J. Physiol. (1972), 225, pp. 637-656 637 With 1 plate and 11 text-figure8 Printed in Great Britain A COMPARISON OF CHEMICAL AND ELECTRICAL SYNAPTIC TRANSMISSION BETWEEN SINGLE SENSORY CELLS AND A MOTONEURONE IN THE CENTRAL NERVOUS SYSTEM OF THE LEECH BY J. G. NICHOLLS AND D. PURVES* From the Department of Neurobiology, Harvard Medical School, Boston, Ma8ssachusetts 02115, U.S.A. (Received 24 March 1972) SUMMARY In leech ganglia, three sensory cells of different modality converge on a motoneurone, where they form chemical and electrical synapses. Each of these synapses behaves in a characteristic manner and the nature of the transmission mechanism has significant functional consequences for the operation of the reflexes. An analysis has been made of the effects of trains of impulses on synaptic transmission through these pathways, using frequencies that correspond to natural firing. 1. At the chemical synapse between the nociceptive sensory cell and the motoneurone, two opposing events occur: facilitation and depression. Thus, with trains of impulses, the synaptic potentials first increase in amplitude and then decrease. The two processes could be separated by altering the Mg and Ca content of the bathing fluid. In concentrations of Mg that reduced the amplitude of a single control chemical synaptic potential, pure facilitation occurred during a train. Depression predominated during brief trains in raised concentrations of Ca, although synaptic potentials were initially larger. These results suggest that changes in the amount of transmitter released by each presynaptic action potential can account for the changes observed in chemical synaptic transmission. 2. In contrast, electrical transmission between the sensory cell responding to touch and the same motoneurone did not show facilitation or depression. The electrical coupling potential in the motoneurone was relatively constant when the touch cell fired at high or low frequencies in normal Ringer fluid, high Mg, or high Ca fluid. * Present address: Department of Biophysics, University College London, England.
638 J. G. NICHOLLS AND D. PURIVES 3. Further differences between chemical and electrical synapses were apparent when the preparation was cooled to 40 C. In the cold the latency of chemically evoked synaptic potentials in the motoneurone increased and their amplitude declined drastically with repetitive stimulation, while electrical coupling potentials were unaffected. 4. A brief hyperpolarization of the presynaptic cell by injected current produced a marked and prolonged increase in chemically evoked synaptic potentials, but did not influence electrical synaptic transmission. 5. The synapses of the sensory cell responding to pressure, which are both chemical and electrical, behaved as expected: the chemical synaptic potentials showed facilitation and depression while electrical transmission remained relatively constant. 6. These experiments emphasize the different functional consequences of electrical or chemical synapses in reflex pathways for the transmission of signals that arise as a result of natural sensory stimuli. INTRODUCTION The integrated reflex response of an animal to a sensory stimulus depends on a number of factors including adaptation by the sensory nerve endings, inhibitory and excitatory influences converging from different pathways, and the previous history of activity. The physiological properties of the synapses involved in transmitting the signals from neurone to neurone also play an important role. At chemical synapses, transmission can become more effective with trains of impulses, but it can also decline progressively or remain relatively constant (see del Castillo & Katz, 1954; Mallart & Martin, 1968; Bennett, 1968). One might therefore expect that the particular characteristics of a chemical or an electrical synapse in a reflex pathway could determine how well the response was maintained with long-lasting or repetitive stimulation. Thus, electrical synapses might not only transmit more rapidly, but could also be more constant and less affected by preceding impulses (Martin & Pilar, 1963; Auerbach & Bennett, 1969a, b). In the present investigation we have examined the effects of trains of impulses on synaptic transmission through reflex pathways in leech ganglia. Here, it has been shown (Nicholls & Purves, 1970) that three types of sensory cell, driven by cutaneous mechanical stimuli, make excitatory monosynaptic connexions on a pair of motoneurones (the large longitudinal motoneurones or L cells) that control the length of the segment (Stuart, 1970). The positions of these cells in a ganglion are shown in P1. 1. For each sensory cell the modality and the type of synaptic connexion are specific: sensory cells responding to noxious stimulation of
CHEMICAL AND ELECTRICAL SYNAPSES 639 the skin (N cells) act on the motoneurone by a chemical synapse, cells responding to touch (T cells) by a rectifying electrical synapse, and cells stimulated with pressure (P cells) by a combination of both mechanisms. These connexions are the basis for segmental shortening reflexes which occur when the skin is mechanically deformed. To determine how these chemical and electrical synapses vary in effectiveness, single sensory cells were stimulated with intracellular microelectrodes at different frequencies comparable to those initiated by natural stimuli, while recording synaptic potentials produced in the motoneurone. Marked differences were found in the properties of the chemical and electrical synapses: the chemically mediated synaptic potentials are highly dependent on previous activity of the presynaptic cell, whereas electrically mediated coupling potentials remain relatively constant in a wide variety of experimental situations. The experiments suggest that the changes observed at chemical synapses in the c.n.s. of the leech occur as a result of alterations in transmitter release. METHODS Most of the methods have been fully described in a previous paper (Nicholls & Purves, 1970). In summary, an isolated ganglion was pinned in a shallow bath with the dorsal surface uppermost. In this position the large longitudinal motoneurones are easily seen, since they are located on the dorsum of the ganglion, while the three most lateral mechanoreceptor cells on each side of the ganglion can also be seen through a variable thickness of tissue, even though they are located on the ventral aspect (see P1. 1). Glass micro-electrodes filled with 3M-KCI or 3 M K acetate, with resistances of 40-100 Mfl, were introduced into the pre- and post-synaptic cells for simultaneous recording and injection of current. The sensory cells and the motoneurones can be unambiguously recognized by the size and position of their cell bodies, by their electrical properties (in particular, the time course of the action potential) and in addition, for the motor cell, by a characteristic synaptic potential elicited by the presynaptic cell (see Nicholls & Baylor, 1968; Nicholls & Purves, 1970). The presynaptic cells cause excitatory synaptic potentials in the ipsilateral large longitudinal motoneurone and also in its homologue on the opposite side of the ganglion. The two motor cells are electrically coupled by non-rectifying junctions (Stuart, 1970). In all the experiments described here we have used the ipsilateral motoneurone, since the synaptic potentials recorded in it are consistently larger. The amplitudes of synaptic potentials were measured from the peak to the projected tail of the preceding synaptic potential (see Mallart & Martin, 1967). The composition of Ringer fluid was NaCl 115 m, KC1 4 mm, CaCl2 1-8 m, Tris-maleate 10 mm, and dextrose 9-5 mm. In this solution, the motor cell fires continuously at a rate of 1-3/sec because of synaptic input from 'spontaneously active' cells, and its relatively low threshold after impalement by the micro-electrode. Recordings made from its axon indicate that it is usually silent before penetration. The occurrence of action potentials in the motoneurone makes it difficult to determine accurately the amplitude of a synaptic potential; to prevent firing, the motor cells were hyperpolarized by 5-10 my in experiments made in normal Ringer fluid. In a few of the penetrations
640 J. G. NICHOLLS AND D. PURVES made in normal Ringer fluid the impalement of the motor cell did not cause firing. The results for these cells were similar to those obtained with DC hyperpolarization. In solutions containing high concentrations of Ca or Mg (10-15 mm) the amount of NaCl was correspondingly reduced. In these solutions the motoneurone does not fire, since its threshold is raised and the synaptic bombardment from other cells upon it is reduced (Nicholls & Purves, 1970). The bathing fluid could be changed through a flow system controlled by a tap without removing the intracellular electrodes. Most of the experiments were made at room temperature (20-25' C). The bath could be rapidly cooled by thermoelectric units, and the temperature monitored to within 0.50 C by a small thermistor. Before use, all leeches were kept at 40 C in a cold room. RESULTS Chemical synaptic transmission between the N cell and the motoneurone Brief trains of impulses in normal Ringer fluid. At the chemical synapse between the N cell and the L motoneurone, the synaptic potentials are initially facilitated during a brief train of impulses, but at frequencies of 10-15/sec they eventually become smaller than the control amplitude ('depression'). These phenomena are illustrated in Text-fig. 1, which also shows the changes in the synaptic potentials that occur after the train. Single test stimuli were applied to the sensory cell once every 10 or 20 sec before and after trains lasting 3-5 see; tests at this frequency did not on their own produce significant residual changes in synaptic transmission. To evaluate transmission immediately after trains a few tests were sometimes given at shorter intervals. Text-fig. 1 shows that at a low frequency of 5/sec the initial facilitation persisted throughout the train, although it reached a maximum and then declined. At higher frequencies of 10-15/sec or with longer trains, the synaptic potential became progressively smaller. After the end of the train the synaptic potential remained smaller for a few seconds, but then again increased in amplitude to more than the control value, during a second phase of facilitation. This 'post-activity' facilitation also was frequency dependent. In the examples of Text-fig. 1, it was barely noticeable after a train at 5/sec but after stimulation at 15-20/sec, the synaptic potential could double in size and return to control values gradually over the next 1-3 min. Another feature of the period after the train was a maintained depolarization of the L motoneurone, lasting for several sec. This was consistently seen when trains were delivered in Ringer fluid or high Ca but not in Mg (see Text-figs. 2 and 4 and Discussion). The amplitude of chemically evoked synaptic potentials is, therefore, determined by opposing processes, possibly in the presynaptic axon (see Discussion). During and after a train of impulses in the presynaptic cells,
CHEMICAL AND ELECTRICAL SYNAPSES 641 facilitation tends to augment the synaptic potentials and other mechanisms tend to depress them. In the standard Ringer fluid, 'spontaneous activity' makes an analysis of these antagonistic mechanisms difficult. However, in altered Ca and Mg the processes could be separated (see below). N call - 5/sec Ringer fluid......... N cill 10/sec ~ ~~~~~~~ A- Lcell_- W..-, _ Is/sec Ncell. 15/.sdc ]100 mv 4mV 3.5 sec Text-fig. 1. Effects of repetitive stimulation at different frequencies on transmission at the chemical synapse between the N sensory cell (upper traces) and the large longitudinal motoneurone (L cell, lower traces) in Ringer fluid. During trains, synaptic potentials are initially facilitated, but at higher frequencies (10, 15/sec) they become smaller than the control amplitude. Following the train, the synaptic potential again increases in size (post-activity facilitation); after a 3-5 sec train at 15/sec this can persist for up to 2 min. All records are from the same cell pair with intervals of several minutes between trains to allow for complete recovery. Increased synaptic activity in other cells presynaptic to the L cell probably accounts for the maintained depolarization after the train; this activity is greater with higher frequency trains and indicates that the N cell also makes polysynaptic connexions on the L cell. Trains of impulses in high Mg Ringer fluid. Text-fig. 2 shows an experiment made in Ringer fluid containing 10 mm-mg to reduce the release of transmitter. In this solution the synaptic potentials are smaller than in normal Ringer fluid, and the base line is quieter, owing to decreased synaptic bombardment and an increase in the threshold of the motoneurone (see Methods). Compared with Text-fig. 1, one sees a marked facilitation throughout the train at 10/sec. Following the train, the synaptic potentials continued to be increased in amplitude for some time, usually for 30 see to 3 min (Text-fig. 2). This is roughly the same as the postactivity facilitation seen in Ringer fluid. With higher frequencies of stimulation (e.g. 20/sec) the synaptic potential was at first more facilitated but 24 PHY 225
642 J. G. NICHOLLS AND D. PUR VES then tended to decline during the train. To avoid this complication, trials were made at still lower frequencies; for example, at 1/sec stimulation could be continued for 1 min or longer during which the facilitation was maintained. A simple interpretation of these results would be that Mg acts presynaptically to reduce the amount of transmitter released, and that this counteracts the processes tending to decrease the synaptic potential. In this respect the facilitation in Ringer fluid and high Mg appears similar to that observed at other chemical synapses, such as the neuromuscular junction of the frog (del Castillo & Katz, 1954). 10/sec 10 mmmg2m Lcell>...... 21 sec 31 sec 41 sec 3-5 sec Text-fig. 2. Facilitation of chemical synaptic transmission between the N sensory cell and the L motoneurone in Ringer fluid containing 10 m-mg. The lower trace is a record of the current pulses injected into the N cell to initiate impulses, but these are not shown. The synaptic potentials in the motoneurone increase in size progressively and remain larger for more than 40 sec after the end of the train. The degree of facilitation depends upon the frequency; at higher frequencies, however (l/sec or more), the synaptic potential begins to decrease in amplitude during trains of this duration. The effect of Mg also serves to reduce or block background activity; thus in comparison with Text-fig. 1 there is no increase in synaptic bombardment of the L cell via other pathways and the membrane potential rapidly returns to the base line. Facilitation could also be observed in high Mg fluid at this synapse following a single impulse. Individual records are illustrated in Text-fig. 3A and the time course is shown in Text-fig. 3B where each point shows the mean and se. of eight or more observations. With short intervals between stimuli to the N cell, the synaptic potential in the motoneurone increased to about twice its original amplitude. The facilitation decayed rapidly at first, with a half-time of about 100 msec, but in this and other Legend to Text-fig. 3. Text-fig. 3. Facilitation after a single impulse at the chemical synapse between the N sensory cell and the L motoneurone. Intracellular recordings from the N and L cells are shown in A with three different intervals between stimuli. The facilitation is shown in B as % ofthe controlsynapticpotentials. Facilitation was measured (as in Mallart & Martin, 1967) from the extrapolated tail of the first synaptic potential. Each point is the mean ± s.e. of 8-25 measurements.
CHEMICAL AND ELECTRICAL SYNAPSES A 10 mm-mg2+ 643 N cell * 1 L cefl N cell L cell N cell I I ] 50 mv Lcell ; l mv B 40 msec 200 150 F- 0_! 0* In 100.....-5 910.........5.. 0-5 1-0 1-5 sec Text-fig. 3. For legend see opposite page. 24-2
644 J. G. NICHOLLS AND D. PURVES experiments of the same type, appreciable facilitation could still be detected for up to 1P5 sec. The later part of the curve is difficult to determine accurately because of the large scatter, but it is clear that the curve as a whole cannot be fitted by a simple exponential. This result suggests that there are at least two components to the process of facilitation. Brief trains in high Ca solution. In 15 mm-ca, the synaptic potential evoked by an impulse in the N cell is larger than in normal Ringer fluid or in high Mg fluid, as though more transmitter were being released (Nicholls A 15 mm Ca2+ 35 impulses N cell 10/sec 2IV20 mv LoJ L cell _.,, -~~~~~~~~I. 10 mv B 3-5 sec Hell 1 20 sec 3-5 sec Text-fig. 4. Effects of 35 impulses at 10/sec and 1/sec on synaptic transmission between a pair of N and L cells in Ringer fluid containing 15 mm-ca. A shows intracellular recordings from an N cell (upper trace) and an L cell (lower trace) with stimulation at 10/sec for 3-5 sec. The action potentials in the N cell are not visible at this speed and gain of recording. Note that the N cell becomes hyperpolarized by more than 15 mv. The synaptic potentials in the L cell increase slightly but then become progressively smaller. Synaptic activity on the L cell from other cells is increased after the train. B, effect of stimulation of the same N cell at 1/sec (the intracellular recording from the N cell is not shown in this record). The synaptic potentials recorded in the L cell decrease in amplitude without facilitation. They remained depressed for more than 2 min after the train (see Text-fig. 5). In Mg, stimulation at a frequency of 1/sec does not lead to depression. & Purves, 1970). Text-fig. 4A shows synaptic potentials in the motoneurone before, during, and after a train at 10/sec in the N sensory cell. Comparing the responses with those in Ringer fluid or Mg, one major difference is in the rate of development and the degree of depression. Another feature in high Ca fluid is that the presynaptic cell became markedly hyperpolarized (Baylor & Nicholls, 1969; J. K S. Jansen & J. G. Nicholls, unpublished work). The initial facilitation was slight and was followed by a pronounced decline, so that the synaptic potentials were rapidly depressed to a point where it became impossible to distinguish
CHEMICAL AND ELECTRICAL SYNAPSES 645 them from the base line. At the same time, a steady level of depolarization was maintained in the L cell, declining over several seconds after the train. A similar maintained depolarization was seen in normal Ringer fluid although depression is less striking in that case (see Text-fig. 1). The marked depression in high Ca suggested it might be possible to find a frequency of stimulation at which a train produced depression of transmission without facilitation. Text-fig. 4B shows a record from the same Trains 15 mm-ca 15 }. 10/sec *** * **Os _ E 10 o~l o... 11sec 5 0 2 4 min Text-fig. 5. Time course of recovery of the synaptic potentials after trains of 35 impulses at 10/sec and 1/sec in Ringer fluid containing 15 mm-ca (same cells and trains as in Text-fig. 4). After the train at 1/sec (open circles joined by interrupted line) the amplitude of the synaptic potential in the L cell is reduced and recovers gradually over several min. After a train at 10/sec to the same N cell (filled circles, continuous line) the synaptic potentials are first depressed, then briefly facilitated and finally depressed again before recovering. The balance of post-activity facilitation and depression depends on the frequency and duration of the train. cell illustrated in Text-figs. 4A and 5 during and after a train of 35 impulses at 1/sec. There is no evidence of facilitation, but only a progressive decline in the synaptic potentials during the train. Subsequently they recovered only gradually over a period of minutes. In Mg solution, no such depression occurs at 1/sec, even when stimulation continues for several minutes. In the period following a train at higher frequency (10-20/see) a series of changes occurred. These are illustrated in Text-fig. 5. First, depression of transmission continued for a few see after the train had ended. Next, synaptic potentials transiently increased and then declined again before returning to their control size. Results like those of Text-fig. 5 were consistently seen in a number of experiments made in high Ca fluid; they
646 J. G. NICHOLLS AND D.PUR VES suggest that the processes which tend to increase and decrease the synaptic potential continue to interact for some minutes after a brief train. Facilitation after a single impulse in high Ca fluid was present but less marked than in Mg. The time course of the decay was otherwise similar to that observed in Mg with an initial half-time of about 100 msec and a slow tail lasting for over 1 sec. In contrast to the results obtained with Mg, high Ca appears to increase release of transmitter and enhance depression. Electrical synaptic transmission between the touch cell and the motoneurone; comparison with chemical transmission In addition to the chemical synapse from the N cell, the same large longitudinal motoneurone receives monosynaptic connexions from two other modalities of sensory cell, touch cells and pressure cells. The touch cells make electrical synapses with the motor cell, while the pressure cells affect the post-synaptic cell through a combination of electrical and T cell L cell Mg2+-Rlnger, 2O/sec, electrical synapse ]lo1mv 2M 35 sec Text-fig. 6. Electrical synapse between a touch sensory cell and the L motoneurone. The touch cell was stimulated at 20/sec for 3-5 sec through the intracellular micro-electrode (upper trace) while coupling potentials were recorded in the L cell (lower trace). Note the absence of facilitation or depression compared to the chemical synapse between the N cell and the motoneurone. This experiment was made in Ringer fluid containing 10 mm-mg. chemical mechanisms (Nicholls & Purves, 1970). In contrast to the complex effects seen at the chemical synapse between N and L cells, coupling potentials mediated through electrical junctions were stable and showed little change. Text-fig. 6 shows a train of impulses at 20/sec for 3-5 see in the touch cell and the coupling potentials recorded in the motor cell in high Mg Ringer fluid. Only small fluctuations in the size of the coupling potentials occur during and after the train of stimuli. This result is representative of other experiments at frequencies of 1-30/sec. At higher frequencies (40/sec) and with prolonged firing there was a small decrease in the amplitude of the coupling potentials during the train. However, the decline developed slowly and was far less than that occurring during a train of
CHEMICAL AND ELECTRICAL SYNAPSES 647 chemical synaptic potentials. There was no obvious facilitation after the train in high Mg solutions or enhancement of depression in high Ca. In contrast to the findings at the chemical synapse it was also evident that little or no change occurred in coupling potentials after a single impulse to the presynaptic T cell. Sample records with different intervals T cell L cell T cell L cell Tcell 4 6 < ]5Q mv 40 msec Text-fig. 7. Absence of facilitation after a single impulse at the electrical synapse between the T cell (upper trace) and the L cell (lower tram) with three different intervals between stimuli (cf. Text-fig. 3A). Bathing fluid contained 10 mm-mg. between stimuli are shown in Text-fig. 7. In this pair of cells, eighty-four trials were made with intervals ranging from 25 to 350 msec with no significant variations in the amplitude of the coupling potential. This result is strikingly different from that shown in Text-fig. 3 for the chemical synapse.
648 J. G. NICHOLLS AND D. PUR VES Comparison of the effect of cooling on chemical and electrical synapses. Leeches live well in the cold and continue to respond to external stimuli, albeit more sluggishly. At 40C, sensory impulses could still be initiated in T, P and N cells by appropriate mechanical stimulation of the skin. The frequency of firing was lower and the duration of the action potential markedly increased. 240 C N cell J0 ]JOmV L cell l _ l2mv 40C 20 msec N cell Text-fig. 8. Effect of cooling on chemical transmission between the N sensory cell (upper traces) and the L motoneurone (lower traces). The upper pair of records show the synaptic potential in the L cell at room temperature (240 C). In the lower records the preparation had been cooled to 40 (C. The delay between the peak of the action potential in the N cell (interrupted line) and the onset of the synaptic potential in the L cell was increased. No such change occurred at electrical synapses between T or P cells and the L cell (see Text-fig. 10). The duration of action potentials in the sensory cells is also increased by cooling. Pronounced changes occurred at the chemical synapses between N cells and the large longitudinal motoneurone when the preparation was cooled. In contrast, the properties of the electrical connexion between the T or P cells and the same motoneurone were little affected. Text-figs. 8 and 10 demonstrate these differences in behaviour. The most obvious effect was in the delay between the peak of the action potential in the N sensory cell and the synaptic potential in the motoneurone, which increased from 2-4 msec to about 20 msec when the temperature was reduced from 24 to
CHEMICAL AND ELECTRICAL SYNAPSES 649 40 C. Another effect of the cold was that the synaptic potentials were initially large but declined after very few impulses. For example, when the presynaptic N cell was stimulated at a rate of only 1/sec or even less frequently, the synaptic potential decreased in amplitude with each successive shock and disappeared entirely after 6-10 impulses (Text-fig. 9). This is a far more pronounced reduction than that occurring at the same frequency at room temperature (see Text-fig. 4). Text-fig. 9 further shows that although the decline occurred more steeply in the cold, the synaptic potentials recovered relatively rapidly after the train of stimuli had ended. 40 C 27 impulses N cell I I 10s J 20s 30 100 mv ~ ~J%~ J%_)10 my 1 sec Text-fig. 9. Effect of repetitive stimulation on chemical synaptic transmission between N and L cells at 40C in 15 mm-ca fluid. The upper trace shows the impulses in the N cell and the lower trace the synaptic potentials in the L cell. At a frequency of l/sec the post-synaptic response became progressively smaller. Recovery after the train of 27 impulses is shown at the right. The depression during the train is more pronounced than at room temperature (cf. Text-fig. 4B). Similar results were obtained with preparations in Ringer fluid at low temperatures. Similar results were obtained at lower frequencies of stimulation (0-5/sec) and at 3/sec, the fastest rate that the N sensory cell would follow in the cold. For convenience, most of these experiments were made in fluid containing 15 mm-ca in order to enhance the amplitude of the synaptic potential. The same results were obtained in a few experiments made in normal Ringer fluid. At the electrical synapse between the touch cell and the large longitudinal motoneurone no increases in delay and no depression were observed in the cold. With repeated stimulation at a frequency of 1/sec at 40 C the amplitude of the electrical coupling potential remained constant, as at room temperature. Depolarizing current spread directly from the sensory to the motor cell, but, as at room temperature, in only this direction. A clear demonstration of the different effects of low temperatures upon chemical and electrical transmission was provided by the connexions of the sensory P cell upon the large longitudinal motoneurone. These synapses work by both mechanisms (Nicholls & Purves, 1970). At room temperature one can demonstrate the direct spread of current and also a chemical component of the synaptic potential, which is blocked by a high concentration of Mg. When the preparation was cooled to 2 C, the separation between the two components of the synaptic potential became obvious,
650 J. G. NICHOLLS AND D. PUB VES owing to the increase in latency at the chemical synapse (Text-fig. 10). As with the N cell, after the pressure cell had been stimulated a few times at 1 sec intervals in the cold, the chemically mediated component disappeared, leaving only the electrical coupling potential (Text-fig. 10). At room temperature the chemical component of the P cell synapse also behaved like that of the N cell, showing facilitation and depression that were influenced by Ca and Mg. The effects of presynaptic hyperpolarization on chemical and electrical synap)8e8. Another contrast in the properties of the chemical and electrical synapses was provided by experiments in which the presynaptic sensory 220C l 20C 20C Pcell So-50mV 3? 3) 50mV L cell 3 1mV. 305mV 20 msec Text-fig. 10. Effect of cooling on the combined chemical and electrical synapse between a P cell (upper trace) and an L cell (lower trace). At 220 C the presynaptic action potential evokes a synaptic potential that begins with no appreciable delay. Part of this potential is depressed by high Mg and augmented by high Ca (see Nicholls & Purves, 1970). On cooling to 20 C a notch appears between the two components, presumably because of an increase in latency at the chemical synapse (see Text-fig. 8). Subsequently, with stimulation at 1/sec for a few sec, the delayed chemical component disappeared leaving only the electrically mediated coupling potential. The presynaptic action potential increased in duration with repetitive stimulation in the cold. All these changes were reversed by re-warming the preparation to room temperature. In this experiment the bathing fluid contained 15 mm-ca. cell was hyperpolarized by current injected through the micro-electrode in the cell body. A large, brief hyperpolarization of the N sensory cell led to an increase in the size of the chemically mediated synaptic potentials recorded in the motoneurone. An example is shown in Text-fig. 11A. A remarkable feature of this effect was that it appeared after a brief delay and persisted for several sec (Text-fig. 11 B). An augmentation of this type was seen in both high Ca and in high Mg fluid, provided that the current was of the order of 5 x 10- to 10-7 A in the hyperpolarizing direction and lasted for 50 msec or longer; briefer currents (5 msec) and depolarizing currents were ineffective. The hyperpolarization of the cell body of the sensory neuron resulting from the injection of 5 x 10 A was approximately 200 mv. With such large currents the input impedance of the cell decreases during the pulse owing to anomalous rectification. The experiments could be repeated many times on the same cell with no irreversible changes in its properties, although the action potential recorded in the cell
CHEMICAL AND ELECTRICAL SYNAPSES 651 body and the input impedance were often reduced in amplitude for the first seconds after the pulse. A possible explanation of this result is that current injected into the cell body hyperpolarizes the presynaptic nerve terminals and that this leads to an increase of release as at the chemical synapse in the stellate A a before b pulse c 2 sec later B 8_ N cell - lo** 4-l m- 9 LLI<_4mV Lcellll current ~- J 15x10-7A 50 msec 7 6 5 *4.4..ft~~~~~~~~~~~~.4... 2 50 msec pulse 1-8x10-8A 4 1 1 1 1 1 1 I I I 8 12 16 20 24 28 32 36 40 U 44 sec Text-fig. 11. Effect of presynaptic hyperpolarization on transmission at the chemical synapse between the N cell and the L motoneurone. A shows intracellular recordings from the N cell (upper traces) and the motoneurone (middle traces) before, immediately after and 2 see after injecting a brief (50 msec) hyperpolarizing pulse (bottom traces) through the microelectrode in the N cell. In this experiment the Ringer fluid contained 15 m - Ca; similar facilitation also occurred in 10 m -Mg. B is a graph of the relation between the amplitude of the synaptic potential recorded in the L cell and the time after the current pulse. In this, as in most other experiments, there was a delay of about 1 sec before the facilitation reached its peak. No facilitation was seen after depolarizing pulses or with pulses of less than 5 x 10-8 A (see text). Electrical synapses between T or P cells and the L cell did not show facilitation, but the chemical component of the P cell synapse did.
652 J. G. NICHOLLS AND D. PUB VES ganglion of the squid (Katz & Miledi, 1967). We have shown in an earlier paper that hyperpolarizing current injected into the cell body can reach some of the terminals (Nicholls & Purves, 1970; see also below). However, it is not possible to say what fraction of the potential change reaches terminals that are tens or hundreds of microns away from the soma. At the electrical synapse between the touch cell and the large longitudinal motoneurone no comparable change in the coupling potential occurred after a hyperpolarizing pulse. A further example of the different behaviour of chemical and electrical synapses is provided by the pressure cell connexion on to the motoneurone. In high Ca fluid, where the chemical component is enhanced, there is a persistent increase in the synaptic potential after a large hyperpolarizing pulse to the cell body. On the other hand in 15 mm-mg fluid, in which only the electrical coupling potential remained, no obvious augmentation occurred after the pulse. This result provides additional evidence that the chemical component of the connexion between the pressure sensory cell and the motoneurone is monosynaptic; if an interneurone had intervened, the chemical synaptic potential would not be expected to vary in a graded manner (see Nicholls & Purves, 1970). DISCUSSION Light touch, sustained pressure and noxious stimuli all give rise to segmental shortening reflexes through monosynaptic pathways that excite the large longitudinal motoneurone. Electrical transmission between the touch sensory cell and the longitudinal motoneurone is relatively stable. This is in sharp contrast to the chemical synapse between the N sensory cell and the same motoneurone, where the effectiveness of transmission depends critically on the preceding history of activity. A single impulse in the N cell leaves behind changes that can be discerned for up to 1-5 sec; with trains, the synaptic potentials can first double in size and then become vanishingly small. The mixed chemical and electrical synaptic connexion of the pressure cell shows both types of behaviour, one component fluctuating while the other remains stable. The changes at these synapses during and after repetitive firing resemble in many respects those described by Martin & Pilar (1963, 1964) in the chick ciliary ganglion, where electrical coupling potentials remain constant, while facilitation occurs at the chemical synapse. Opposing processes contribute to the changes observed at the chemical synapse between the N sensory cell and the large longitudinal motoneurone. They can be separated almost completely by using various concentrations of Ca and Mg. The results suggest that the principal phenomena of facilitation and depression can be explained by assuming that they
CHEMICAL AND ELECTRICAL SYNAPSES 653 depend on modifications of the release of transmitter. Presently we know of no analogous effects that depend on post-synaptic changes, and it is hard to see what post-synaptic mechanism could give rise to the slow decline in amplitude observed at low frequencies of stimulation in high Ca, or in the cold. Nevertheless, without knowledge of the identity of the chemical transmitter, it is not possible to rule out desensitization as a contributing factor to depression. Furthermore, the maintained plateau of depolarization of the L cell after trains in high Ca may represent residual transmitter diffusing away only slowly. This effect was far less apparent in Mg. The complex changes that occur at the chemical synapse could be explained if the terminals of the N cell behaved like those of the motor nerve at the frog neuromuscular junction. There, the probability of release is enhanced after each impulse. With brief trains, the effects are cumulative and lead to longer lasting facilitation (Mallart & Martin, 1967). With prolonged trains less transmitter is released by each impulse, so that progressively smaller synaptic potentials are set up (del Castillo & Katz, 1954; Mallart & Martin, 1968). At the neuromuscular junction there is another distinct phase of facilitation called post-tetanic potentiation, which only occurs after prolonged trains at high frequencies. We cannot at present say whether this also plays a part at the chemical synapse between the N and L cell, or whether there is only one process giving rise to both facilitation and post-activity facilitation. Other presynaptic mechanisms might play a part in the progressive depression of transmission. With repetitive firing, impulses might fail to invade the terminals owing to conduction block. D. C. Van Essen (to be published) has shown that natural frequencies of firing can eventually lead to failure at branch points and that this is enhanced in high Ca. However, the absence of changes in the electrical coupling potentials and the effects of low frequencies in Ca argue against this idea. Accordingly, while we cannot rule out the possibility that changes in the post-synaptic cell may be involved in depression, it is hard to see how they could account for many of the changes in synaptic efficacy that we observed. The functional significance of chemical and electrical synapses in leech ganglia. One of the principal questions arising from these experiments concerns the way in which the properties of a synapse influence the performance of a reflex movement. There are many instances in which it has been shown that certain electrical synapses occur in pathways where speed of action is required. One is the rapid escape reaction of fish, which is performed without the delay involved in transmitter release (Furshpan, 1964; Auerbach & Bennett, 1969b). Another known function of electrical synapses is to ensure synchrony (Bennett, 1968). For example, in the leech, the two large longitudinal motoneurones which are coupled electrically
654 J. G. NICHOLLS AND D. PUR VES fire together and produce a symmetrical shortening of the body wall (Stuart, 1970). The electrical synapse between the touch cell and the large longitudinal motoneurone does not appear to serve either purpose. The synaptic potential produced by an impulse in the T cell is small and several must usually sum before threshold is reached. Hence, even though the synaptic delay is short, the reflex builds up only slowly in time (see Fig. 10 in Nicholls & Purves, 1970). The function of this electrical synapse may instead be related to constancy of action. The touch cells are activated by small displacements of the skin (20 /z or less) or by the movement of water over the surface of the animal. The cells presumably fire while the animal is crawling along or swimming, and the presence of an unfailing electrical synapse ensures a maintained synaptic drive to the L cell. In contrast, the N cells presumably fire rarely in the natural course of events as they respond only to severe mechanical deformation of the skin. At room temperature they produce large synaptic potentials in the L cell that are usually above threshold. It is not known what function is served by the facilitation or by the failure that becomes so obvious in the cold. It seems that the N cell makes synaptic connexions with neurones other than the L cell, either directly or by way of interneurones. This is strongly suggested by increased synaptic bombardment on the L cell from other unknown pathways following brief trains of impulses in the N cell. Such connexions might provide the pathways whereby the initial shortening reflex is superseded by more complex movements involving other segments of the animal acting in concert. If depression occurred at one synapse while facilitation was building up at another, the nervous system could switch from one pattern of activity to the next in response to a continued stimulus. However, we have not yet traced connexions from the N cells to other motoneurones or interneurones and the functional role of depression and facilitation at these chemical synapses remains obscure. Neither is it clear what function is served by the same presynaptic cell (P cell) making both chemical and electrical synapses. The leech lives in temperate climates and survives for long periods at 40C. At this temperature the touch cells still fire in response to gentle stimuli and still produce the shortening reflex. The electrical coupling at this junction was not noticeably changed in the cold unlike crayfish synapses which become uncoupled (see Payton, Bennett & Pappas, 1969). The leech synapses still rectified so that depolarization of the sensory cell, but not hyperpolarization, spread to the motoneurone. The chemical synapses fatigued rapidly in the cold. This was presumably not caused by conduction block since the electrical coupling potentials were still transmitted. It will be of interest to determine whether other chemical synapses
CHEMICAL AND ELECTRICAL SYNAPSES 655 in the leech C.N.s. behave in the same way or whether they can continue to transmit effectively. At these synapses, at least, the reflex response of the animal to mechanical stimuli will vary with the temperature of the environment and will depend on whether a chemical (N cell) or electrical (T cell) synaptic mechanism is the transmitting link in the pathway. Presynaptic hyperpolarization and transmitter release. A consistent and reproducible increase of the chemical synaptic potential occurred in response to injection of a brief but large hyperpolarizing current pulse into the soma of N or P sensory neurones. A feature of the facilitation was its relatively long duration: shortly after a 50 msec current pulse the synaptic potential remained larger for 20 see or more. Measurements made at the combined chemical and electrical synapse of the P cell indicate that the amplitude of the action potential at the terminal did not change with the accuracy of measurement of the coupling potential in the L motor cell (± 15%). The interpretation of the effect of hyperpolarization is not certain because we cannot estimate the extent of the membrane potential change at the terminals that secrete transmitter. One possibility is that a preceding step of hyperpolarization allows Ca to enter, leading to an increased probability of release (see Katz & Miledi, 1967). It is natural to wonder whether the maintained hyperpolarization of the presynaptic cell that follows repetitive firing could also lead to post-activity facilitation (see, for example, Text-fig. 4), especially as the time courses of the two processes are similar. This hyperpolarization in the P and N cells can be attributed to the activity of an electrogenic pump, and to a long-lasting increase in potassium conductance (Baylor & Nicholls, 1969; J. K. S. Jansen & J. G. Nicholls, unpublished work). On the other hand the hyperpolarization is greater in high Ca fluid, where depression is enhanced. One obvious experiment would be to prevent the hyperpolarization from occurring after a train of impulses and determine whether transmission is altered. We have so far not been able to do this experiment successfully since agents like strophanthidin and high K that reduce the hyperpolarization also change the properties of the post-synaptic cell. The link between post-activity facilitation and changes in the membrane potential of the presynaptic terminal therefore remains speculative (see also Martin & Pilar, 1964). Supported by USPES Grant Numbers 5 R01 NS 08277-03 and 5-TI-MH 07084. Dale Purves was supported for part of this work by a Grass Fellowship. We wish to thank Mr David Freeman for valuable help with the electronics.
656 J. G. NICHOLLS AND D. P UR VES REFERENCES AUERBACH, A. A. & BENNiTT, M. V. L. (1969a). Chemically mediated transmission at a giant fiber synapse in the central nervous system of a vertebrate. J. gen. Physiol. 53, 183-210. AUERBACH, A. A. & BENNETT, M. V. L. (1969b). A rectifying electrotonic synapse in the central nervous system of a vertebrate. J. gen. Physiol. 53, 211-237. BAYLOR, D. A. & NICHoLLs, J. G. (1969). After-effects of nerve impulses on signalling in the central nervous system of the leech. J. Physiol. 203, 571-589. BENNETT, M. V. L. (1968). Similarities between chemically and electrically mediated transmission. In Physiological and Biochemical Aspects of Nervous Integration, ed. CARL5ON, F. Englewood Cliffs, New Jersey: Prentice-Hall. DEL CASTILLO, J. & KATZ, B. (1954). Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124, 574-585. FusRsiPAN, E. J. (1964). Electrical transmission at an excitatory synapse in a vertebrate brain. Science, N.Y. 144, 878-880. KATZ, B. & MLEDI, R. (1967). A study of synaptic transmission in the absence of nerve impulses. J. Physiol. 192, 407-436. MALLART, A. & MART=N, A. R. (1967). An analysis of facilitation of transmitter release at the neuromuscular junction of the frog. J. Physiol. 193, 679-694. MALLART, A. & MARTIN, A. R. (1968). The relation between quantum content and facilitation at the neuromuscular junction of the frog. J. Physiol. 196, 593-604. MARTIN, A. R. & PnAR, G. (1963). Dual mode of synaptic transmission in the avian ciliary ganglion. J. Physiol. 168, 443-463. MARTIN, A. R. & PLAR, G. (1964). Presynaptic and post-synaptic events during posttetanic potentiation and facilitation in the avian ciliary ganglion. J. Physiol. 175, 17-30. NICHouTs, J. G. & BAYLOR, D. A. (1968). Specific modalities and receptive fields of sensory neurones in the C.N.S. of the leech. J. Neurophysiol. 31, 740-756. NiCHOLLS, J. G. & PuRvEs, D. (1970). Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech. J. Physiol. 209, 647-667. PAYTON, B. W., BENNExr, M. V. L. & PAPPAS, G. D. (1969). Temperature-dependence of resistance at an electrotonic synapse. Science, N. Y. 165, 594-597. STUART, A. E. (1970). Physiological and morphological properties of motoneurones in the central nervous system of the leech. J. Phy8iol. 209, 627-646. EXPLANATION OF PLATE Dorsal view of leech ganglion showing the position of the L motoneurones. The most lateral sensory cells that respond to touch (T), pressure (P), and noxious stimulation (N) of the skin can also be seen through the ganglion. Only a small part of the upper T cell, the lower N cell and both P cells is visible in this photograph. One can usually impale these sensory cells from the dorsal surface of a ganglion.
*T The Journal of Physiology, Vol. 225, NAo. 3 Roots I *: (pr. M'1 so'-... *... _. ;... *. :,.;... e*...... _L --4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~;f7'-s- MX5 Plate 1 _s,;*, St,, ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~5 t---e-. '--L-- J. G. NICHOLLS AND D. PURVES (Facing p. 656)