J. Phyasol. (1978), 285, pp. 231-255 With 12 text-figure. Printed in Great Britain

231

GRADED SYNAPTIC TRANSMISSION BETWEEN LOCAL INTERNEURONES AND MOTOR NEURONES IN THE METATHORACIC GANGLION OF THE LOCUST

BY M. BURROWS AND M. V. S. SIEGLER From the Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ

(Received 16 March 1978) SUMMARY

1. In the metathoracic ganglion of the locust some neurones can effect changes in the membrane potential of identified post-synaptic motor neurones without themselves spiking. 2. These 'non-spiking' neurones have processes only within the metathoracic ganglion, and therefore are local intraganglionic interneurones. 3. The absence of spikes in the interneurones reflects their normal physiological state and is not due to the experimental conditions. 4. When the interneurones are depolarized by the injection of current pulses lasting several hundred milliseconds, post-synaptic motor neurones are either depolarized, or hyperpolarized, for the duration of the pulse. 5. The magnitude of the change in post-synaptic voltage is graded according to the amount of presynaptic current. 6. A number of physiological tests indicate that the graded effects upon motor neurones are mediated by chemical synaptic transmission. For example, an evoked hyperpolarization of a motor neurone can be reversed in polarity by simultaneously hyperpolarizing the motor neurone with injected current. 7. At their resting potential some interneurones tonically release sufficient transmitter to have a measurable post-synaptic effect. The injection of depolarizing and hyperpolarizing currents into these interneurones effects opposite changes in postsynaptic potential. 8. Other interneurones must be depolarized from resting potential before a postsynaptic effect is observed, and hyperpolarizing currents have no post-synaptic effect. In these interneurones it is estimated that a depolarization of only 2 mV is sufficient to effect the release of transmitter. 9. The membrane potentials of non-spiking interneurones can fluctuate by as much as 15 mV during active movements of the hind legs and individual p.s.p.s as large as 5 mV can be recorded. Therefore, summed p.s.p.s or even single ones are expected to be the electrophysiological signals effecting transmitter release from these interneurones.

232

M. BURROWS AND M. V. S. SIEGLER INTRODUCTION

Some neurones lack action potentials and use only graded electrical signals for intracellular communication (for a review, see Pearson, 1976). A variety of primary and secondary sensory cells, in vertebrates and invertebrates alike, were first demonstrated to function in this way (e.g. Gwilliam, 1963; Ripley, Bush & Roberts, 1968; Werblin & Dowling, 1969). More recently, central neurones that do not produce action potentials have been described in crustaceans (Mendelson, 1971; Maynard & Walton, 1975), and insects (Pearson & Fourtner, 1975; Burrows & Siegler, 1976). Graded changes in the membrane potential of these non-spiking neurones imposed by the intracellular injection of current affect, in a graded way, the frequency of action potentials recorded from neurones one or more synapses away (Mendelson, 1971; Bush & Cannone, 1973; Baylor & Fettiplace, 1976; Pearson & Fourtner, 1975). The assumption is that the non-spiking neurones effect intercellular communication by graded synaptic transmission. In the locust, it is possible to record simultaneously and intracellularly from a non-spiking local interneurone and an identified post-synaptic motor neurone (Burrows & Siegler, 1976). It should therefore be feasible to examine synaptic transmission between neurones where the absence of spikes has not been artificially induced. This has only been possible in one other preparation, the stomatogastric ganglion of the lobster, where graded inhibitory transmission has been shown (Maynard & Walton, 1975; Graubard 1975; Graubard, Raper & Hartline, 1977). Here it will be shown that small changes in the membrane potential of non-spiking interneurones are able to effect an hyperpolarization or a depolarization of motor neurones of the hind leg by the graded release of transmitter. The graded effects upon the motor neurones produce co-ordinated movements of the leg similar to those which occur during normal behaviour. It is argued that graded synaptic transmission in the absence of action potentials is the normal mode of functioning for these neurones. METHODS

Adult male and female locusts Schiatocerca gregaria (renamed SchIitocerca americana gregaria by Dirsh, 1974) were obtained from our own crowded cultures. Locusts were mounted with the dorsal side up and the pro- and mesothoracic legs restrained. The metathoracic femora were rotated through 1800, and held so that the tibiae and tarsi could move freely. One tibia could be forcibly moved by a mechanical vibrator driven by a function generator. The locust was opened by a dorsal mid-line incision that avoided cutting the underlying dorsal longitudinal muscles. The gut, some air sacs, and fat body were removed, leaving the main longitudinal tracheae intact, so that the pumping movements of the abdomen could provide adequate oxygenation to the central nervous system and to the muscles. The two sternal apophyses lateral to the meso- and metathoracic ganglia, and the spina between the meso- and metathoracic connectives were cut, and the attached ventral longitudinal muscles removed. The meso- and metathoracic ganglia were stabilized on a wax covered platform and bathed with a constant flow of saline (Usherwood & Grundfest, 1965) at 19-20 0C. To facilitate the passage of the micro-electrodes across the ganglionic sheath, it was usual, though not necessary, to treat the ganglion with a 1 % (w/v) solution of protease (Sigma type VI) in saline for 3 min. No difference was observed in the activity of neurones in treated ganglia compared with those in untreated ganglia. Six pairs of 50 ,um diameter stainless-steel wire electrodes, insulated but for the tip, recorded the extracellular activity of known muscles of the hind leg and hind wing. The same wires

GRADED SYNAPTIC TRANSMISSION

233

could be used to stimulate the terminals of the motor neurones in the muscles and evoke antidromic spikes. Intracellular recordings were made from the metathoracic ganglion with glass micro-electrodes filled with 2 M-potassium acetate and with resistances of 30-50 MCI. Current could be injected through the recording micro-electrodes by means of a bridge circuit and measured by a virtual ground amplifier. Intracellular recordings were made either from the somata or neuropilar processes of motor neurones, but exclusively from neuropilar processes of interneurones. The somata of most of the motor neurones studied here are on the dorsal or lateral surfaces of the ganglion. To penetrate those located ventrally, the electrode had to pass through the neuropile, from the dorsal surface. Motor neurons could be identified as individuals according to criteria previously set out (Hoyle & Burrows, 1973), so that the same ones could be penetrated in different locusts. Neurones were stained by the intracellular injection of cobalt (Pitman, Tweedle & Cohen, 1972) and the subsequent intensification of the sulphide precipitate with silver (Bacon & Altman, 1977). RESULTS

Physiological and anatomical properties of non-spiking interneurones Effects of non-spiking and spiking neurones upon motor neurones In the metathoracic ganglion, intracellular micro-electrode recordings can be made simultaneously from motor neurones and from the neuropilar processes of a variety of different presynaptic neurones. Some of these neurones are able to effect changes- in the membrane potential of motor neurones without apparently producing spikes. In one such non-spiking neurone, an imposed step of depolarizing current, 600 msec in length, fails to evoke spikes, but merely enhances the amplitude of any hyperpolarizing synaptic potentials (Fig. 1A). Nevertheless a post-synaptic motor neurone is gradually depolarized throughout the current pulse to a level sufficient to evoke a spike. Similarly, when another apparently non-spiking neurone is depolarized, no spikes occur, but a maintained hyperpolarization of a postsynaptic motor neurone is evoked (Fig. 1 B). The effects in the motor neurone are not the result of the extracellular spread of current, because equal or stronger stimuli have no effect upon the motor neurone when the stimulating micro-electrode is just outside the presynaptic neurone. Although no spikes are recorded when such presynaptic neurones are depolarized by injected current, they might occur at sites remote from the micro-electrode and therefore not be detected. This possibility seems unlikely, for two reasons. First, intracellular recordings made simultaneously with two micro-electrodes in different neuropilar regions of the same neurone fail to reveal spikes (Fig. 1 C). Depolarizing current, injected at one recording site does not evoke spikes at that site, or at a site 200 #sm away, but is, nevertheless, able to alter the frequency of spikes in a post-synaptic motor neurone of the hind leg (Fig. 1 C). Synaptic transmission can therefore occur in the absence of presynaptic spikes. Even if the injected current is sufficient to depolarize the distant recording site by 8 mV, spikes are not elicited. Furthermore, no spikes are recorded when the neurone is depolarized by 15 mV, during voluntary or imposed movements of the hind leg. Secondly, when current is injected into an apparently non-spiking neurone, the resulting depolarization or hyperpolarization of a post-synaptic motor neurone is relatively smooth, if due allowance is made for the fact that it is. superimposed

234 M. BURROWS AND M. V. S. SIEGLER upon other spontaneous synaptic inputs (Fig. 1 A, B). This contrasts with the discontinuous change in the membrane potential of motor neurones produced by spikes in other presynaptic neurones. For example, an imposed step depolarization in the latter type of presynaptic neurone evokes a series of spikes, that are each followed with a constant latency of 1P5 msec, by a discrete excitatory post-synaptic potential (e.p.s.p.) in a motor neurone (Fig. 1 D). When more current is injected, the frequency of spikes increases, and the resulting e.p.s.p.s sum to give a maintained shift in the membrane potential of the motor neurone (Fig. 1 E). No matter what the frequency of spikes in the presynaptic neurone, discrete e.p.s.p.s are still recorded in the motor neurone. Likewise, other spiking neurones can evoke discrete inhibitory post-synaptic potentials (i.p.s.p.s) in motor neurones. A

~~~~~~~~~StR ~int.

A

W

VVV11_ ___

B

C

200 msec D

.t.

E

mn. t

Fig. 1. A comparison of the effects of non-spiking and spiking neurones upon motor neurones. A, B, C, non-spiking neurones. A, depolarization of this neurone (int., 1st trace) by injecting current (3rd trace) evokes a slow depolarization in a neuropilar process of a flexor tibiae motor neurone (mn., 2nd trace) which eventually leads to a spike (at arrow). B, depolarization of another neurone evokes an hyperpolarization of a flexor tibiae motor neurone, recorded from its soma. C, two electrodes in the same non-spiking neurone fail to detect spikes. 2-5 nA of current (4th trace) injected through electrode (1st trace) depolarizes the membrane by 2 mV at the recording site 200,um away (2nd trace), but does not evoke spikes. Synaptic transmission is nevertheless effected, because the frequency of potentials in the extensor tibiae muscle is increased (3rd trace). D, E, a picking neurons. D, depolarization of this neurone (1st trace) by injection of current (3rd trace) evokes a series of spikes, which are each followed by an e.p.s.p. in the soma of a flexor tibiae motor neurone (2nd trace). E, a stronger current pulse leads to an increased frequency of spikes. The resultant e.p.s.p.s in the motor neurone summate. Calibration: voltage 1st traces (A-C) 20 mV, (D, E) 50 mV, 2nd traces (A-E) 4 mY, current 26 nA.

The non-spiking state is not due to damage by micro-electrodes Depolarization of some non-spiking neurones can alter the output of several motor neurones and thereby produce co-ordinated movements of the hind leg. The role of these neurones in the production of motor patterns would therefore seem crucial. For a future understanding of the role of these neurones in behaviour, it is necessary to show that their effects upon motor neurones in the absence of spikes are due to their normal mode of functioning, and are not one induced by the experimental conditions. For example, it is known that in the vertebrate retina,

GRADED SYNAPTIC TRANSMISSION 235 ganglion and amacrine cells may sometimes be damaged and depolarized upon penetration with micro-electrodes so that they no longer spike. If some of these neurones are hyperpolarized sufficiently by the intracellular injection of current, spikes may again be generated (Werblin, 1977; Murakami & Shimoda, 1977). Neurones in the lobula of the blowfly Calliphora also do not spike at the membrane potential normally recorded by Hengstenberg (1977), but can be induced to spike during sustained intracellular injection of hyperpolarizing current. In the locust, however, it seems unlikely that the 'non-spiking' neurones are simply damaged neurones that normally spike, for the following reasons. Fir8t, all 100 intracellular stains of neurones identified physiologically as 'nonspiking' proved to be of local, intraganglionic interneurone8 (Siegler & Burrows, 1978). Two examples are shown (Fig. 2A, B). A single process emerges from a cell body 1030 ,sm in diameter, and gives rise in the neuropile, to an extensive arborization, the largest branches of which are 3-8 /zm in diameter. There are no processes which leave the metathoracic ganglion, either through peripheral roots or central connectives. Such a consistent anatomical picture would not emerge were the absence of spikes simply the result of damage to any of the neurones in the ganglion, caused by the micro-electrodes. Secondly, there is no change in the membrane potential of a post-synaptic motor neurone when a non-spiking interneurone is penetrated by a micro-electrode (Fig. 3). Were penetration of the interneurone to inflict damage, then the effect should be registered by a change in the membrane potential of the motor neurone. At the beginning of the record in Fig. 3A, one micro-electrode is outside the process of an interneurone in the neuropile, and a second is in the soma of a motor neurone. With one sharp tap on the manipulator, the micro-electrode enters the non-spiking interneurone, the downward deflexion registering a membrane potential of 38 mV. There is no sustained change in the membrane potential of the motor neurone as a result of the penetration. A few depolarizing synaptic potentials occur in this motor neurone soon after penetration, but neither the resting potential of the motor neurone nor its average level of synaptic inputs is changed. Injection of 0.5 nA of depolarizing current into the interneurone subsequently depolarized the motor neurone. Similarly, at the beginning of Fig. 3B, one micro-electrode is outside another non-spiking neurone, and a second is in the soma of another motor neurone. The first tap results in a small deflexion of the interneurone trace; with the second tap there is a larger but transient deflexion of the trace, indicating that the microelectrode has briefly entered the interneurone. With the third tap, the microelectrode enters the interneurone, to register a membrane potential of 37 mV. Throughout, there is no change in the membrane potential of the motor neurone that would indicate damage to the presynaptic neurone. Injection of 0 5 nA of depolarizing current into this interneurone subsequently hyperpolarized the motor neurone. Thirdly, stable recordings of non-spiking neurones can be maintained for as long as 1-2 hr, throughout which time consistent post-synaptic effects are observed. In contrast, penetrations of damaged motor neurones, which show a high frequency of spikes, can rarely be maintained for longer than 30 sec-1 min. Fourthly, the synaptic inputs recorded from neuropilar processes of non-spiking

M. BURROWS AND M. V. S. SIEGLER 236 neurones are not qualitatively different from those seen in neuropilar recordings from spiking neurones. This contrasts with the observations of Pearson & Fourtner (1975) on the cockroach, who report that non-spiking neurones show a characteristic high-frequency 'noise'. Hengstenberg (1977) also observes 'noise' in some lobular

a

A

1

3

iI

I

I

I

I

5

II I

I

II

I

100 pim

11I

Dorsal

L.J

Ventral

Posterior

Fig. 2. The morphology of two non-spiking interneurones as revealed by the intracellular injection of cobalt and subsequent intensification of the sulphide precipitate with silver. When depolarized, both interneurones inhibit a tonic coxal motor neurone, the anterior adductor of the coxa. Drawings are made from whole mounts of the metathoracic ganglion. A, one interneurone is shown in side view. Its cell body is in the ventral rind of cell bodies. The outline of the ganglion is shown as it appears from the antero-posterior mid line. The dashed line shows the extent of the neuropile, as it appears in line with the origins of nerves 1 and 5. The anterior and posterior connectives and nerve 1 are shown, but all other nerve roots are omitted. B, the other interneurone is viewed from the dorsal surface of the ganglion. Its cell body is dorsal. The mid line of the ganglion is on the left edge of the drawing. The dashed line indicates the anterior, lateral and posterior extent of the neuropile. Nerve roots posterior to nerve 5 are omitted.

GRADED SYNAPTIC TRANSMISSION 237 interneurones in the blowfly, and presents evidence that this 'noise' is due to abortive spikes. Similar 'noise' is not evident in recordings from non-spiking neurones in the locust. V

A

200 msec

Motor neurone

He

,~~~~~~~~~~~~~~~~n

Fig. 3. Penetration of a non-spiking interneurone has no effect upon the membrane potential of a post-synaptic motor neurone. A, at the start of the trace an electrode is outside an interneurone in the neuropile (1st trace) and a second electrode is in the soma of the lateral fast flexor tibiae motor neurone (2nd trace). One tap (arrow) of the micro-manipulator causes the electrode to penetrate the interneurone, but there is no effect upon the membrane potential of the motor neurone. B, three taps (arrows) are needed to penetrate this interneurone, but again there is no effect upon the membrane potential of this tergotrochanteral motor neurone. Calibration: vertical, interneurone 16 mV; motor neurone (A) 8 mV, (B) 4 mV.

Fifthly, when the membrane potential of a non-spiking neurone is increased by injection of steady hyperpolarizing current, no spikes are revealed. This test was prompted by the observation that non-spiking neurones, as a group, have transmembrane potentials that are significantly lower (P < 0-001) than those of spiking neurones, measured in the neuropile. The membrane potentials of non-spiking neurones range from -35 to -60 mV, with a mean + S.E. of - 47-7 + 0-5 mV; by contrast spiking neurones have transmembrane potentials ranging from -45 to -75 mV with a mean + S.E. of - 63-5 + 0-7 mV. Although these measurements are made when the locust is quiescent, they cannot .be considered as true 'resting potentials'because theneurones are continuously bombarded by synaptic inputs. The low resting potential could have inactivated the spike mechanism so that only small abortive spikes remain. In one example, imposed flexion of the tibia (from a femoraltibial angle of 90-60O) evokes a sequence of between four and six depolarizing potentials at the normal resting potential of an interneurone (Fig. 4B). To test whether these potentials are abortive spikes or e.p.s.p.s, the imposed flexion movement is repeated and the interneurone is hyperpolarized progressively with injected current (Fig. 4C-E). The same pattern of depolarizing potentials is evoked but they progressively increase in amplitude and duration, until with 6 nA of current they are more than twice their original amplitude (Fig. 4E). At this membrane potential spontaneously occurring synaptic potentials that are hyperpolarizing at the normal resting potential have reversed to become depolarizing. The imposed flexion nevertheless fails to elicit spikes, even when the hyperpolarization is maintained for 20 min. A sustained depolarization of the interneurone diminishes the amplitude

238 M. BURROWS AND M. V. S. SIEGLER of the depolarizing potentials that occur with each flexion, and again fails to reveal spikes (Fig. 4A). Neither depolarization nor hyperpolarization of the interneurone altered the frequency of the depolarizing potentials, as would be expected if they were spikes. It is concluded that the depolarizing potentials evoked by flexion are e.p.s.p.s, rather than abortive spikes or remnants of spikes. This test has been repeated on a wide variety of suspected non-spiking interneurones, always with

the above result. Flexi, Extend A

+3nA

C

-3 nA

D

-45 nA -6 nA

200 misc Fig. 4. Altering the membrane potential of a non-spiking interneurone fails to reveal spikes. The tibia is rhythmically flexed and extended by an imposed movement (1st trace) and the membrane potential of an interneurone is altered by the injection of current. The interneurone is at its normal resting potential in B, depolarized in A, and hyperpolarized in C, D and E. Each flexion evokes a series of depolarizing potentials but no spikes. Calibration: vertical, 16 mV and 600.

Is oxygenation of the ganglion adequate? A second potential source of damage to neurones is that due to metabolic changes resulting from hypoxia. For example, invertebrate photoreceptors become depolarized, and ultimately fail to respond to light when their supply of oxygen is low (Baumann & Mauro, 1973). Moreover, brief (10 min) exposure of insect ganglia to carbon dioxide or nitrogen, presumably causing hypoxia, can have long-lasting effects upon the metabolism of neurones (Argiro, Pelikan, Wood & Cohen, 1977). In order to minimize possible damage caused by hypoxia, the tracheal supply of the metathoracic ganglion of the locust was always left intact. All thoracic and abdominal spiracles had unobstructed access to the air, and the pumping movements of the abdomen, continuous throughout an experiment, ensured that air was circulated to the metathoracic ganglion. The main tracheation of the ganglion enters from the ventral surface, so that it is possible that the platform, upon which the ganglion rests, occludes some of the air supply. To circumvent this possible

GRADED SYNAPTIC TRANSMISSION 239 problem, locusts were dissected from the ventral side, so that the dorsal surface of the ganglion rested upon the stabilizing platform. No pronase was used. The tracheae and their associated air sacs investing the ganglion were left intact and could be seen to inflate rhythmically with each cycle of ventilation. These movements continued uninterrupted throughout the 20 min that it took to encounter the first non-spiking interneurone. There were no discernible differences in the properties of interneurones recorded in this way and those studied by dorsal penetrations of the ganglion. Does temperature affect the properties of the interneurones? This question is prompted because the properties of some motor neurones in the locust change with temperature. Most notably, the membrane potential is increased, and the threshold for spike initiation is lowered as the temperature in the body cavity is increased from 18 to 35 0C (Heitler, Goodman & Rowell, 1977). These temperatures are within the range recorded from locusts in the wild. To test whether changes in temperature could qualitatively alter the properties of non-spiking neurones, perhaps to induce spikes, non-spiking interneurones were first recorded in locusts at 18 'C. The saline was then heated, just before it entered the body cavity, and the temperature of the thorax monitored by a thermistor 1 mm away from the metathoracic ganglion. After the temperature had stabilized at 28 00 for 15 min suspected non-spiking interneurones did not produce spikes, either in response to injection of 20 nA of depolarizing current, or upon sudden release of 10 nA of hyperpolarizing current. They also did not spike in response to a variety of sensory inputs that induced vigorous movements of the hind legs. When depolarized with injected current, the interneurones evoked graded changes in the membrane potential of motor neurones. The temperature was then raised to 35 0C and maintained at that level for 90 min. Again, no spikes could be induced in the interneurones, and graded transmission was still observed. All of the above lines of evidence indicate that these local interneurones are able to affect their post-synaptic motor neurones without themselves producing spikes.

Properties of synaptic transmission between non-spiking interneurones and motor neurones Graded synaptic transmission An abrupt depolarization of a non-spiking interneurone, induced by an intracellularly applied pulse of current, evokes a more slowly rising change in the membrane potential of a post-synaptic motor neurone. The shape of the postsynaptic potential resembles that in the presynaptic interneurone. For example, a ramp function injected into the interneurone produces a more slowly rising ramp in the motor neurone. The amplitude of the post-synaptic potential is continuously graded with respect to the amount of current applied to the presynaptic interneurone. Two examples of such effects are shown, the first of an interneurone that depolarizes a motor neurone (Fig. 5), the second of an interneurone that hyperpolarizes a different motor neurone (Fig. 6). At low current strengths the post-synaptic response to stimulation of an interneurone is just discernible above the background of other

240 M. BURROWS AND M. V. S. SIEGLER synaptic inputs to the motor neurone (Figs. 5A, 6A), but can be accentuated by signal averaging. A certain amount of current must be injected into some interneurones before there is a measurable effect in the post-synaptic motor neurone. For example in Fig. 5 almost 1 nA is necessary to produce a detectable post-synaptic effect. A

int. inn.

Current

+3_ _~~~~~~~~~~~ E e*

4-1

+2

C.,

M

C

o~~~~~d

4-1

Presynapticcurn~A E~~~~~~ ~~~ ~~~~~

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-

Fig. 5. Graded synaptic transmission from a non-spiking interneurone that depokarize8 a motor neurone. The motor neurone is the lateral fast flexor tibiae and the recordings are from its soma (A-E). Depolarizing current (3rd trace) injected into a neuropilar process of the interneurone (int., 1st trace) causes graded depolarizations of the motor neurone (inn., 2nd trace), which spikes (arrow in E) when sufficiently depolarized. The bridge circuit is unbalanced in D and E. F, the relationship between the amount of current injected into the intemneurone and the resulting change in potential of the motor neurone. The letters on the graph correspond to the records on the left. Calibration: current 29 nA; voltage, motor neurone 3 mV, interneurone 33 mV.

The more current injected into an interneurone, the greater is the change in the membrane potential of the motor neurone (Figs. 5B-E, 6B-E). A depolarization can be sufficient to evoke a spike (Fig. 5E), or a series of spikes whose frequency can be accurately adjusted by varying the level of presynaptic current. As a result, co-ordinated movements of the leg can occur similar to those in normal behaviour. Typically the relationship between presynaptic current and post-synaptic voltage is approximately linear for currents up to 5 nA (Figs. SF, 6F). With larger currents,

GRADED S YNAPTIC TRANSMISSION 241 the post-synaptic voltage reaches a plateau probably due to it reaching the equilibrium potential (Fig. 6F). The peak change in the post-synaptic potential is maintained throughout a 700 msec pulse injected into the presynaptic interneurone. A

int. mn.

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Fig. 6. Graded synaptic transmission from a non-spiking interneurone that hyperpolarizes a motor neurone. The motor neurone innervates branch d of the tergotrochanteral muscle, that elevates the hind wing during flight and depresses the trochanter of the hind leg during walking. Recordings are from its soma. A-E, depolarizing current (3rd trace) injected into a neuropilar process of the interneurone (int., 1st trace) causes graded hyperpolarizations of the motor neurone (mn., 2nd trace). F, the relationship between the amount of depolarizing current injected into the interneurone and the resulting hyperpolarization of the motor neurone. Hyperpolarizing current injected into the interneurone results in a depolarization of the motor neurone, indicating that at rest the motor neurone is held continuously hyperpolarized by this interneurone. The letters on the graph correspond to the records on the left. Calibration: current 29A; voltage, motor neurone 3mV, interneurone 33 mV. some motor neurones the potential can be maintained without decrement during 2-3 sec pulses. With still longer pulses lasting several minutes, there is a gradual

In

decline in the amplitude of the post-synaptic potential. Nevertheless, after 7 min of maintained depolarization of the interneurone, there is still a post-synaptic effect.

242

M. BURROWS AND M. V. S. SIEGLER

Chemical synaptic transmission The transmission between all combinations of non-spiking interneurones and motor neurones so far encountered is thought to be mediated by chemical and not electrical synapses, for the following reasons. First, there is no change in the membrane potential of a presynaptic interneurone when a post-synaptic motor neurone is hyperpolarized or depolarized by injection of current into its soma or into one of its neuropilar processes, as would be expected if the synapse were electrical. Furthermore, spikes in a motor neurone are not directly reflected into a presynaptic interneurone. However, spikes in a motor neurone can activate pathways that evoke synaptic potentials in interneurones. Secondly, the amplitude of a post-synaptic potential, evoked by stimulation of a non-spiking interneurone, is dependent upon the membrane potential of the motor neurone. For hyperpolarizing potentials, a reversal potential can be demonstrated. In one example an interneurone is repeatedly depolarized with 300 msec long pulses of constant current, which hyperpolarize the motor neurone (Fig. 7A). At the same time, the membrane potential of the motor neurone is independently manipulated by the injection of current. When the motor neurone is depolarized the amplitude of the evoked hyperpolarization increases. Conversely, when the motor neurone is progressively hyperpolarized, the evoked hyperpolarization decreases in amplitude, until at an apparent reversal potential there is no voltage change (Fig. 7 A). Hyperpolarizing the motor neurone further results in a reversal of the polarity of the evoked post-synaptic potential. Similarly, a depolarizing post-synaptic potential in the motor neurone, evoked by stimulation of a non-spiking interneurone, is accentuated by hyperpolarization of the motor neurone, and diminished by depolarization. These changes in the size of a post-synaptic potential are expected if the membrane potential of the motor neurone is being altered relative to the equilibrium potential of a chemically mediated p.s.p. Thirdly, known chemical e.p.s.p.s in motor neurones are decreased in amplitude when they occur at the same time as a post-synaptic potential of opposite sign evoked by a non-spiking interneurone. In one example, a spike in the fast extensor tibiae motor neurone evokes a compound e.p.s.p. in a fast flexor tibiae motor neurone (Fig. 7Bi; Hoyle & Burrows, 1973; Burrows & Horridge, 1974). A 600 msec long depolarizing pulse of current is delivered to an interneurone that hyperpolarizes the flexor motor neurone and is timed to begin 100 msec before the compound e.p.s.p. (Fig. 7Bii). As the amplitude of the hyperpolarization of the motor neurone increases, there is a decrease in the size of the compound e.p.s.p. (Fig. 7Biii, iv). This 'shunting' of the e.p.s.p. would be expected if the non-spiking interneurone evokes a conductance change in the motor neurone. It would not be expected if the hyperpolarization of the motor neurone were electrotonic in origin. The effect is not caused by rectification of the motor neurone: changing the membrane potential of the motor neurone to the same level by the direct injection of current increases rather than diminishes the amplitude of the e.p.s.p. Fourthly, injection of brief depolarizing pulses of current into non-spiking interneurones evokes changes in the membrane potential of post-synaptic motor neurones

GRADED SYNAPTIC TRANSMISSION 243 that far outlast the stimuli. A 12 msec pulse that depolarizes a non-spiking neurone evokes a depolarization of a flexor motor neurone and sometimes a spike (Fig. 8Ai, ii). The time course of the depolarization is seen more clearly in averaged records A

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Fig. 7. Evidence that graded transmission is chemically mediated. A, injection of a 300 msec constant current pulse of 5.6 nA, into the neuropilar process of an interneurone evokes an hyperpolarization of a tergotrochanteral motor neurons. Direct current, shown in the left-hand scale, is then injected into the soma of the motor neurone to alter its membrane potential. Depolarization of the motor neurone accentuates the evoked hyperpolarization, and hyperpolarization reduces it, eventually resulting in a reversal of polarity. B (i), a compound e.p.s.p. in the lateral fast flexor tibiae motor neurone (flex. mn., 2nd trace) results from activation of a central pathway when an antidromic spike occurs in the fast extensor tibiae motor neurone (ext. mn., 3rd trace). (ii-iv), increasing amounts of current (4th trace) are injected into a neuropilar process of an interneurone (int., 1st trace) that hyperpolarizes the flexor motor neurone, whereupon the amplitude of the e.p.s.p. is decreased. Calibration: (A) 3 6 mV; (B) interneurone 33 mV, flexor motor neurone 6 mV, extensor motor neurone 14 mV; current (A) 28 nA, (B) 66 nA.

(Fig. 8A iii). The potential rises to a peak in 15-20 msec, and has a duration of 70-80 msec. These long-lasting potentials are not the result of charging the membrane of the motor neurone by an electrotonic junction, since similar pulses, injected directly into the motor neurone evoke changes in potential that reach their peak

M. BURROWS AND MI. V. S. SIEGLER 244 during the stimulus, and have a much shorter decay time. A sustained effect of chemical transmitter is therefore implicated. Fifthly, the depolarization of a motor neurone evoked by a non-spiking interneurone is often characterized by an increase in the number of voltage fluctuations in the motor neurone. The more the motor neurone is depolarized, the more frequent are the voltage fluctuations. If the motor neurone is depolarized to the same level by A

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Fig. 11. The current-voltage relationship in two interneurones. Two electrodes, linearly separated by 200 #sm at their tips, were placed in neuropilar processes of the same interneurone. A, an interneurone that excites the slow extensor tibiae motor neurone to cause a slow extension of the tibia. Open circles represent the voltage change recorded by the first electrode when current was passed through the second; closed circles the reverse. B, an interneurone that excites many flexor tibiae motor neurones to cause a strong flexion of the tibia. The dotted lines indicate the voltage change necessary in the interneurone to effect a change in the frequency of spikes in the post-synaptic motor neurones.

electrode. Measurements at low current strengths when the bridge was balanced at the stimulating electrode, indicate that the voltage is attenuated by 60-70 % between the two electrodes. They were placed about 200 gm apart in the neuropile, though the intracellular distance this represents is not known. From similar bridgebalanced records, on the linear portion of the curve, the input resistances of these non-spiking interneurones are estimated to be 3-5 MU. This is no more than a minimum value, since it is measured in neurones with considerable background synaptic inputs. Hyperpolarization of the two interneurones had no effect on post-synaptic motor neurones (Fig. 11). In both interneurones, a depolarization of only about 2 mV, measured at the recording electrode, was sufficient to change the frequency of spikes of post-synaptic motor neurones (Fig. 11). At the stimulating electrode, it took 2 nA of current to effect this change. Were a more sensitive measure of the post-synaptic response to have been used, the voltage change in the interneurone

249 GRADED SYNAPTIC TRANSMISSION necessary to effect transmission would be seen to be lower. In interneurones with similar post-synaptic effects in other experiments (e.g. Figs. 5, 6), 2 nA of current caused a post-synaptic change of about 2 mV at the soma of a motor neurone.

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Fig. 12. Voltage changes in interneurones during behaviour should be adequate to effect the graded release of transmitter. Movements of the hind legs were elicited by visual or tactile stimulation, and recordings made simultaneously from the soma of a motor neurone (2nd trace) and from a neuropilar process of an interneurone (1st trace) presynaptic to it. There are corresponding voltage changes in the interneurone and the motor neurone. A, an interneurone that when depolarized results in a depolarization of the lateral fast flexor tibiae motor neurone. Depolarizations occur in both during evoked flexions of the tibia (arrows). B, an interneurone that when depolarized results in an hyperpolarization of the same flexor motor neurone. Repolarization of the flexor after each evoked flexion of the tibia (arrows) is preceded by a depolarization of the interneurone. C, an interneurone that when depolarized results in an hyperpolarization of a coxal motor neurone. Evoked movements are accompanied by a depolarization of the interneurone and a concomitant hyperpolarization of the motor neurone. Myograms from the flexor tibiae muscle (A, B) and from the coxa (C) are on the third traces. Calibration: (A) 8 mV, (B) 14 mV, (C) 7 mV.

Voltage ch2nge8 in interneurones during behaviour To establish a behavioural role for non-spiking interneurones it is necessary to show that during behaviour they undergo voltage fluctuations large enough to influence the rate of transmitter release. In non-spiking interneurones of quiescent locusts, individual synaptic potentials can be as large as 5 mV. During active movements of the legs, the membrane potentials of the interneurones may fluctuate

M. BURROWS AND M. V. S. SIEGLER 250 by as much as 15 mV (Fig. 12). Moreover, the fluctuations in the membrane potential of the interneurones correspond with those of post-synaptic motor neurones. For example, depolarization of one interneurone depolarizes a motor neurone; during active movements of the hind legs, there are fluctuations of the same polarity in the two, with depolarization of the interneurone preceding that of the motor neurone (Fig. 12A). Depolarization of other interneurones causes hyperpolarization of some motor neurones; during active movements there are fluctuations of opposite polarities in an interneurone and a motor neurone, with depolarization of the interneurone preceding the hyperpolarization of the motor neurone (Fig. 12B, C). Since individual motor neurones receive synaptic inputs from many sources, an exact correspondence between the wave forms in the two neurones is not to be expected. Therefore it is not possible to say to what extent the post-synaptic potential is causally related to the presynaptic one; any one interneurone will only be partly responsible for the changes in membrane potential of a particular motor neurone. Nevertheless, the voltage changes in the interneurones are far in excess of those estimated to be necessary to effect graded synaptic transmission. DISCUSSION

Local interneurones within the metathoracic ganglion of the locust can effect graded changes in the membrane potential of post-synaptic motor neurones without themselves producing spikes. The non-spiking state of these neurones appears to be their normal mode of functioning and not the result of the experimental conditions. When depolarized by injected current, some interneurones effect post-synaptic depolarization, while others effect post-synaptic hyperpolarization. These postsynaptic changes can be of sufficient magnitude either to evoke spikes in motor neurones, or to suppress spikes effected by other synaptic inputs. By several different electrophysiological tests it has been shown that the graded post-synaptic effects are mediated by chemical synaptic transmission. It is estimated that changes of only a few millivolts in membrane potential of the interneurones are sufficient to change the rate of release of transmitter. Therefore, in these non-spiking interneurones, summed p.s.p.s, or even single p.s.p.s, are expected to be the normal electrical signals effecting transmitter release. The physiological state of the non-spiking neurones Some possible causes for the suppression of spikes in the interneurones during the course of the experimental procedure have been eliminated. First, penetration of a non-spiking neurone does not appear to inflict damage. There is no shift in the membrane potential of a post-synaptic neurone that would indicate that the rate of transmitter release from the interneurone has been changed. The membrane potential of the non-spiking neurone remains stable for as long as the penetration is maintained, and this can sometimes exceed 1 hr. Secondly, adequate oxygenation of the ganglion makes it unlikely that hypoxia occurs with its concomitant effects upon the metabolism of the neurones. Thirdly, raising the body temperature of the locust from 18 to 35 0C does not qualitatively affect the properties of the nonspiking neurones. At the higher temperature (35 0C), the neurones still do not

GRADED SYNAPTIC TRANSMISSION

251 produce spikes, but effect graded changes in the membrane potential of motor neurones. The morphology of the neurones identified physiologically as non-spiking is consistent; all neurones that did not spike were seen to be local, intraganglionic interneurones. It seems unlikely that such a consistent picture would be obtained if the non-spiking state were the result of damage inflicted by the micro-electrode upon any neurone penetrated in the neuropile. Of course the above tests do not exclude the possibility that under some as yet untested conditions, the interneurones might be able to spike. Nonetheless, these neurones are able to perform integration without spikes under conditions that are, as far as we are able to determine, entirely physiological. This conclusion is further supported by the observation that the behaviour of the dissected locust is very similar to that of the intact locust. For example, the frequency of spikes in identified tonic motor neurones that maintain posture, and the changes of frequency that occur in these neurones during resistance reflexes, are the same in both the dissected preparation and the normal locust. Even complex motor patterns such as those that bring about jumping and kicking are indistinguishable in the two situations (Heitler & Burrows, 1977; Pfluger & Burrows, 1978). All of the identified motor neurones involved in these movements are known to be affected by nonspiking interneurones. Throughout 4 hr long experiments, the locust continues to make co-ordinated movements in response to visual, tactile and auditory stimuli. At the end of many experiments, a locust once released will walk or jump from the experimental dish. It seems likely that such directed movements could only be performed by an animal whose nervous system is in a normal physiological state.

Input current and output voltage relationships of interneurones and motor neurones Interneurones have been studied that depolarize or hyperpolarize tonically spiking motor neurones, such as those which maintain posture, and phasically spiking ones, such as those used only in flying or jumping. Despite these differences, the curves that relate the strength of current injected into an interneurone to the change in potential in a motor neurone, have the following features in common (see Figs. 5, 6 and 10). In the region where an interneurone is most hyperpolarized, small current steps effect no apparent change in the post-synaptic membrane potential. As the interneurone is depolarized, there is an inflection in the curve, before it enters a region where there is a linear relationship between presynaptic current and post-synaptic voltage. The linear region is the steepest portion of the curve, and by inference, it is the region where changes in presynaptic voltage are most effective in releasing transmitter. Finally, with further depolarization of the interneurone, the curve reaches a plateau where changes in presynaptic current produce no further change in post-synaptic voltage. This plateau can be attributed to the rectification of the interneurone (cf. Fig. 11) and to the decrease in size of the post-synaptic potential as it approaches its equilibrium potential. The input current-output voltage curves presented here are similar in shape to those relating pre- and post-synaptic voltages for synapses between spiking neurones where spikes have been blocked by TTX (Katz & Miledi, 1966, 1967; Martin & Ringham, 1975; Charlton & Atwood, 1977), and for graded transmission between

M. BURROWS AND M. V. S. SIEGLER 252 neurones in the lobster stomatogastric ganglion (Graubard et al. 1977). It is possible therefore that all chemical synapses, whether activated by spikes or not, have similar curves relating input voltage to output voltage. The low resting potentials of non-spiking interneurones in the locust can then be seen as a device for shifting the neurone near, or into, the linear portion of the release curve. Small voltage fluctuations such as those caused by p.s.p.s would then be able to influence the rate of transmitter release.

Membrane potentials of interneurones and transmitter release No appreciable amount of transmitter is released from some interneurones at their 'resting' membrane potential. In others sufficient transmitter is released to have a detectable postsynaptic effect. Hyperpolarizing current injected into these latter interneurones evokes post-synaptic potentials which are of opposite polarity to those evoked by depolarizing current. At a given time, which interneurones are 'quiescent', and which are releasing significant amounts of transmitter, could depend upon diverse factors, such as the posture, state of arousal, and sensory inputs to the locust. When, for example, the set position of the hind leg is changed, the membrane potentials of some interneurones are shifted by up to 10 mV to new maintained levels. It is likely that such changes in membrane potential would be sufficient to shift the interneurones to, or from, the tonically releasing state. Therefore, in these interneurones at least, and possibly all non-spiking interneurones, the 'resting potential', and the input current-output voltage relationship depend upon the behavioural context in which they are measured. Are connexions between interneurones and motor neurones monosynaptic ? Synaptic delays between non-spiking interneurones and motor neurones in the locust vary widely. They can be as short as 1-2 msec, comparable to delays at presumed monosynaptic connexions between spiking neurones within the meta-

thoracic ganglion (Fig. 1 and Burrows, 1975), or as long as 12-5 msec. However, care must be taken in inferring from these data that the connexions are, or are not, monosynaptic. To judge whether connexions between spiking neurones are monosynaptic, a number of different physiological measures can be made (see Berry & Pentreath, 1976 for a review). But these measures fail to indicate whether connexions between non-spiking interneurones and motor neurones are monosynaptic. For example, tests that rely on the ability of the presynaptic neurones to produce spikes, such as the following frequency of p.s.p.s to spikes, clearly cannot be used. Nevertheless, it is unlikely that spiking neurones are interposed between the nonspiking neurones and motor neurones studied here. Graded changes in presynaptic voltage evoke graded changes in post-synaptic voltage, with no discontinuities as might be expected if an interposed interneurone had spiked. This is true even for stimuli as brief as a single action potential. But it cannot necessarily be concluded from this that the connexions between the interneurones and motor neurones are monosynaptic, because some non-spiking interneurones also interact amongst themselves by graded chemical transmission (M. Burrows, in preparation). It is therefore possible that some of the post-synaptic effects of stimulating a single interneurone may arise by graded transmission through an interacting network of non-spiking

GRADED SYNAPTIC TRANSMISSION 253 interneurones. This problem is of more general significance, for similar arguments would apply to supposedly monosynaptic connexions between spiking neurones: available tests would fail to reveal whether a non-spiking neurone, or other neurones using graded chemical transmission were interposed. The possibility also remains that graded electrical interactions could be involved as well, although no such connexions have been revealed so far. The difficulty of detecting interposed electrical connexions has been discussed by Berry & Pentreath (1976) with regard to spiking neurones; similar problems obtain when investigating the connexions between non-

spiking neurones. Concluding remarks While one of the notable features of the interneurones here described in the locust is that they do not produce action potentials, it is instead their capability for graded synaptic transmission that should be emphasized. A simple division of neurones into 'spiking' and 'non-spiking' may lead to the perhaps erroneous conclusion that there is a similar dichotomy of synaptic function. Even in spiking neurones, it is apparently not the action potential per se, but changes in membrane potential that constitute the electrical signal for transmitter release. This is implicit from studies on spiking neurones showing that the rate of transmitter release can be altered by depolarization of synapses, in the absence of spikes (e.g. del Castillo & Katz, 1954). Of course, in most spiking neurones, under normal circumstances, transmitter release is closely associated with the occurrence of action potentials. Exceptions have, however, been found. For example, receptors in the dragonfly ocellus respond to light with a graded depolarizing potential and a superimposed spike. When the spikes are blocked by TTX, the response of post-synaptic neurones is unchanged, suggesting that it is the slow wave which is responsible for synaptic transmission (Chappell & Dowling, 1972). In the lobster stomatogastric ganglion a spiking neurone can also transmit slow changes in membrane potential, which sum with discrete impulse-mediated p.s.p.s (Maynard & Walton, 1975). In the vertebrate olfactory bulb, analysis of extracellular field potentials has provided indirect evidence that neurones which generate spikes may also, in some regions, release transmitter in response to graded and local inputs (Rall & Shepherd, 1968). This general theoretical proposition has been argued elsewhere by Ralston (1971), and Shepherd (1974). It would appear that whether a neurone spikes or not tells us how it transmits electrical information internally, from one part to another. But, at synaptic sites, the distinction between spike-engendered changes in membrane potential, and the graded signals of non-spiking neurones, may be one merely of degree. This work was supported by a Nuffield Foundation grant to M.B. REFERENCES

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