demonstrates decreased activity during the NREM stages of sleep and exhibits the lowest levels in REM, but is at a high level in wakefulness; 5HT occurs in the awake state and decreases in the REM stage; acetylcholine in the reticular activating system stimulates activity in the awake state, but is also highly active during REM (in NREM it stimulates connectivity of the hippocampus and cortex); dopamine has an involvement sometimes in the sleep state and sometimes in the awake state; and orexin which is only produced in the hypothalamus triggers wakefulness, but at night low levels of it might drive sleep and this is linked to the action of GABA in the hypothalamus. The action of the neurotransmitters directly affects neuron firing and stimulation and therefore, different levels of neurotransmitter and the mechanisms associated with those neurotransmitters in brain regions even at the microscale (eg. the rafts described above by Ball) could influence how an anaesthetic agent can work. This is because, although neurotransmitters bind to specific receptors on the neuronal cell membrane surface, anaesthetic agents are believed to have other effects.

Ball described in his article some of the biochemical affects that anaesthetic agents are supposed to elicit in the neuronal cell. It has been reported that anaesthetic agents in general affect only certain neurotransmitter receptor types, eg. GABA A type receptors as target for the agents propofol and etomidate and NMDA R receptors in the cortex, thalamus and brainstem regions. Ball suggested one effect of the binding of the anaesthetic agent was its effect on the ´critical temperature` of the cell membrane. This is an important feature in action potentials and cell firing where localized changes in membrane fluidity can affect the physiological structures of membrane components, movement of components within the membrane and vital firing mechanisms such as clustering of receptors, ion channel opening and exocytosis of neurotransmitters through vesicle binding to the cell membrane. ´Critical temperature` is defined as the optimal membrane temperature (or critical membrane energy state) which would allow the vital functions to take place and although tempting to think this might be over the whole cell membrane it is more likely that it occurs locally in small nano-domains at certain times during the cell firing process and cell recovery.

The ´critical temperature` (or ´critical membrane energy state`) is likely to occur through the activity of molecules within the membrane and by being active then a higher temperature or state is achieved. With reference to the action of the anaesthetic agents, activity is likely to be via the lipid polarity of the membrane molecules that make up its physiological structure, eg. phospholipids and their electron status achieved through the biochemical groups eg hemes, Fe-S bridges of which they are composed. Just like Turin suggested, the anaesthetic agents would provide electrons through their binding. Binding of groups, molecules, hydrogen ions and electrons cause configuration changes in the proteins and other molecules of the membrane with each tertiary and quartenary conformational change giving different activity to its normal state. This supports the modern lipid hypothesis that lateral pressure distribution can be changed by anaesthetic binding, ie. conformational changes are elicited that affect the activity of the molecule or area in question. Interaction could increase the ´temperature` or energy status of the molecules so that the cell firing is either depressed or activated. It is likely that critical changes occur in small micro-areas and this supports the proposed nano-domains or lipid rafts associated with neuronal firing. It also supports the idea of the action potential stage of neuronal firing only being achieved when a threshold of firing has been reached. It is likely that lots and lots of small nano-domains fire which reach a group effect. Naturally, this is difficult to measure although patch clamping of single channels can demonstrate a limited area of the cell membrane due to the size of the pipette, but it is not possible to say how many nano-domains are present.

Although the idea of membrane lipid binding and changes in the critical temperature of nano-domains appears to be a suitable solution to the action of anaesthetic agents, studies have shown that low temperature changes in membranes are not sufficient to cause a change in consciousness. Therefore, the idea of a membrane fluidity effect by anaesthetic agents is more likely and this is supported by the observation that as chain length of the anaesthetic agent grows, there is an increased effect, but only to a maximum length of 6-10 units after which there are no effects. This can be explained by the anaesthetic agent binding at specific points of the membrane, but once reaching a particular size, this binding cannot occur.

Aside from the anaesthetic agent effect on membrane fluidity (or ´critical temperature`), Ball also pinpointed their action on particular ion channels. Ion channels and the flow of ions are important in the action potential mechanism and cell firing and anaesthetic agents have been shown to have a preference for a particular one, the sodium ion channel. Work by Strichartz and colleagues have demonstrated that anaesthetic agents bind to sodium channels preventing an increase in membrane permeability. They report no change in the calcium ion channels, another important channel in depolarization and recovery mechanisms. Experiments have shown that anaesthetic agents bind to the inner side of the channel after normal depolarization preventing sodium ion influx. Therefore, the block is increased with the frequency of nerve impulses and leads to larger refractory periods. Proteins have been shown to have what is described as ´buried cavities`, since binding of isoflurane to sodium channels occurs even if the channel is closed. Since many neurotransmitters have receptors that are linked to sodium ion channels, eg NMDA receptors, acetylcholine receptors this hypothesis supports the loss of firing and ultimately, loss of consciousness observed. However, anaesthetic agents have also been observed to bind to GABA receptors especially the A type and also have been shown to affect G-protein coupled receptors. In the case of GABA A receptor binding as observed with both propofol (Yip) and etomidate (Li), the anaesthetic agent has been shown to bind within the beta subunit at the interface between the transmembrane domain. It may act by promoting the binding of the receptors agonist, GABA which mediates most synaptic inhibition in the brain or indirectly by positively affecting the associated chloride channel resulting in higher influx of chloride ions and hyperpolarization of the cell. In the case of the G-protein receptors, the anaesthetic agents may not bind to or affect directly the membrane bound receptor or G protein complex, but may produce its affect by affecting the activity of an enzyme further down in the cascade mechanism, that of protein kinase C. A reduction in effective protein kinase C would prevent activation of the post-synaptic mechanisms that control calcium ion release and hence, correct functioning of the neuron would not occur. Anaesthetic agents have also been shown to affect other non-membrane bound molecules eg luciferase, cytochrome p450 and even the microtubular proteins, beta actin and beta tuberlin and therefore, although it is tempting to think that their effect is purely membrane related, their actual function may not be so clear-cut, eg. Turin suggested  their action lies at the mitochondrial membrane.

What has been discussed so far are ´pure` biochemical mechanisms for the action of anaesthetic agents, but work by Turin gave alternative suggestions that were described as ´zombie theories`, but should not be lightly dismissed. The idea of electron transfer within molecules causing changes in conformation and with ion channels involved in the transport of positive or negative ions has already been explained and these are ´accepted` biochemical mechanisms, eg. electron transfer in photosynthesis or ATPsynthase function. Modulation of such factors can lead to firing inhibition or stimulation and hence, anaesthetic agents can produce their effects by altering the electron status of molecules or the firing environment. The breakdown of the mechanism to electrons and electron flow is further supported by the observation that the application of a DC electric current can cause a loss of consciousness from lower to higher (back to front) brain areas and can be observed by monitoring brain wave changes and connectivity of areas as the electric current is applied. A return to consciousness may involve an increase of electron transfer above the baseline of normal wakefulness. This electric current observation has been utilized in the use of electric anaesthesia as early as 1961 (Chappel) by vets and more lately, the application of electric current as a local anaesthetic in dentistry (January 2016).

Therefore, we conclude this examination of how anaesthetic agents work by voicing two thoughts: the first, that anaesthetic agents may provide a means of examining cellular mechanisms in more detail especially if fluorescently labelled molecules (or optogenetics, radioactive tags) can be used; and secondly, does the use of anaesthetic agents in experiments actually affect the results being observed and therefore, can in some cases, definitive explanations to neuronal mechanisms be made if they are present?

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

…………….if ECT is carried out under general anaesthesia, is there a ´double electron whammy`  effect on neuronal cell functioning?

…………….as the patient emerges from general anesthesia, the EEG patterns proceed in approximately reverse order from Phases 2 or 3 of the maintenance period to an active EEG that is consistent with a fully awake state. Therefore, would the administration of certain neurotransmitter blockers at these stages indicate how the neuronal firing mechanism re-boots itself?

……………..work by Varin and colleagues showed that the administration of glucose induced SWS by activating the VLPN neurons and leading to the closure of ATP sensitive potassium channels. Are the same channels affected with the administration of anaesthetic agents?

………………..work by Pigeat and colleagues showed that LTD of intrathalamic GABA A synapses during SWS involved the T-type calcium channel and metabotropic glutamate receptors. Is the same mechanism employed by anaesthetic agents?

………………..application of a GABA agonist (Mesbata) leads to thalamus receptor binding and an increase in theta brain oscillations (1-4HZ) in wakefulness, plus increased REM, decreased sleep spindles and increased the speed of transitions in the NREM stages. It is known that anaesthetic agents affect the GABA A receptor and therefore, would the same observations be seen with them as for the GABA agonist?

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