Department of Physiology, University College London, Gower Street, London WC1A 6BT, UK ... ÃThe Board of Management and Trustees of the British Journal of ..... with a calcium-sensing dye (Oregon green BAPTA-1), which then diffused ...
British Journal of Anaesthesia 89 (1): 79±90 (2002)
Anaesthetic modulation of synaptic transmission in the mammalian CNS C. D. Richards Department of Physiology, University College London, Gower Street, London WC1A 6BT, UK Br J Anaesth 2002; 89: 79±90 Keywords: anaesthesia, general; brain, synapses; nerve, transmission
Although they affect many different organ systems within the body, general anaesthetics are administered primarily to render patients unconscious during surgical procedures. While unconscious, patients do not experience pain and the events surrounding the surgery are largely forgotten. As the brain and spinal cord are the organs that give rise to our individual and personal perceptions of the world, they are clearly the sites at which general anaesthetics exert their chief actions. Immobility, the other desirable characteristic of anaesthesia, is nowadays usually achieved by the use of muscle relaxants, which block neuromuscular transmission. It is an unfortunate fact that, whereas the pathways that are responsible for many important re¯exes are now relatively well understood, those that are involved in the emergent properties of central nervous system (CNS) function (i.e. consciousness and perception) are much less well de®ned. Although spinal pathways are involved both in re¯ex behaviour (such as limb ¯exion in response to a nociceptive stimulus) and in the transmission of information about the state of the body to the brain, it is the brain that is responsible for the formation of perceptions about the world and the setting down of memory. Therefore, to understand fully how general anaesthetics work, it will be necessary to understand the neural mechanisms that underlie consciousness. This is a dif®cult problem and it may appear from this analysis that any attempt to elucidate the mechanisms involved in general anaesthesia is unlikely to succeed. However, as with many troublesome issues, a reductionist approach provides a useful starting point. The changes that are responsible for the state we call general anaesthesia must ultimately be an expression of the activity of the neurones that make up the nervous system. This activity depends both on the excitability of the constituent neurones and on their synaptic physiology. For this reason, much effort has been devoted to understanding how anaesthetics modulate synaptic transmission in de®ned neural pathways. This review, which is an update of an earlier one on the same topic,73 will explore the mechanisms
that underlie these effects. It will present evidence that anaesthetics act mainly by modulating synaptic transmission. These effects are the result of actions on a small number of different types of ion channel, notably ligandgated channels.
Basic synaptic mechanisms Since the original description of synaptic transmission by Sherrington84 in the early part of the last century, the detailed mechanisms that underpin synaptic transmission have gradually become clearer. In mammals, including man, synapses generally operate by the secretion of a small quantity of a chemical (a neurotransmitter) from the nerve terminal. Such synapses are known as chemical synapses. However, some synapses operate by passing the electrical current generated by the action potential to the postsynaptic cell via gap junctions between adjacent neurones (electrical synaptic transmission). This type of synaptic transmission is not found in mammals but is important in some invertebrates, for example cray®sh.36 Nevertheless, gap junctions between adjacent neurones do occur in some areas of the mammalian CNS, although their precise function remains unknown in most cases. Gap junctions do not exhibit the directional transmission characteristic of chemical synapses. A well-known example of gap junction connections between neurones is the coupling between the horizontal cells of the retina, which facilitates the lateral spread of activity between the dendrites of adjacent neurones.11 As the wider importance of electrical synapses in the activity of the mammalian CNS is uncertain, the action of anaesthetics on this mode of synaptic transmission will not be discussed further. Before proceeding, it may be helpful to remind the reader of the basic organization of neurones. Their cell bodies are very varied in both size and shape but all CNS neurones have extensive branches called dendrites, which receive information from many other neurones. Each nerve cell
Ó The Board of Management and Trustees of the British Journal of Anaesthesia 2002
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Fig 1 Diagrammatic representation of a typical CNS synapse showing the main ultrastructural characteristics. This type of synapse is called an en passage synapse and an axon makes many such contacts as it courses through the cerebral cortex. Note the presence of endoplasmic reticulum in the boutons and dendritic spines, which appears to act as an intracellular calcium store.
gives rise to a single axon, which subsequently branches to contact a number of different target cells. The axon branches are called collaterals. As they make contact with other neurones they form synapses, which act as one-way valves for the passage of information from one neurone to another. An axon may form a synaptic contact with a dendrite, a cell body or another axon. Occasionally, synapses are formed between two dendrites (dendrodendritic synapses), as in the nigrostriatal pathway.41
small, round, membrane-delimited features lacking any electron-dense material in their centre (as is the case for the majority of synaptic contacts in the CNS) or they may contain electron-dense material of some kind.57 Vesicles of the latter type are referred to as dense-cored vesicles and are found typically on the noradrenergic nerve ®bres of the sympathetic nervous system. The membrane immediately under the nerve terminal is called the postsynaptic membrane; it often contains electron-dense material that makes it appear thicker than that of the plasma membrane outside the synaptic region. This is known as the postsynaptic thickening. The postsynaptic membrane contains speci®c receptor molecules for the neurotransmitter released by the nerve terminal.
The structure of chemical synapses When axons reach their target cells, they form small swellings known as synaptic boutons. In the CNS, a single axon frequently makes contact with a number of target neurones as it courses though the tissue. Such synapses are known as en passage synapses, and similar synaptic contacts also occur between autonomic nerves and their target cells (Fig. 1).69 In other cases, an axon branch ends in a small swelling called a nerve terminal, which contacts its target cell. The classical example of this type of synaptic contact is the neuromuscular junction.9 In this account, the terms `synaptic bouton' and `nerve terminal' will be used interchangeably as the functions of these contacts are identical (although their properties may differ in detail). The synaptic bouton, or nerve terminal, together with the underlying membrane on the target cell, constitutes a synapse. The nerve terminal is the presynaptic component of the synapse and is usually closely attached to the target (or postsynaptic) cell, leaving only a small gap of about 20 nm between the two elements. This small gap is known as the synaptic cleft, and it isolates electrically the presynaptic and postsynaptic cells. The nerve terminals contain mitochondria, cytoskeletal elements and a large number of small vesicles known as synaptic vesicles (Fig. 1). Under the electron microscope these may appear either as
The main stages of chemical transmission The principal events during the operation of a chemical synapse are as follows. Action potentials travel along the axon of the presynaptic neurone and invade the synaptic boutons, which become depolarized. This depolarization opens voltage-gated calcium channels, allowing calcium ions to enter the nerve terminal. The consequent increase in the free calcium concentration triggers the secretion of a neurotransmitter (such as acetylcholine, glutamate or GABA) from the nerve terminal into the synaptic cleft. This process occurs via the fusion of a synaptic vesicle with the presynaptic membrane of the bouton, during which the contents of one or more vesicles become discharged into the synaptic cleft (exocytosis). The neurotransmitter diffuses across the synaptic cleft and binds to speci®c receptor molecules on the postsynaptic membrane. As a result, ion channels open and change the permeability of the postsynaptic membrane, so modulating the excitability of the postsynaptic neurone (Fig. 2). For many synapses within the CNS, the link between the arrival of an action potential 80
Anaesthetic modulation of CNS synaptic transmission
Fig 2 Flow chart illustrating the principal stages of synaptic transmission in the CNS. Although anaesthetics affect many aspects of cell function, only those that appear to be of most importance in modulating synaptic transmission are indicated here.
Action potentials in the presynaptic neurone may lead to excitation or inhibition of the postsynaptic cells according to the type of synaptic contact. If the transmitter activates an ion channel directly, synaptic transmission is usually both rapid and short-lived. This type of transmission is called fast synaptic transmission and is typi®ed by the action of acetylcholine at the neuromuscular junction. If the neurotransmitter activates a G-protein-linked receptor, the change in the postsynaptic cell is much slower in onset and lasts for a much greater period. An example is the excitatory action of norepinephrine on a1-adrenoceptors in the peripheral blood vessels. The secreted neurotransmitter is removed from the synaptic cleft by diffusion, by enzymatic activity or by uptake into the nerve terminals of surrounding glial cells. From this bare outline, it is clear that synaptic
at a bouton and the secretion of neurotransmitter is probabilistic. Thus, the action potential increases the probability that a synaptic vesicle will empty its contents into the synaptic cleft, but not every action potential will result in the secretion of neurotransmitter (i.e. the probability of vesicle release is less than 1). This interpretation is an extension of the classical work of Bernard Katz and his colleagues on synaptic transmission at the neuromuscular junction.47 Confocal imaging of small projections on dendrites, known as dendritic spines, has recently provided direct evidence of the probabilistic nature of transmitter release in the CNS32 and legitimizes the use of probabilistic analysis of synaptic transmission at visually identi®ed CNS synapses. (This powerful analytical tool is often called quantal analysis.) 81
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effect on excitation, general anaesthetics have been found to enhance inhibitory synaptic transmission both in the spinal cord27 and in the brain.37 64 66 Nevertheless, transmission at some inhibitory synapses is depressed.31 35 From this it is apparent that different synapses show different degrees of susceptibility to modulation by anaesthetics. Different synapses are specialized to perform different functions. Some are concerned with reliable onward transmission of information (e.g. the synapses of the cuneate nucleus and those of primary afferents ending on spinal motor neurones), whereas others are concerned with more integrative activity in which individual synaptic contacts may be relatively weak and plastic (e.g. those of the cerebral cortex and hippocampus). It is therefore not surprising that different synapses respond in different ways to anaesthetic agents. In all cases it is necessary to determine to what extent the effect of a particular anaesthetic is presynaptic and to what extent it is postsynaptic. This can only be established by a detailed study of the effects of a variety of anaesthetics on speci®c synaptic systems. In what follows, the various mechanisms by which anaesthetics modulate fast excitatory and inhibitory synaptic transmission in the brain and spinal cord will be explored. Discussion of their effects on slow synaptic transmission in the CNS is limited by a paucity of experimental data.
transmission can be divided into two main stagesÐa presynaptic stage, which is concerned with the mechanisms involved in controlling the secretion of the neurotransmitter, and a postsynaptic stage, which is concerned with the processes that occur in the postsynaptic cell after the secreted neurotransmitter has bound to its receptor.
Anaesthetic modulation of synaptic transmission To understand precisely how general anaesthetics modulate the activity of the CNS, it is necessary to answer the following questions. First and foremost, do anaesthetics affect synaptic transmission or do they modulate the excitability of the neurones themselves? If they affect synaptic transmission, are their main effects presynaptic or postsynaptic? If their action is presynaptic, what is the mechanism? Do they interfere with action potential propagation into the presynaptic nerve ®bres? If so, is the mechanism responsible for this effect a partial blockade of voltage-gated sodium channels or an increase in potassium conductance in the resting membrane? Alternatively, do anaesthetics reduce the probability of transmitter release? If so, do they act by reducing the amplitude of the calcium transients in the presynaptic nerve terminals or do they have a direct effect on the cellular apparatus responsible for the secretion of neurotransmitter? If their effect is on the calcium channels, which of the various subtypes is affected? If their action is postsynaptic, do they modulate the excitability of the postsynaptic neurones or are their effects explicable in terms of modulation of the postsynaptic receptors? If the latter, which speci®c receptors are involved? The ®rst detailed studies of the action of general anaesthetics on synaptic transmission were carried out by Bremer and Bonnet10 12 on the frog spinal cord and by Larrabee and Posternak52 in mammalian sympathetic ganglia. Both pairs of authors found that many general anaesthetics depressed excitatory synaptic transmission without signi®cantly depressing the propagation of action potentials in nerve axons. These early studies gave rise to the idea that anaesthetics exert a selective depressant action on the process of synaptic transmission. Subsequent investigations established that a wide variety of general anaesthetics depress excitatory synaptic transmission both in the spinal cord85 86 93 and in the brain.31 74±76 78 79 Not all excitatory synapses, however, are easily blocked. Whereas those of, say, the olfactory cortex or hippocampus are readily depressed by modest concentrations of general anaesthetics, the excitatory dendrodendritic synapses of the olfactory bulb are relatively resistant to most anaesthetics.64 Synaptic transmission through the cuneate nucleus has even been reported to be facilitated by clinically effective concentrations of general anaesthetics.63 In contrast to their predominantly depressant
Presynaptic effects of general anaesthetics Matthews and Quilliam61 provided the ®rst direct evidence that anaesthetics could depress the amount of transmitter secreted in response to nerve impulses. They found that the amount of acetylcholine secreted in response to stimulation of preganglionic sympathetic nerves was decreased by amylobarbital and a number of other central depressants. A decrease in the amount of transmitter secreted in response to each nerve impulse could result from a number of different factors: anaesthetics could prevent action potentials fully invading the axonal arbour and thereby decrease the synaptic drive to the postsynaptic neurones.8 52 92 This would lead to the silencing of a proportion of the normal synaptic contacts and result in decreased excitation of the postsynaptic neurones. Alternatively, as discussed above, their effects might result from direct effects on the process of exocytosis itself, either by inhibiting calcium entry into the presynaptic bouton or by direct action on the exocytotic machinery. Until recently, the small size of CNS axons and their collaterals made it dif®cult to determine whether general anaesthetics could affect adversely the propagation of action potentials into all the branches of an axon. To determine whether this was indeed the case required direct measurement of action potentials through identi®ed branch-points in central axons. In the mammalian CNS this is impossible with classical electrophysiological methods. However, recent advances in confocal32 and two-photon microscopy22 49 have provided a way of following the action 82
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sium-evoked secretion of glutamate from slices of rat brain is inhibited by pentobarbital.21 More recent work83 has shown that volatile anaesthetics also depress the secretion of glutamate by isolated synaptic boutons (synaptosomes). Some authors have reported that anaesthetic concentrations of barbiturates slightly increase the secretion of the inhibitory amino acid neurotransmitter GABA,21 which could explain why barbiturates potentiate synaptically mediated inhibition. Other workers, however, have found that GABA secretion is inhibited by anaesthetics.46 48 62 Because the fundamental steps of neurosecretion are not thought to be different for different neurotransmitters, the balance of the evidence must favour the idea that neurosecretion is either unaffected or inhibited by anaesthetics. What is the mechanism by which anaesthetics inhibit neurosecretion? The small size of most nerve endings and the heterogeneity of brain tissue make this question dif®cult to answer for synapses in the brain. Instead, it has proved more useful to study the effects of anaesthetics on the secretion of epinephrine and norepinephrine from adrenal medullary chromaf®n cells. These endocrine cells are, like neurones, derived from neural crest tissue and share many of the properties of sympathetic postganglionic neurones. Their neurosecretory properties have been studied intensively and correspond closely to those of nerve endings. GoÈthert and his colleagues,38 39 using perfused adrenal glands, showed that anaesthetics depress the secretion of catecholamines evoked by nicotinic agonists and depolarization with high concentrations of potassium. Subsequently, Charlesworth and colleagues18 and Pocock and Richards70 71 used isolated chromaf®n cells to investigate the action of a wide variety of anaesthetics on neurosecretion. They found that all the general anaesthetics they investigated could inhibit catecholamine secretion evoked by direct depolarization with high concentrations of K+. For most clinically useful agents, the inhibition of secretion occurred within the range of concentrations expected during general anaesthesia. A number of mechanisms could account for this depression of secretion: anaesthetics could decrease the amount of catecholamine in each secretory granule; they could disrupt the mechanism of exocytosis itself in some way, for example by inhibiting the docking of secretory granules at the plasma membrane; or they could inhibit the entry of calcium in response to depolarization by blockade of the voltage-activated calcium channels. First, do anaesthetics decrease the catecholamine content of chromaf®n granules or the transmitter content of synaptic vesicles? A thorough study by Akeson and Deamer2 showed that anaesthetics did not deplete isolated chromaf®n granules of their catecholamines. Furthermore, Pocock and Richards72 found that a variety of anaesthetics were without effect on the leakage of catecholamines from isolated chromaf®n cells rendered leaky to small molecules by exposure to high-voltage electrical discharge (electro-
Fig 3 Evidence that pentobarbital has no effect on action potential invasion of axon branches. To visualize the invasion of an axon and its collateral branch by an action potential, the parent neurone was loaded with a calcium-sensing dye (Oregon green BAPTA-1), which then diffused into the dendrites, axon and axon collaterals. The passage of the action potential led to a transient increase in intracellular calcium concentration, which is revealed as a change in the ¯uorescence intensity of the calcium indicator. Calcium transients are seen in both axon branches, showing that both are invaded by the action potential. DF/F is the relative change in the ¯uorescent signal, which is roughly proportional to the increase in calcium concentration.5a
potential invasion of small axons and their collaterals. The method depends on the fact that the arrival of an action potential at a given point along the axon will cause voltagegated calcium channels to open, resulting in calcium in¯ux and an increase in intracellular free calcium concentration. Localized measurements of intracellular free calcium concentration with ¯uorescent calcium indicators can therefore reveal whether an action potential has invaded an axon, collateral or synaptic bouton. Using this approach, Baudoux and colleagues5 5a have shown recently that neither pentobarbital nor etomidate prevent action potentials invading the axonal arbour (Fig. 3). Perhaps more surprisingly, they also found that concentrations of procaine suf®cient to reduce the amplitude of the action potential by about 20% did not prevent action potential propagation through branchpoints. This strongly suggests that axonal propagation into the axonal arbour has a high safety factor and is therefore reliable. If failure of the action potential to invade the synaptic boutons is not the mechanism by which anaesthetics depress the secretion of neurotransmitter, what is responsible? Direct effects on the neurosecretory process itself have been implicated. Measurement of the effects of anaesthetics on the chemically evoked release of various neurotransmitters has shown that the secretion of neurotransmitters is depressed by a variety of anaesthetics. Thus, the potas83
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adrenal chromaf®n cells, N- and L-type calcium channels are present and both appear to contribute to normal catecholamine secretion. Direct measurement of the calcium transients recorded from synaptic boutons has shown that the calcium channels are predominantly of the P/Q and N subtypes49 and this is in agreement with pharmacological studies of synaptic transmission.88 Do anaesthetics inhibit the activity of voltage-gated calcium channels? Gross and Macdonald40 examined the action of pentobarbital on speci®c subtypes of calcium current in primary sensory neurones. They found that pentobarbital affected the high-threshold N or L calcium currents but had little effect on the low-threshold T current. In adrenal chromaf®n cells18 a variety of anaesthetics inhibit the currents as a result of activation of calcium channels of the N and L subtypes, although halothane has little inhibitory effect. There was no evidence to suggest a selective action of the other anaesthetics on either N or L channels. P-type calcium channels are also relatively insensitive to a variety of anaesthetics.42 Given that the secretion of neurotransmitter from synaptic boutons depends on the P/Q- and N-type calcium channels, these results suggest that the depressant effects of anaesthetics on excitatory synaptic transmission are more likely to be caused by direct effects on the postsynaptic receptors. Before accepting this conclusion, however, it is important to recognize that we do not know the relationship between the increase in calcium concentration within the presynaptic bouton and the probability of transmitter secretion. Three studies using quantal analysis have provided evidence that anaesthetics depress excitatory synaptic transmission by decreasing the action potential-evoked secretion of neurotransmitter.50 93 96 This conclusion has been supported recently by direct measurement of the effect of pentobarbital on the amplitude of the calcium transients recorded in the presynaptic boutons.5 5a This shows a greater reduction in the calcium transients than that expected from the known action of this anaesthetic on calcium channels, and probably re¯ects the involvement of calcium-induced calcium release in the presynaptic boutons.33 As this is a positive feedback process, a small reduction in calcium entry could lead to a substantial decrease in the probability of transmitter release from a given synaptic bouton. Such an eventuality would lead to a decrease in the excitatory synaptic drive to the postsynaptic neurone. This attractive proposition should be treated with caution, however. The transients recorded occur after transmitter release (release occurs on a submillisecond timescale, whereas the transients reach their maximal amplitude in a few milliseconds). To resolve the matter fully will therefore require closer examination of the relationship between the presynaptic calcium transients and the release of neurotransmitter. Is the decrease in calcium entry brought about by block of calcium currents suf®cient to account for the inhibition of secretion in a model system such as the chromaf®n cell? To investigate this question, the in¯ux of 45Ca2+ into freshly
Fig 4 Effects of three anaesthetics on catecholamine secretion by electropermeabilized adrenal chromaf®n cells. Catecholamine secretion was stimulated by increasing concentrations of calcium. For each anaesthetic, the left column shows the control condition ([Ca2+] 10±8 M), the centre column shows the secretion elicited by [Ca2+] 10±6 M (around threshold for activation of secretion) and the right column shows maximal secretion (obtained in response to [Ca2+] 10±4 M). The concentrations of all three anaesthetics would be suf®cient to inhibit depolarization-induced secretion by 30±50%. These data show that anaesthetics do not increase the basal leakage of transmitter (in this case epinephrine and norepinephrine) and do not alter signi®cantly the threshold or the maximal secretion that can be attained. From Pocock and Richards70 71 and the same authors' unpublished experiments.
permeabilized cells) (Fig. 4). Equally, the content of neurotransmitter in brain tissue does not decline in anaesthesia. It is either unchanged or is increased depending on the transmitter studied.73 Secondly, do anaesthetics inhibit the intracellular events that lead to exocytosis? To investigate this possibility Pocock and Richards72 examined the effects of three types of anaesthetic on the activation by calcium of secretion from electropermeabilized chromaf®n cells. Anaesthetics did not alter the basal secretion of catecholamines or the secretion evoked by calcium (Fig. 4). Consistent with this, Schlame and Hemmings83 reported more recently that halothane did not inhibit the release of glutamate evoked by ionomycin (which directly raises the intracellular calcium concentration) from isolated synaptic terminals, although it did inhibit the secretion evoked by 4-aminopyridine (which induces secretion by depolarizing the nerve terminals). Because calcium is an important second messenger and regulates neurosecretion and neuronal excitability, in addition to a host of other important actions, the nature of the channels concerned with controlling the entry of calcium into the cell has been the subject of intense interest. It is now thought that there are at least ®ve major classes of voltagegated channel, which are known as the L, N, P/Q, R and T subtypes. These have now been cloned and their primary sequences determined. In living cells, they can be distinguished both by their electrophysiological characteristics and by their sensitivity to various inhibitors and toxins. In 84
Anaesthetic modulation of CNS synaptic transmission
AMPA receptors discussed above), attention has focused on the action of anaesthetics on these receptor subtypes. Ketamine, phencyclidine, ethanol and diethyl ether have all been shown to depress responses of the NMDA subclass preferentially.3 Kendig and her colleagues13 20 82 94 have investigated the effects of several different classes of anaesthetic on glutamatergic synaptic transmission in the spinal cord. They found that the AMPA receptor-mediated component of the synaptic potentials was depressed by iso¯urane, ethanol and urethane but not by barbiturates, ketamine or propofol. All of these agents did, however, depress the NMDA receptor-mediated component. To determine the effects of anaesthetics on speci®c subtypes of glutamate receptor, a number of experiments have been conducted with recombinant glutamate receptors expressed in oocytes. These have shown that the receptors formed by different subunits have different sensitivities to anaesthetic agents.25 Nevertheless, the present evidence suggests that recombinant AMPA receptors are, in general, rather insensitive to volatile anaesthetics. Studies of recombinant GABAA receptors have revealed that the effects vary with subunit composition.44 For example, the effect of etomidate depends on the presence of the speci®c b subunit present.6 Point mutation studies have revealed that the r1 subunit, which is not normally sensitive, can be made sensitive to anaesthetics.7 While these observations have little to do with the mechanism of anaesthetic action in the CNS, they clearly imply that anaesthetic effects depend on very speci®c structural features in their targets.
isolated chromaf®n cells was measured and compared with the amount of catecholamine secreted. The depression of potassium-evoked secretion was closely paralleled by the inhibition of the calcium in¯ux. This led to the conclusion that the anaesthetic-induced depression of catecholamine secretion evoked by direct depolarization could be completely explained by the inhibition of calcium in¯ux.70±72 This reinforces the conclusion that anaesthetics do not have a signi®cant depressant effect on the intracellular machinery responsible for exocytosis. Nevertheless, it is important to recognize that not all anaesthetics inhibit neurosecretion at low concentrations. Halothane and ketamine have little effect on the potassium-evoked secretion of catecholamines at concentrations likely to be found in the brain during anaesthesia.71 89 Moreover, ketamine and halothane are poor inhibitors of potassium-evoked secretion of excitatory amino acids from brain tissue slices and synaptosomes.68 83 Further work on systems that are more physiologically intact is clearly required to resolve the various contradictions.
Postsynaptic effects of general anaesthetics Although there is good evidence that many anaesthetics depress neurosecretion, the fact that inhibitory synaptic transmission is often augmented by anaesthetics indicates that other processes are at work. It has long been known that anaesthetics modulate the responses of neurones to arti®cially applied neurotransmitters, such as glutamate, GABA, glycine and acetylcholine. Since the early work in this area, the receptors for many of these substances have been cloned and their amino acid sequences determined. This has greatly facilitated the characterization of their responses to anaesthetic agents and has been taken a step further with the use of point mutations to examine the nature of anaesthetic± receptor interactions. The principal excitatory neurotransmitter of the brain is now generally agreed to be glutamate, which acts on speci®c ionotropic receptors that show little structural homology to other neurotransmitter receptors. As with the voltage-gated calcium channels, it is now known that there are several different types of glutamate receptor±ion channel complexes. The primary division is between receptors that respond to AMPA (a-amino-3-hydroxy-5-methylisoxazole4-proprionic acid) and those that respond to NMDA (N-methyl-D-aspartate). There are also G-protein-linked glutamate receptors (metabotropic glutamate receptors; mGlu1-6). Early experiments23 showed that barbiturates decreased the sensitivity of cortical neurones to iontophoretically applied glutamate. Halothane had no signi®cant depressant effect. Later studies76 77 found that several other anaesthetics, including diethyl ether, methohexital and the steroid anaesthetic alphaxalone, decreased the sensitivity of olfactory neurones to glutamate. Once again, halothane had little effect. Since the recognition that there are two major types of ionotropic glutamate receptor (the NMDA and kainate/
Effects on the kinetics of ion channels Why do anaesthetics depress the activity of the ion channels responsible for excitation but increase the activity of those responsible for inhibition? Initially, studies of the effects of anaesthetics on ion channels concentrated on the nicotinic receptors of the neuromuscular junction, but other ion channels have received attention more recently. Many years ago, Adams1 showed that barbiturates and procaine depressed the second of a pair of closely spaced endplate potentials much more effectively than the ®rst. He proposed that this occurred because the anaesthetics blocked ion channels preferentially when they were open. He went on to suggest that the blocked channel must then unblock and return to the open state before it can close. This simple scheme, which has guided the investigation of the action of anaesthetics on ion channel kinetics since its introduction, is known as the sequential blocking model. It makes a number of important predictions about the way anaesthetics may interact with ion channels that can readily be tested using patch-clamp recording techniques. Many anaesthetics have been shown to reduce the mean open time of activated nicotinic receptors, as expected for the sequential block model. However, the duration of channel burst activity also falls, in violation 85
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processes. Anaesthetics could modulate the time course of synaptic potentials by interfering with one or more of these processes. For example, many general anaesthetics prolong the decay of GABA-mediated inhibitory postsynaptic potentials (IPSPs; see above) and GABA uptake blockers are reported to have similar, though weaker, effects.81 Thus, it is possible that the prolongation of inhibitory currents by anaesthetics might result from a blockade of GABA uptake. This possibility has been investigated extensively. Although the results are somewhat contradictory, it seems that any such action is minor compared with the known effects of anaesthetics on other aspects of synaptic transmission. In general, volatile anaesthetics have no effect on GABA uptake, whereas i.v. agents affect uptake only at excessively high concentrations.53 60
of the model's predictions (a burst is a group of openings). Instead, it has been proposed that the blocked channel enters a further, long-lived blocked state.26 This extended block model accounts for much of the action of anaesthetics on the muscle nicotinic receptor, including the apparent reduction in single-channel conductance seen with certain agents. The effects of anaesthetics on neuronal nicotinic channels17 70 71 have been studied in chromaf®n cells, in which it has been shown that the catecholamine secretion evoked by nicotinic agonists is more susceptible to inhibition by anaesthetics than that evoked by depolarization with potassium. Pentobarbital,45 methohexital, methoxy¯urane, etomidate19 and procaine15 all reduced the time for which the nicotinic channels were open in a concentrationdependent manner. However, neither the sequential blocking model nor the extended block model provided an adequate explanation of how the anaesthetic reduced the lifetime of the open channel. It appears that a blocked channel can return to the closed state without passing through an open state. This implies that these anaesthetics do not directly occlude the channel pore but presumably modulate the channel kinetics via an allosteric binding site. The details of the mechanisms by which anaesthetics block AMPA receptor channels are largely unknown, but there have been some recent studies of the action of anaesthetics on NMDA channels. Pentobarbital depresses the function of native NMDA receptors by blocking the open channel.16 This results in a shortening of the time for which the channels remain open and a decrease in the inward current. The unitary conductance of the open channel was not affected, so that the anaesthetic did not change the ionic selectivity of the channel. Dissociative anaesthetics, such as ketamine, appear to block open NMDA channels preferentially and this block is both voltage- and use-dependent.43 58 67 In this case, the channel closes while the anaesthetic is bound and dissociation of the anaesthetic from the blocked state is very slow. Consequently, the number of blocked channels accumulates with repeated activation. One of the most dramatic effects of many anaesthetics is their ability to potentiate the action of the neurotransmitter GABA at inhibitory synapses. It is now clear that this potentiation results from a direct action of anaesthetics on GABA-activated channels themselves. Detailed investigation has shown that GABAA channels have three different modes of opening and that pentobarbital increased the relative frequency of the opening mode with the longest duration.91 This has the effect of increasing the mean open time of the channel, prolonging the inhibitory effect of GABA.
Modulation of neuronal excitability The excitability of a neurone is a qualitative measure of the ease with which it can generate an action potential. It is determined by two factors: the membrane potential and the intrinsic excitability of the cell (i.e. its threshold for the generation of action potentials). Early studies, directly or indirectly, examined the ease of action potential generation and, in the main, general anaesthetics had little effect.74±76 78 86 93 Subsequently, it emerged that some neurones are hyperpolarized by anaesthetics,65 and this has since been con®rmed many times. Two mechanisms appear to be involved. Some anaesthetics have a direct GABAmimetic effect and hyperpolarize neurones by this mechanism. Unsurprisingly, this hyperpolarization can be blocked by GABAA receptor antagonists.59 80 95 Nevertheless, for many agents (e.g. pentobarbital) this effect is observed only at relatively high anaesthetic concentrations. The second mechanism involves sensitization of the GABA receptor.29 30 In the presence of diazepam or pentobarbital, the GABAA receptor is activated by concentrations of GABA (around 0.5 mM) that are much lower than the concentration in the synaptic cleft of an activated inhibitory synapse. Such low GABA concentrations approach the background concentrations in the extracellular ¯uid. If this sensitization were to occur in vivo it would result in tonic hyperpolarization of some CNS neurones and decrease their excitability. In addition to these effects on the GABAA receptor, it is now clear that volatile anaesthetics can activate some potassium channels. The ®rst unambiguous example was an anaesthetic-activated potassium conductance in a subset of molluscan neurones.34 Subsequent studies have shown that halothane causes hyperpolarization of some thalamic neurones by increasing their potassium conductance.87 Small effects on those potassium channels that are responsible for setting the resting membrane potential are also likely to play a role in the modulation of excitability. This is an area that is likely to repay further study.
Effects on neurotransmitter uptake systems Removal of many transmitters from the region immediately adjacent to the synaptic cleft is dependent on speci®c uptake 86
Anaesthetic modulation of CNS synaptic transmission
dominate because the amount of transmitter secreted would have to be reduced signi®cantly before it failed to saturate the available receptor population. If, however, the neurotransmitter did not saturate the available pool of receptors, a reduction in secretion would lead to a proportionate reduction in the amplitude of the EPSP even though the receptors themselves might be unaffected by the anaesthetic. Such considerations may go some way to explaining the differences in the susceptibility of different synapses to modulation by anaesthetic agents. A modest reduction in the secretion of neurotransmitter together with a decrease in open channel lifetime of the postsynaptic receptors would lead to depression of excitation. A similar reduction in neurosecretion at an inhibitory synapse could be offset by an increase in open channel lifetime (cf. the effects of barbiturates on the GABAA channels) and result in the prolongation of an IPSP.
Do anaesthetics preferentially affect presynaptic or postsynaptic events? In the CNS, most anaesthetics have more than one action. For this reason, their effects may vary from one type of synapse to another. Nevertheless, they can exert two basic kinds of action on synaptic transmission: they may act primarily on the mechanisms involved in neurosecretion or on the receptors of the postsynaptic membrane. These two sites of action are not mutually exclusive. As discussed above, neurotransmitters are secreted into the synaptic cleft by exocytosis. At each synaptic contact, therefore, the magnitude of the postsynaptic response is related to the number of vesicles released and the amount of transmitter in each vesicle. As anaesthetics do not affect the quantity of neurotransmitter present in the synaptic vesicles, the depression of synaptic transmission by an anaesthetic must re¯ect either a decrease in the probability that a given vesicle will release its contents or a decrease in the sensitivity of the postsynaptic membrane to the released transmitter, or both. To enhance synaptic transmission, either more transmitter must be secreted or the effect of the released transmitter must be increased in some way. Several attempts have been made to determine the relative contributions of presynaptic and postsynaptic effects of anaesthetics during the depression of excitatory synaptic transmission. By examining the effect of anaesthetic on the ¯uctuations in the amplitude of the synaptic potentials, three studies50 93 96 on spinal motor neurones have produced evidence to show that the quantal content of excitatory postsynaptic potentials (EPSPs) is reduced by general anaesthetics. These studies also concluded that quantal size was unaffected. These data would suggest that anaesthetics exert their effects primarily by decreasing the secretion of neurotransmitter in response to an action potential. The principal weakness of this work is the assumption that quantal size re¯ects the exocytotic release of neurotransmitter by a single vesicle. In the CNS, however, there is evidence that quantal size may be determined by the number of postsynaptic receptors available rather than by the number of vesicles secreted.28 51 This casts doubt on the notion that anaesthetics work primarily by depressing neurosecretion at the synapses between the 1A afferents and the spinal motor neurones. More work is required to resolve this issue. The relative importance of presynaptic and postsynaptic mechanisms may well vary between different types of synapse, even for a single anaesthetic. Consider a synapse where the amount of transmitter secreted exceeds the number of receptors available by a considerable margin. When the synapse is activated, all the receptors will bind the neurotransmitter and participate in the generation of the synaptic potential (EPSP or IPSP). If the anaesthetic were to affect both neurosecretion and the postsynaptic receptors equally, the effect on the postsynaptic receptors would
Can anaesthesia be explained completely by effects on the GABAA receptor? The pronounced effect of many general anaesthetics on inhibitory synaptic transmission has led some enthusiasts to propose that anaesthesia can be explained fully by the modulatory effect of anaesthetics on the GABAA receptor. While it is undeniable that many general anaesthetics do have pronounced effects on inhibition, there are problems with such an extreme reductionist view. First, not all agents that are potent agonists at GABAA receptors are effective as general anaesthetics. The benzodiazepines are a case in point. They are useful adjuncts to anaesthetics but on their own they are employed as anxiolytics. Only at concentrations far in excess of those required to activate the GABAA receptor14 91 are they able to produce a state resembling anaesthesia.54 Secondly, a number of effective general anaesthetics have little discernible effect on GABAergic transmissionÐfor example ketamine13 56 and xenon.24 Finally, administration of the GABAA agonist muscimol to an intact animal does not result in general anaesthesia, nor can anaesthesia be reversed simply by administering a GABAA antagonist, such as bicuculline.55 These caveats should not be taken as evidence that anaesthetic interactions with the GABAA receptor are unimportant in causing anaesthesia. For many anaesthetics, the potentiating effect of anaesthetics on the GABA receptors is a major component of their mode of action. Evidence in support of this comes from work on agents such as etomidate and iso¯urane, which have optical isomers with different anaesthetic potencies. Their potency in vivo parallels that on GABAA-mediated synaptic currents.4 92
Conclusions Earlier, I posed a number of questions that needed resolution before we could account for the action of general anaesthetics on synaptic transmission in the CNS. We 87
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8 Berg-Johnsen J, Langmoen IA. The effect of iso¯urane on unmyelinated and myelinated ®bres in the rat brain. Acta Physiol Scand 1986; 127: 87±93 9 Birks R, Huxley HE, Katz B. The ®ne structure of the neuromuscular junction of the frog. J Physiol (Lond) 1960; 150: 134±44 10 Bonnet V, Bremer F. Analyse oscillographique des depressions fonctionelles de la substance grise spinale. Arch Int Physiol 1948; 56: 97±9 11 Borst A, Engelhaaf M. Dendritic processing of synaptic information by sensory interneurons. Trends Neurosci 1994; 17: 257±63 12 Bremer F, Bonnet V. Action particuliere des barbituriques sur la transmission synaptique centrale. Arch Int Physiol 1948; 56: 100±102 13 Brockmeyer DM, Kendig JJ. Selective effects of ketamine on amino acid-mediated pathways in neonatal rat spinal cord. Br J Anaesth 1995; 74: 79±84 14 Callachan H, Cottrell GA, Hather NY, Lambert JJ, Nooney JM, Peters JA. Modulation of the GABAA receptor by progesterone metabolites. Proc R Soc Lond B Biol Sci 1987; 231: 359±69 15 Charlesworth P, Jacobson I, Pocock G, Richards CD. The mechanism by which procaine inhibits catecholamine secretion from bovine chromaf®n cells. Br J Pharmacol 1992; 106: 802±12 16 Charlesworth P, Jacobson I, Richards CD. Pentobarbital modulation of NMDA channels in neurons isolated from rat olfactory brain. Br J Pharmacol 1995; 418: 543±53 17 Charlesworth P, Pocock G, Richards CD. The action of anaesthetics on stimulus±secretion coupling and synaptic activity. Gen Pharmacol 1992; 23: 977±84 18 Charlesworth P, Pocock G, Richards CD. Calcium channel currents in bovine adrenal chromaf®n cells and their modulation by anaesthetic agents. J Physiol (Lond) 1994; 481: 543±53 19 Charlesworth P, Richards CD. Anaesthetic modulation of nicotinic ion channel kinetics in bovine chromaf®n cells. Br J Pharmacol 1995; 114: 909±17 20 Cheng G, Kendig JJ. En¯urane directly depresses glutamate AMPA and NMDA currents in mouse spinal cord motor neurons independent of actions on GABAA or glycine receptors. Anesthesiology 2000; 93: 1075±84 21 Collins GCCS. Release of endogenous amino acid neurotransmitters from rat olfactory cortex: possible regulatory mechanisms and effects of pentobarbitone. Brain Res 1980; 190: 517±23 22 Cox CL, Denk W, Tank DW, Svoboda K. Action potentials reliably invade axonal arbors of rat neocortical neurons. Proc Natl Acad Sci USA 2000; 97: 9724±8 23 Crawford JM. Anaesthetic agents and the chemical sensitivity of cortical neurones. Neuropharmacology 1970; 9: 31±46 24 de Sousa SL, Dickinson R, Lieb WR, Franks NP. Contrasting synaptic actions of the inhalational general anesthetics iso¯urane and xenon. Anesthesiology 2000; 92: 1055±66 25 Dildy-May®eld JE, Eger EI 2nd, Harris RA. Anesthetics produce subunit-selective actions on glutamate receptors. J Pharmacol Exp Ther 1996; 276: 1058±65 26 Dilger JP, Brett RS, Lesko LA. Effects of iso¯urane on acetylcholine receptor channels. 1. Single-channel currents. Mol Pharmacol 1992; 41: 127±33 27 Eccles JC, Schmidt RF, Willis WD. Pharmacological studies on presynaptic inhibition. J Physiol (Lond) 1963; 168: 500±30 28 Edwards FA, Konnerth A, Sackmann B. Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J Physiol (Lond) 1990; 430: 213±49
now have answers to some of these questions. Do anaesthetics modulate synaptic transmission? Clearly they do. Do they do so by preventing the action potential reaching all the synaptic boutons? No. Do they affect neurosecretion? If so, how? Many general anaesthetics do depress neurosecretion, but this action appears to be much more important for some anaesthetics (e.g. volatile anaesthetics, barbiturates) than others (e.g. etomidate). Their depressant action on neurosecretion can be fully accounted for by their effects on the calcium channels that regulate transmitter release. It is not necessary to postulate additional effects on the intracellular machinery responsible for exocytosis. Do anaesthetics affect the postsynaptic receptors? Yes, but different anaesthetics have different pro®les of action. For some, the effects on GABAA receptors appear to be dominant (e.g. etomidate, barbiturates), whereas for others the effects on glutamate receptors (particularly the NMDA receptor) are more important (ketamine, xenon). Finally, do anaesthetics affect the excitability of neurones? A quali®ed yes: some neurones are affected and others not. Some of this action can be explained by the direct activation of GABAA receptors by anaesthetics. In other cases, the effects of anaesthetics on potassium channels may play a signi®cant role. Despite their varying pro®les of action, it is becoming clear that general anaesthetics act on a relatively small number of molecular targets within the CNS. The next challenge will be to determine where within the CNS anaesthetics exert their principal pharmacological actions.
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