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Synaptic Plasticity and the Modulation of the Ca2+ Current

1980; The Company of Biologists; Volume: 89; Issue: 1 Linguagem: Inglês

10.1242/jeb.89.1.117

1477-9145

Marc Klein, E Shapiro, Eric R. Kandel,

Photoreceptor and optogenetics research

The study of the mechanisms of neuronal plasticity and the attempt to relate these mechanisms to actual instances of learning has accelerated in recent years as a result of the application of the techniques of biophysics and cell biology to central neurones and their interconnexions. Although support for it has been obtained only recently, the idea that learning might involve changes in the effectiveness of the connexions between neurones actually had its origins at the turn of the century. Following an earlier suggestion by E. Tanzi (1893), Ramon y Cajal postulated, in his Croonian Lecture to the Royal Society of London in 1894 (a lecture to which he was invited through the intervention of Charles Sherrington), that learning might involve changes in strength of connexions between neurones. An almost identical idea was put forth by Sigmund Freud also in 1894 in a fragmentary manuscript published only in 1950. A first requirement of this postulate is that some synapses have plastic properties, that they can change their efficacy following simple use or following more complex patterns of stimulation. This basic requirement has now been fully satisfied. A variety of experiments have shown that chemical synapses can undergo changes in effectiveness as a result of activity or inactivity in a given pathway (homosynaptic change) or as a result of activity in other pathways (heterosynaptic change). Some of the best evidence has come from studies of simple synaptic systems such as the synapses between vertebrate motor neurones and skeletal muscle (for reviews, see Katz, 1962; Eccles, 1964; Kandel & Spencer, 1968).Soon after the initial discovery of the end-plate potential by Gopfert & Schaefer (1938), Schaefer, Scholmerich & Haass (1938) and subsequently Feng (1941) found that repeated (tetanic) stimulation increased the amplitude of the end-plate potential without producing any obvious changes in the action potential of the presynaptic axon. The increase in the end-plate potential was not restricted to the period of tetanic stimulation but persisted for several minutes after the tetanus. Feng called the synaptic enhancement that persisted after the tetanus post-tetanic potentiation. He also found that longer tetani produced greater potentiation than did shorter ones. In 1947, Larrabee and Bronk found post-tetanic potentiation at a peripheral neurone-to-neurone synapse between preganglionic and postganglionic cells of the stellate ganglion. In 1949, Lloyd described similar potentiation in the monosynaptic reflex of the spinal cord, thereby showing that these plastic changes also occur in central neurones. Lloyd found (as had Larrabee and Bronk) that post-tetanic potentiation produced by stimulating one afferent pathway did not increase the response of the postsynaptic cell to synaptic activation via another, unstimulated, afferent pathway. These experiments indicated that post-tetanic potentiation is homosynaptic: it is restricted to the stimulated pathway and results from a change in the synapse itself. Lloyd (1949) also described a second type of plastic change when he found that low frequencies of stimulation produced a decrease in synaptic effectiveness. This form of plastic change he called low-frequency or homosynaptic depresssion.Analysis remained at this stage for a number of years because it was difficult to determine whether these changes were due to a presynaptic mechanism (a change in transmitter release) or to a postsynaptic mechanism (a change in receptor sensitivity). The solution to this problem was facilitated in 1954 when del Castillo & Katz (1954a) demonstrated that release of acetylcholine at the nerve muscle synapse is not graded but quantized. An action potential releases about 200 multimolecular packets of transmitter-called quanta -and each quantum contains several thousand molecules of ACh. Quantal transmission was soon found to be the general mode of transmitter release at chemical synapses (see Dudel & Kuffler, 1961a; Eccles, 1964). The discovery of quantal transmission not only established important new insights into the nature of transmitter release from the terminals but also provided a method for analysing the relative contribution to synaptic transmission of changes in presynaptic and postsynaptic mechanisms. Del Castillo & Katz (1954a, b), Liley (1956a, b) and subsequently others, analysed alterations in synaptic effectiveness in terms of quantal transmission and found that both homosynaptic depression and post-tetanic potentiation represented a presynaptic alteration in the number of transmitter quanta released per impulse. The sensitivity of the receptor seemed not to be affected.This work was extended in a major direction in 1961 when, following the earlier suggestion of Frank & Fuortes (1957), Dudel and Kuffler described the first clear instance of a heterosynaptic interaction : presynaptic inhibition at the crayfish nervemuscle synapse. Dudel & Kuffler (1961a) found that in the crayfish the inhibitory axon to muscle has a double function: (1) it produces an inhibitory postsynaptic potential in the muscle, and (2) it depresses the excitatory postsynaptic potential produced by the excitatory axon. By applying a quantal analysis Dudel & Kuffler (19616) found that presynaptic inhibition reduces the number of transmitter quanta released by the excitatory axon without affecting the sensitivity of the receptor molecules. These experiments provided the first evidence that the membrane of the presynaptic terminals contains receptors to transmitter molecules, and that these receptors can control transmitter release. Subsequently, Kandel & Tauc (1964), Epstein & Tauc (1970), and Castellucci and his colleagues (1970) presented suggestive evidence for presynaptic facilitation of transmitter release in the synapses of Aplysia. Recently again based upon a quantal analysis, Castellucci & Kandel (1976) provided direct evidence for a presynaptic mechanism.In 1967 Katz and Miledi advanced the study of synaptic plasticity still one important step further by finding that transmitter release is dependent on the influx of Ca2+ that occurs with each action potential. They proposed that Ca2+ allows the synaptic vesicles, the subcellular organelles that store transmitter (and which are thought to represent individual quanta), to bind to release sites. In addition, Katz & Miledi (1967 a, b) found that the presynaptic terminal contained a high density of voltagegated Ca2+ channels.These findings suggested to Katz & Miledi (1968) and to Rahamimoff (1968) that changes in the intracellular level of free Ca2+ might be important for short-term synaptic plasticity. Rahamimoff (1968; Alnaes & Rahamimoff, 1975) proposed that the intracellular concentration of Ca2+ is controlled by intracellular organelles that buffer Ca2+-the mitochondria and endoplasmic reticulum -and that aspects of synaptic plasticity might depend upon this control. In support of this idea, Rahamimoff found that short-term homosynaptic facilitation seems to be due to residual Ca2+, the Ca2+ that remains in the terminal following a series of action potentials, and that is taken up slowly by the buffering organelles. A similar mechanism also seems to be operative in Aplysia (Kretz, Shapiro & Kandel, 1980).Another factor that controls the free Ca2+ levels in the terminals is the Ca2+ influx. It seemed to us (as it had earlier to Zucker (1974) and to Stinnakre & Tauc (1973)) that influx might not be constant but might be modulated. In the past, however, this idea has proven difficult to examine in central neurones showing plastic changes, since the most direct test of the hypothesis would require recording of Ca2+ current in presynaptic terminals simultaneous with monitoring of transmitter release. This problem can be overcome, to a certain degree, in Aplysia neurones. First, the membrane of the cell body of Aplysia neurones contains Ca2+ channels whose properties seem to resemble those of the terminal membrane (Geduldig & Junge, 1968 ; Geduldig & Gruener, 1970; Stinnakre & Tauc, 1973; Llinas, Steinberg & Walton, 1976). Moreover, in certain cases changes in the calcium current of the cell body parallel the changes in transmitter release at the terminals (Klein & Kandel, 1978). In addition, in some of these neurones the presynaptic terminals controlling transmitter release appear to be sufficiently close to the cell body electrically so that it is possible to modify transmitter release from the terminals by injecting current into the cell body (Shimahara & Tauc, 1975; Shimahara & Peretz, 1978; Shapiro, Castellucci & Kandel, 1980a). The observations suggested to us that we might be able to examine the relationships between transmitter release and specific ionic currents of the presynaptic membrane. To this end we have combined analysis of ionic currents of the cell body of the presynaptic neurone, using voltage-clamp analysis and pharmacological blockade of specific ion channels, with assay of transmitter release from the presynaptic cell, using intracellular recordings of the synaptic potential in the postsynaptic cell (Fig. 1). These combined techniques provide a powerful method for studying changes in specific ionic conductances associated with various presynaptic mechanisms for synaptic plasticity.We have applied these techniques to two identified synaptic connexions in the bdominal ganglion of Aplysia: (1) the connexions made between the multi-action cholinergic cell L10 and its identified follower cells, and (2) the connexions between the mechanoreceptor sensory neurones of the gill-withdrawal reflex and their follower cells.The situation was particularly favourable for L10 and its synapses. When we blocked the Na+ channels with tetrodotoxin and axotomized L10 we found that graded depolarizing commands applied to the membrane of the presynaptic cell body under the voltage clamp caused graded transmitter release from the terminals, as determined by intracellular recording from postsynaptic cells (Figs. 2 A, 2 B, and 3 A). We obtained further evidence that release sites were being controlled by the clamp currents in the cell body by finding that transmitter release is also a graded function of the duration of the depolarizing step (Fig. 2B2). Voltage control can be further improved by adding pharmacological agents that block each of the three known outward K+ currents: tetraethylammonium ions (TEA+), which block the delayed K+ channel; 4-aminopyridine (4-AP), which blocks the early K+ channel; and substitution of Ba24- ions for Ca24-, which block the Ca2+-dependent K+ channel (see Adams, Smith & Thompson, 1980; Shapiro, Castellucci & Kandel, 1980a, b). Blocking these K+ channels also lengthens the effective space constant of the neurone. MUsing these K+ channel-blocking agents we could now examine the relationship between transmitter release and presynaptic Ca2+ current (Fig. 3 A). We found that in the range of depolarizing voltages in which the Ca2+ current was increasing, transmitter release increased in a linear fashion (Fig. 3B). These results suggest that in this voltage range (from about —40 to zero mV) we could control transmitter release from the terminals with voltage clamp of L10’s cell body.Although in many cases (particularly in the studies of the connexions made by sensory neurones), we lacked ideal voltage control of the terminals, these procedures nonetheless allowed sufficient control to study transmitter release while at the same time examining ionic currents in the soma of the presynaptic neurone. Moreover, we repeated all experiments except those involving presynaptic inhibition (where the transmitter is not known) in mechanically isolated presynaptic cell bodies where optimal space clamp control can be achieved.Using this approach, we have examined four types of synaptic plasticity, including two directly involved in simple nonassociative forms of learning: (1) the control of transmitter release in spike generating neurones by the membrane potential of the presynaptic neurones; (2) presynaptic inhibition; (3) homosynaptic depression (the mechanism underlying short-term habituation); and (4) presynaptic facilitation (the mechanism of behavioural sensitization). We have found that each of these involves modulation of the Ca2+ current, although the details of the mechanisms differ in each case.In 1975 Shimahara and Tauc described a simple form of synaptic plasticity whereby the membrane potential of the presynaptic neurone exerts a powerful influence over the effectiveness of the connexions made by that neurone. Hyperpolarizing the presynaptic cell decreased the synaptic potential elicited by the presynaptic action potential whereas depolarizing the presynaptic cell enhanced the synaptic action. Although this effect was opposite to that described in the giant synapse of the squid (Hagiwara & Tasaki, 1958; Takeuchi & Takeuchi, 1962) and at first seemed paradoxical, similar potential dependent control of transmitter release by membrane potential has now been described in the leech (Nicholls & Wallace, 1978 a). A form of this plasticity occurs between the identified multiaction cholinergic cell L10 and its follower cells (Waziri, 1977; Shapiro, Castellucci & Kandel, 1980a).Intracellular stimulation of neurone L10 produces EPSPs in some follower cells (R15 and RB cells) and IPSPs in others (Li to L6, LBVC, LDHI, etc.; see Kandel et al. 1967; Wachtel & Kandel, 1967, Kehoe, 1972; Koester et al. 1974; Koester & Kandel, 1977). When the membrane potential of L10 is progressively increased, its ability to release transmitter at its various branches is reduced (Fig. 4). Even a slight increase in membrane potential of 5 mV produces significant depression of transmitter release. With larger hyperpolarizing changes of 20 – 40 mV, transmitter release can be completely blocked (Fig. 4 A). Conversely, depolarizing the cell’s membrane potential increases transmitter release. The changes in membrane potential affect the duration and height of the presynaptic action potential: spikes become narrower and shorter with hyperpolarization and broader and taller with depolarization (Fig. 4B). both of these potential induced alterations are likely to alter transmitter release because hey alter the Ca2+ current (Horn & Miller, 1977; Klein & Kandel, 1978). However.However, as we shall discuss below, these alterations account for only part of the effects of Membrane voltage on release. Part of the effect of membrane voltage is independent of changes in spike height and duration.The ability to alter transmitter release from the terminals by injecting current into the cell body implies that at least some release sites are electrically close to the soma. If the axon of the cell is cut close to the ganglion and the preparation is treated with TTX, graded depolarizing steps under voltage clamp lead to graded release of transmitter (Figs. 2, 3 and 5). The sigmoid function relating transmitter release to presynaptic depolarization is similar to that reported at the squid giant synapse (Katz & Miledi, 1969; Llinas et al. 1976). As is the case with the squid giant synapse the function relating transmitter release to presynaptic depolarization overlaps the voltage sensitivity of the presynaptic Ca2+ current (Fig. 3). However, in contrast with results in squid, hyperpolarizing the presynaptic cell decreases the size of the synaptic potential elicited by step depolarization to a given level (Fig. 5 B).At depolarized holding potentials, currents elicited by step depolarizations under voltage clamp are less outward than at hyperpolarized holding potentials (Fig. 5 A and Connor & Stevens, 1971b; Adams et al. 1980). This difference in net current presumably accounts for the difference in the configuration of the action potential at the two holding potentials in unclamped cells (Fig. 4B). In addition, the finding that the synaptic potentials are graded with the duration of the command pulse (Fig. 2B2) provides further evidence that the K+ currents, which control spike duration and amplitude, could modulate transmitter release. However, the ability of the membrane potential to modulate transmitter release under voltage-clamp conditions, in which the duration and amplitude of the presynaptic command pulse is held constant, suggested that mechanisms other than modulation of the K+ current contribute to this form of plasticity. One possible additional mechanism is a change in transient Ca2+ current. To test this idea we blocked the several outward K+ currents which are responsible for alterations in the shape of the spike to see whether the membrane potential was still capable of modulating the transmitter release caused by a depolarizing command, and whether this modulation was due to a direct action on the transient Ca2+ current.Three pharmacologically separable K+ conductances have been described in mol-luscan somata (Thompson, 1977; Adams et al. 1980). Two of these are voltagedependent; an early, rapidly inactivating K+ current sensitive to 4-AP (Connor & Stevens, 1971b; Byrne et al. 1979; Thompson, 1977), and a delayed K+ current sensitive to TEA+ (Connor & Stevens, 1971 a; Thompson, 1977; Byrne et al. 1979). A third K+ conductance is thought to be not dependent on voltage but on intracellular Ca2+ concentration, and can be blocked by agents which block the Ca2+ current (e.g. Ca2+, EGTA) and by substituting Ba24- ions for Ca Ca2+ ions (Meech, 1972; Meech & standen, 1975; Eckert & Lux, 1976; Thompson, 1977; Adams et al. 1980; Shapiro et al. 1980a). Blocking the voltage-dependent K+ channels caused peak transies inward currents to appear relatively unchanged from different holding potentials (Fiji 6 A). With all K+ channels blocked pharmacologically (so that there is no voltagesensitive outward current with depolarizing command) and in a solution that was free of both Na+ and Ca2+ (265 mm-TEA+, 60 mm-Ba24-, 10 mm-4-AP) the inward current through the Ca2+ channels, now carried by Ba2+ ions, can be observed directly. When this inward current was activated with a step depolarization it was not increased by changing the holding potential from —62 to —40 mV (Fig. 6C). At the same time, the synaptic potential elicited by depolarization from the two levels was still affected by membrane potential (Fig. 6B). When high concentrations of TEA+ and 4-AP were utilized together, the peak inward Ca2+ current even decreased slightly when evoked from more depolarized holding potentials (Fig. 6 A), perhaps as a result of steady-state inactivation, or because of an increase in the intracellular concentration of Ca2+ at depolarized holding potentials.These results indicate that differences in the transient currents observed with steps from different holding potentials (Fig. 4A) are due to changes in K+ conductances and not to changes in Ca2+ current. The holding potential does not control transmitter release by regulating directly the transient activation of the Ca2+ channel (Fig. 6).Although membrane potential regulation of transmitter release does not result from direct modulation of the transient Ca2+ current, changes in membrane potential could lead to changes in the steady-state activation of the Ca2+ channels (Fig. 6C). Steadystate activation-the contribution of Ca2+ current to the resting current-might be greater at depolarized levels. Such steady-state activation of Ca2+ channels has been described in molluscan somata by Eckert & Lux (1976) and by Akaike, Lee & Brown (1978).A steady-state activation of Ca2+ could lead to an increased concentration of intracellular Ca2+ at more depolarized holding potentials, which would add to the Ca2+ influx during the action potential, resulting in a higher total intracellular concentration of Ca2+ available for transmitter release. In addition, increased intracellular Ca24- could cause relative saturation of intracellular Ca2+ buffering systems that are thought to compete with the release process (Alnaes & Rahamimoff, 1975; Rahamimoff, 1968). A third possibility is that increased intracellular Ca2+ concentration causes changes in screening of surface membrane proteins by changing internal membrane surface charges, and transmitter release efficacy (Bass & Moore, 1966; Van der Kloot & Kita, 1973). In each of these possibilities invasion of the terminal by an action potential would provide a more release-effective Ca2+ concentration.To obtain evidence for steady-state Ca2+-channel activation, we examined the steady-state membrane conductance of L10 at different holding potentials in the range of membrane voltages where modulation of release was occurring. We found that the steady-state conductance showed a region of reduced positive slope that is due to the steady-state Ca2+ channel. The steady-state Ca2+ current in turn activates a Ca2+-dependent K+ conductance that is blocked by substituting Ba2+ ions for extract Ba2+ flows through Ca2+ channels without activating the K+ conductance (see Figs. 6B C and 7B and Adams et al. 1980). In the presence of Ba2+, a voltagedependent steady-state inward current flows through the Ca2+ channels at depolarized levels of holding potential (Fig. 7A2, 7B); this inward current is not present at hyperpolarized levels (Fig. 7B). To activate this K+ conductance the concentration of intracellular Ca2+ in the steady state must be significantly larger at more depolarized than at more hyperpolarized membrane potentials.The role of steady-state Ca2+ current in the modulation of transmitter release by the membrane potential is supported by the observations of Nicholls & Wallace (19786) that spontaneous quantal release of transmitter in leech neurones is increased at depolarized potentials. Our results indicate that an additional mechanism also operates in cell L10 : a decrease of both voltage-dependent K+ currents with depolarization. We have not as yet attempted to evaluate quantitatively the relative contributions of the changes in the steady-state Ca2+ and those in the transient K+ current In an unclamped cell, the decrease in the K+ currents accounts for the increase in the weight and duration of the spike. In this way the transient Ca2+ current can also be Tnodulated, albeit indirectly. Our findings also suggest that despite a transient Ca2+ current that is not directly affected by changes in holding potential, powerful control over synaptic transmission-ranging from total block to enhanced effectiveness-can be achieved by variations in the steady-state Ca2+ current (Fig. 8).These results support the idea first proposed by Shimahara & Tauc (1975) that EPSPs and IPSPs have a dual function. In addition to controlling the probability of firing an action potential, synaptic potentials also set the level for transmitter release. The results further suggest a possible mechanism for long-term regulation of synaptic output by metabolic or hormonal actions which affect resting potential (Thomas, 1972; Mayeri, Brownell & Branton, 1979; Mayeri et al. 1979).Presynaptic inhibition has been described in several vertebrate and invertebrate synapses and has been attributed to a depolarization of the synaptic terminals (reviewed in Burke & Rudomin, 1977; Ryall, 1978). This idea seemed consistent with the release properties described at the squid giant synapse where depolarization reduces transmitter release (Miledi & Slater, 1966). However, as described above, in Aplysia and in the leech, depolarization enhances rather than depresses transmitter release (Shimahara & Tauc, 1975; Nicholls & Wallace, 1978 a; Shapiro et al. 1980a). At these synapses, a different mechanism must account for presynaptic inhibition.In Aplysia the synapses of cell L10 that are modulated by membrane potential can also be modulated by presynaptic inhibition. Stimulation of one of the fibre pathways to the abdominal ganglion causes a depression of both the monosynaptic excitatory and the inhibitory connexions made by L10 (Fig. 9; Waziri, Kandel & Frazier, 1969; Shapiro et al. 1980b). Recently, Byrne (1980) has discovered a group of cells (the L32 cells) which, when stimulated, produce presynaptic inhibition without producing any conductance changes in the postsynaptic follower cells of L10.Each of the ways of producing presynaptic inhibition tends to produce an IPSP in the presynaptic neurone, L10. Thus one change in the presynaptic neurone that clearly contributes to presynaptic inhibition is the hyperpolarization that occurs as a result of the IPSP. This hyperpolarization would reduce the output of transmitter by reducing the steady-state Ca2+ influx and by removing some of the inactivation of the K+ channels. However, presynaptic inhibition outlasts the hyperpolarization and can also be observed in cases in which the cell does not hyperpolarize. To determine whether additional mechanisms are operative we controlled for changes in membrane potential by examining the presynaptic neurone under voltage-clamp conditions.In a solution containing TTX and TEA+, and with the cell held at a membrane potential which inactivates the early outward K+ current, the inward current produced by a depolarizing clamp step is carried by Ca2+ ions, and the residual outward current is mainly due to Ca2+-dependent K+ current and the leakage current. Under these circumstances presynaptic inhibition is correlated with a decrease in transient inward current evoked by the depolarizing command (Fig. 10).The decrease in inward current in voltage-clamp records based on net currents can be the result of an increase in small residual outward K+ current, or a decrease in the inward Ca2+ current. The observation that the decrease in early inward current during presynaptic inhibition is larger than the increase in late outward and leakage currents (Fig. 10, Fig. 11 A) suggests that the Ca2+ current may be modulated directly. But to distinguish between these alternative possibilities directly we performed two types of experiments: pharmacological block of all the K+ channels, and an examination of the voltage dependence of the presynaptic action.To test the involvement of Ca2+ channels in presynaptic inhibition we examined the divalent cation current in isolation after blocking the Na+ channel with TTX and the three known K+ channels with TEA and 4-AP, and with substitution of Ba2+ for Ca2+. under these circumstances we found that connective stimulation reduced the inward rent (Fig. 11B, 11C). This reduction paralleled the presynaptic inhibition.In addition, changes in the steady-state inward current also occurred with connective stimulation (Fig. 11 C). These results imply that the decrease in inward current reflects a decrease in the transient and in the steady-state Ca2+ current during presynaptic inhibition.Since the Ca2+ current is voltage dependent, one would expect that the synaptic transmitter that mediates presynaptic inhibition would have no effect on ionic currents at hyperpolarized levels of membrane potential where the Ca2+ current is not activated. We therefore examined the effects of presynaptic inhibition on currents in the voltage range where the Ca2+ channel was activated and in the range where it was not activated. When cell L10 was held in the voltage range in which the Ca2+ channel was activated, connective stimulation caused decreases in inward current (Fig. 12A). When the cell was held in the voltage range in which the Ca2+ channel was not activated, no conductance change was observed after connective stimulation (Fig. 12) These data support the idea that presynaptic inhibition is due to a direct action on the Ca2+ channel (Fig. 13). A similar mechanism for presynaptic inhibition has been discovered in dissociated dorsal root ganglion cells by Dunlap & Fischbach (1978) and by Mudge, Leeman & Fischbach (1979).In vertebrate heart muscle Giles & Noble (1976) have found that ACh, acting on muscarinic receptors in the heart, decreases the Ca2+ current in the heart muscle. This action of ACh is similar to the mechanism of presynaptic inhibition in Aplysia and in dissociated dorsal root ganglion cells. Reuter (1979; Reuter & Scholz, 1977) has suggested that cGMP, or the ratio of cAMP/cGMP in heart muscle, may control the maximum calcium conductance of the muscle. An alternative possibility (Fig. 27) is that the modulatory transmitter closes a Ca2+ channel-receptor complex directly without the mediation of an intracellular second messenger.In mammals, presynaptic inhibition has been correlated with presynaptic depolarization and with increased excitability in afferent fibres. The observed conductance change at some of these synapses follows the chloride Nernst potential (reviewed in Burke & Rudomin, 1977). However, as indicated above, results similar to those reported here have been obtained by Fischbach and his colleagues (Dunlap & Fischbach, 1978; Mudge et al. 1979) on chick dorsal root ganglia. Whether the presynaptic depolarization and the increased Cl− conductance in vertebrates are epiphenomena, that are not directly related to the mechanism of presynaptic inhibition, and are due only to the artificial modes of activation, or whether there are several distinct mechanisms for presynaptic inhibition, needs now to be determined (for a critical review At the studies of presynaptic inhibition in the mammalian brain, see Ryall, 1978).Homosynaptic depression -a self-induced depression in excitatory transmission at the synapses made by the sensory neurones on their central target cells-is the mechanism for short-term habituation of the siphon and gill-withdrawal reflexes in Aplysia (Fig. 14; Castellucci et al. 1970). This depression, which is very profound and rapid at these sensory neurone synapses, is due to a progressive decrease in the amount of transmitter released by each action potential (Castellucci & Kandel, 1974).As a first step in examining homosynaptic depression, we analysed the Ca2+ cont