Acetylcholine receptors on Renshaw cells of the rat

European Journal of Pharmacology 30 (1975) 252-259 © North-Holland Publishing Company

ACETYLCHOLINE

RECEPTORS

ON RENSHAW

CELLS OF THE RAT

P. Max H E A D L E Y t , David LODGE* and Tim J. BISCOE Department of Physiology, University of Bristol, Medical School, Bristol BS8 1TD, England

Received 5 June 1974, revised MS received 23 September 1974, accepted 8 November 1974 P.M. HEADLEY, D. LODGE and T.J. BISCOE, Acetylcholine receptors on Renshaw cells o f the rat, European J. Pharmacol. 30 (1975) 252-259. Experiments have been performed on Renshaw cells of rats to investigate the receptors mediating both the responses to electrophoretically applied cholinergic excitants and the synaptic excitation following stimulation of the ventral roots. The effects of atropine and dihydro#3-erythroidine, muscarinic and nicotinic antagonists respectively, have been tested on both types of excitation. Most importantly we found that in the rat the ventral root evoked response can be blocked by atropine as well as by DH~3E. It was not possible to categorise the responses to the cholinergic agents in terms of muscarinic and nicotinic receptors. In addition we have failed to find the late (muscarinic) excitation seen in the cat. Acetylcholine

Renshaw cells

1. Introduction The pharmacology of the cat Renshaw cell recurrent collateral synapse was first investigated by Eccles et al. (1954) and Eccles et al. (1956) using close intraarterial injection methods. Their findings were subsequently confirmed and extended by a number of workers using the microelectrophoretic technique (Curtis and Eccles, 1958; Curtis et al., 1961; Curtis and Ryall, 1966a,b,c). In short, the experiments showed that there were two types of receptor-mediating excitatory responses to electrophoretically applied acetylcholine (ACh); the first was nicotinic and was blocked by dihydro-/3-erythroidine which also depressed the early excitation following ventral root (VR) stimulation; the second was muscarinic and was blocked by atropine which also depressed a longer latency excitatory effect of VR stimulation. Many other details of the responses of these cells were ext Wellcome Trust Research Training Scholar. * Animal Health Trust/Wellcome Trust Research Fellow; present address: Department of Pharmacology, John Curtin School of Medical Research, A.N.U., Canberra, Australia.

Atropine

DHt3E

amined by the aforementioned workers and by others (e.g. Kuno and Rudomin, 1966, on ACh release; Quastel and Curtis, 1965, on the effect of hemicholinium). Much of the evidence was reviewed and the independent existence of the Renshaw cell defended by Eccles at the Federation Meetings in 1968 (see Eccles, 1969). We have been experimenting on the spinal cord of the rat and here report differences between the pharmacology of rat and cat Renshaw cells.

2. Materials and methods Adult male rats 3 5 0 - 5 0 0 g were anaesthetised with sodium pentobarbitone, 50 mg/kg i.p.; anaesthesia was maintained with further i.v. doses o f the same drug. The trachea was cannulated and the animal artificially ventilated with humidified oxygen following the administration o f gallamine triethiodide 10 mg/kg i.v. The temperature o f the rat was monitored and kept at 3 7 - 3 8 ° C with a thermistor-controlled heating blanket. A laminectomy was performed extending from L1 to L6 and skin flaps were raised making a

P.M. Headley et al., Rat Renshaw cell ACh receptors

pool which retained liquid paraffin sufficient to cover the spinal cord. The ventral roots (VR) of segments L 4, 5 and 6 were cut and placed on silver stimulating electrodes within this pool. Neurone potentials were recorded from the centre 4 M NaCl-filled barrel of 7 barrel microelectrodes having a total tip diameter of 4 - 7 /am. The outer barrels were filled with various combinations of the following drugs: D,L-hom ocysteate Na, DLH, (KochLight Labs.), 200 raM, pH 8; acetylcholine chloride, ACh, (BDH), 500 mM; carbamylcholine chloride, (BDH), 100 mM; nicotine bitartrate, (BDH), 100 mM; acetyl-/3-methylcholine bromide, (Koch-Light Labs.), 500 raM; atropine sulphate, (Koch-Light Labs.), 10 mM in 165 mM NaC1; dihydro-/~-erythroidine, DH/3E, (Merck, Sharpe & Dohme), 100 raM. The drugs were expelled from the electrode barrels using circuits similar to those described by Curtis (1964), and were retained by applying 0.5 V to each barrel. Renshaw cell action potentials were recorded by conventional means on film. Their frequency was monitored with a ratemeter and recorded continuously on a potentiometric chart recorder. Poststimulus histograms of VR responses were prepared using a Biomac 500.

3. Results 3.1. General

Renshaw cells were excited by stimulation of the ventral roots of the 4th, 5th or 6th lumbar segments. Groups of cells were located from the position of the maximum Renshaw cell field potential, commonly known as the Renshaw ripple. Exploration in this region led to the isolation of single units, which were recognised by their characteristic high frequency discharge following-VR stimulation. Often the first and sometimes the second spike were superimposed upon the field potential generated by antidromic invasion of motoneurones. Two types of experiment were carried out concurrently: in one, the responses of the cells to various electrophoretically applied excitants and acetylcholine antagonists were tested; in the other, the effects of the antagonists upon the synaptic responses evoked by VR stimulation were examined.

253

In the rat this synaptic response is a brief 10-30 msec. discharge at a high frequency as is shown in figs. 1 and 3. In this work we have not been able to evoke consistently the late discharge seen in cats by Curtis and Ryall (1966c), although in an earlier study on rats (Biscoe et al., 1973) this response was seen sufficiently often to investigate the action of pancuronium. In attempting to evoke the late discharge in the present series of experiments a variety of stimulation patterns were used, ranging from a single stimulus to tetani at 30-300 Hz lasting 3 sec. In this way 8 cells were studied and their responses during 32 successive sweeps averaged in the form of poststimulus histograms. In only one case was there any late increase in the spontaneous, or ACh or DLH maintained discharge rate in the 5 sec period following the stimulus or stimulus train; more commonly the discharge rate was markedly depressed throughout the period of stimulation. We have not therefore been able to compare the pharmacology of such a synaptic response in the rat with the late muscarinic excitation seen by Curtis and Ryall (1966c) in the cat. 3.2. The actions o f dihydro~-erythroidine (DH(3E)

With all 26 Renshaw cells on which it was tested, DH/~E ( 0 - 2 0 nA, mean 7 nA) specifically reduced the excitation induced by ACh ( 0 - 5 2 nA, mean 23 hA) without reducing that caused by DLH (3-72 nA, mean 23 nA). During DH/3E ejection by low currents, there was often an early increase in the response to both excitants. In 7 cells the enhanced effect of DLH persisted even when the ACh effect was considerably .reduced or abolished. Because in many cases the antidromically evoked motoneurone field was large, accurate counting of the early VR evoked spikes using a pulse height selector was difficult. For this reason a concurrent study of the evoked response was undertaken in only 5 of these Renshaw cells. With all 5, the total number of spikes in the 20 msec following a submaximal VR stimulus was reduced during DHCJE ejection. Poststimulus histograms were prepared to show peaks in the probability of firing; these represent the first few synchronised spikes following the VR stimulus. Invariably the latency of these peaks was increased and the synchronisation itself became less pronounced. These changes were greater for the later peaks and

254

P.M. Headley et al., Rat Renshaw cell ACh receptors

were not always present for the first Renshaw spike. Data from one Renshaw cell are shown in fig. 1. The ratemeter record in the right hand column of A shows the control responses of the cell to the alternate ejection of acetylcholine (8 nA) and D,L-homocysteate (6 nA). The middle row of these responses (B), recorded during the ejection of DHflE (4 nA), shows that the effect of acetylcholine was considerably reduced. The break in this record represents the time (2 min) taken to prepare the poststimulus histogram and to photograph the evoked response. The lower

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record, taken a few min after the end of the DHflE ejection, shows the full recovery of the ACh response. Throughout these records the action of DLH was essentially unaltered. The left hand column of fig. 1 shows both poststimulus histograms and typical evoked responses, all obtained concurrently with the ratemeter records. In both the control (A) and the recovery (C) records, there are 6 peaks of probability of firing in the 6 msec following the stimulus artefact (large peak at beginning of each histogram). The small number of counts which occur with a latency of about 1 msec are due to occasional false triggering by the antidromically evoked motoneurone field potential. In the poststimulus histogram recorded when the acetylcholine response was reduced by D I ~ E (B) there are only 4 clearly defined peaks. The latency to each peak is increased and (except for the first) each is spread over a large number of bins. The inset photographed records show this change in latency and also the decrease in the total number of VR evoked spikes during DHflE ejection; the evoked responses were however variable, and poststimulus histograms summing such records were essential for an accurate assessment of drug effects. 3.3. The actions o f atropine

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Fig. 1. The actions of dihydro-~-erythroidine on chemically and synaptically evoked excitant responses. A, control; B, during the ejection of DHflE 4 nA; C, recovery. Righthand column: samples of pen recorder traces showing increases in cell discharge rates in response to electrophoretic ejections of ACh and DLH. The horizontal bar above the top trace represents 1 min. During DHflE the ACh responses are markedly reduced. Centre column: photographs of single oscilloscope sweeps of Renshaw cell responses to ventral root stimulation. During DHflE the number of evoked spikes is reduced, and the latency to each spike is increased. These effects are more clearly seen in the poststimulus histograms of the lefthand column. See text for further description.

The effect of atropine (usually 6 - 3 4 nA, mean 13 nA, but up to 68 nA in the experiment of fig. 2) was studied on the ACh ( 0 - 3 0 nA mean 13 nA) and DLH ( 4 - 2 0 nA, mean 11 nA) responses of 13 Renshaw cells. On all 13 neurones, the effect of ACh was reduced or abolished whereas the effect of DLH was somewhat reduced in only 4; one of these latter is illustrated in fig. 3. The specificity of atropine as an ACh antagonist on most cells was good although probably less than that of DI-I~E. For example, on one cell the current of DHflE which just blocked the ACh response could be increased tenfold without substantially changing the response to DLH, and in a later test the atropine current could be similarly increased fourfold. These latter results are presented in fig. 2B which plots the plateau firing rate in response to the two excitants (ACh and DLH) against time during the ejection of atropine with increasing currents. Some of the problems associated with recording from cells for long periods were encountered in

255

P.M. Headley et al., Rat Renshaw cell ACh receptors

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stage when atropine reduced the response to applied ACh the total number of evoked spikes in a given period following the VR stimulus was reduced and the latency of those spikes was increased, as is illustrated in fig. 3 (same neurone as in fig. 1). The ratemeter records on the right hand side show clearly that the ACh plateau response was reduced from 80 to less than 30 spikes/sec during the ejection o f atropine (14 nA). The DLH response was reduced from 65 to 55 spikes/sec. The photographed records of the VR response show that during atropine ejection the number of VR evoked spikes was reduced. The latency change was more rigorously tested by preparing poststimulus histograms. These histograms, shown in the left hand column of fig. 3, indicate that the latencies of the second and more markedly the third peaks in probability of firing were increased during atropine, fig. 3B.

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Fig. 2. (A) The action of dihydro-#-erythroidine on excitant responses of a Renshaw cell to the electrophoretic application of carbachol and acetyl-O-methylcholine, nicotinic and muscarinic agonists respectively. DH#E blocked the actions of both agonists. The points were taken from ratemeter records. (B) The action of atropine on ACh and DLH responses. Atropine blocked entirely the excitant responses to ACh, whilst not affecting the DLH responses, even with an ejection current 4× that which abolished the ACh excitation.

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. •h these tests. F o r example, during the recovery from atropine the response to DLH declined slowly; no electrode movement was attempted to alter this. The decreased sensitivity to DLH suggests that the incomplete recovery o f the ACh response may have been due to a progressive separation o f the electrode tip from the cell. The effect o f DH/3E tested in the same way was equivalent in every respect to that o f atropine. Atropine modified synaptically evoked responses in a similar way to that described for DH/3E. At a

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Fig. 3. The actions of atropine on chemically and synaptically evoked excitant responses. The layout of the figure is equivalent in every way to fig. 1 ; therefore see fig. 1 and text for further details.

256

P.M. Headley et al., Rat Renshaw eell ACh receptors

3.4. The responses to s o m e cholinomimetics

In view of these results, which are at variance with the documented pharmacology of cat Renshaw cells (e.g. Curtis and Ryall, 1966b,c), an investigation was undertaken in the rat of the effects of DH/~E and atropine on Renshaw cell responses to some cholinomimetics. In all, the effects of the antagonists have been examined on 26 Renshaw cells using acetyl-/3methylcholine (9 cells), nicotine (9 cells) and carbamylcholine (17 cells) as excitants. Acetyl-fl-methylcholine was a weak excitant, and with many cells other than these 9 only unsatisfactory and variable long latency responses were seen. Both nicotine and carbachol, however, were found to be potent excitants, but accurate estimates of their potency relative to acetylcholine could not be made because it was usually difficult to establish plateau responses with these excitants; this was due to a combination of the relatively long latency to the onset of the discharge, to the slow but progressive increase in the firing rate, and to the prolonged recovery phase following termination of the ejecting currents. These problems were particularly marked with nicotine, and explain why carbachol was used as an alternative if less specific nicotinic agonist in many of the experiments. The prolonged recovery phase seen with these two cholinomimetics is in marked contrast with that of ACh, and may reflect, at least in part, the role of cholinesterases in terminating the effect of the natural transmitter; a similar explanation may account for the progressive increase in the excitations caused by these cholinomimetics. The actions of other excitants were diminished if they were ejected too soon after the nicotinic agonists. This was particularly true of ACh but also to a lesser extent of DLH. Curtis and Ryall (1966b) attributed these effects to receptor desensitisation. We often found it necessary to leave a period of more than 1 min after these potent substances before reliable responses to the other excitants could be attained. Under these circumstances, DH/3E and atropine only occasionally specifically reduced the effects of the nicotinic and muscarinic agonists respectively. For instance, with 6 cells atropine was tested on the responses to both nicotine and one or two of the other agonists. With only 2 of these 6 cells did atropine fail to affect the nicotine responses at a stage

when the acetyl-fl-methylcholine or ACh rbsponses were markedly reduced. On another 2 of the 6 cells nicotine and ACh were both reduced whilst the DLH responses were unaffected, although in one of these the ACh responses were affected before those to nicotine. On one of the 6 cells nicotine began to be affected shortly after the acetyl-/3-methylcholine responses were reduced, and on the sixth cell the nicotine responses were reduced whilst those to ACh remained unaffected. Similar results were obtained with carbachol, which was tested with atropine on a further 14 cells. Equivalent tests were performed with DH/3E and acetyl-/3-methylcholine on 8 cells. In no case were acetyl-/3-methylcholine responses unaffected at a stage when nicotine, carbachol or ACh responses were significantly reduced. An example of the action of DH/3E on the excitant responses to carbachol 0 nA, and acetyl-/3-methylcholine 44 nA, is shown in fig. 2A, where the frequencies of the plateau responses were obtained from the ratemeter records. The graph shows that the actions of both excitants were suppressed. Fig. 4 illustrates the action of atropine on responses to carbachol, ACh and DLH. The ratemeter record shows that the responses to ACh and also to carbachol were reduced whilst those to DLH were reversibly increased; alAtropine 10nA

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Fig. 4. Pen recorder trace of the firing of a cell in response to ACh, carbachol and DLH. Atropine almost abolished the ACh responses, markedly reduced the area under the carbachol responses, but increased the responses to DLH.

P.M. Headley et al., Rat Renshaw cell ACh receptors

though the maximum firing frequency attained during the carbachol responses was not markedly reduced from control values, the area under the trace, representing the total number of spikes induced by the carbachol ejection, was affected to a greater degree. Nonetheless, in this trace atropine affected the responses to ACh more than those to carbachol. The record also illustrates that the excitatory responses to carbachol ejection were slower in onset than those to ACh or DLH and also that they extended well beyond the period of drug application. One possible explanation for the lack of muscarinic and nicotinic specificity seen with atropine and Dtt/3E would be that the samples used in the present series of experiments were not pure. In order to test this we have performed three experiments on frog (Rana temporaria) rectus muscle, a preparation on which ACh is known to have a principally nicotinic action. Contractions of the rectus muscle, measured with a traditional kymograph system, were induced by 1.5 X 10 -6 M ACh. Two dose levels of each antagonist were chosen such as to give between 30 and 70% inhibition of the ACh-induced contractions, and from these results the doses causing 50% inhibition were estimated. DI-~E (8 × 10 -8 M) was always at least t25 times as effective, and sometimes 1000 times as effective, as atropine (1.0 X 10 -s M to 7.7 X 10 -s M) in reducing the ACh response to 50% of the mean of the control and recovery values.

4. Discussion These results illustrate differences between the acetylcholine receptors of rat and cat Renshaw cells. Firstly, we have not been able to reliably demonstrate the presence of the muscarinic receptor described by Curtis and Ryall (1966b,c) for the cat. Under none of our present conditions could a late muscarinic type of excitation be regularly evoked though in the recent past Biscoe, Duggan and Lodge (1973) did find a late excitatory response to ventral root excitation in the rat. We have no explanation for this discrepancy but, in view of the greater range of stimulus parameters used, we are now inclined to place greater credence upon the results reported here. One possibility to account for this lack of a late

257

response could be that the preparations had deteriorated by the tfme the tests were performed. We do not think that this can be the case since consistent pharmacological and physiological responses were seen throughout extended recording sessions which, from the commencement of recording, lasted for at least 8 hr and often for much longer. If the animals were deteriorating at the time results were first recorded it does not seem likely that they would survive for so long and still produce consistent results. Alternatively, differences in the depth of anaesthesia between different preparations might account for the discrepancy, but this seems unlikely since similar criteria were used in both series of experiments to assess the level of anaesthesia. A further important difference between the two species (cat and rat) concerns the actions of DH/3E and atropine. The most marked difference is that in the rat both antagonists, when electrophoretically applied, block the ventral root activation of Renshaw cells, whereas in the cat electrophoretically applied atropine usually has no effect until non-specific depression is seen (Curtis and Phillis, 1960), and intravenous atropine has no effect on the early response in doses which abolish the late response (Curtis and Ryall, 1966c). In addition, with rat Renshaw cells both DH43E and atropine selectively block ACh responses. The specificity of this atropine effect is illustrated in fig. 2B. In contrast, in the cat, atropine affects DLH responses soon after affecting the action of ACh (Curtis and Ryall, 1966b; see also Curtis and Phillis, 1960). Furthermore, there are marked species differences in the degree of differential antagonism by atropine and DI-I/3E of the excitant actions of muscarinic and nicotinic agonist respectively. Many of these species differences in the actions of atropine could be explained if our atropine were in some' way impure and so not a specific muscarinic blocker. We do not consider this to be likely on two grounds. Firstly the experiments using frog rectus preparations showed a satisfactory differential effect of atropine and DH/3E. Curtis and Ryall (1966b), working on a similar preparation, found a somewhat greater degree of specificity (in excess of 200 × as against our 125 × or more), but it seems unlikely that this alone could account for the differences between our study in rats and that of Curtis and Ryall (1966b,c) in cats. Secondly, further evidence that our

258

P.M. Headley et al., Rat Renshaw cell A Ch receptors

atropine can act differently to DH/3E lies in experiments performed on the paramedian reticular nucleus of rats (Duggan et al., in press). With this nucleus, excitatory responses to ACh were specifically antagonised by D I ~ E or atropine, but n o t by both. The atropine used in these experiments came from the same source and was made up in the same batch of solutions as those used here. The possibility that rat Renshaw cells possess a mixture of the traditional type of nicotine and muscarinic receptors seems to be excluded by the finding that both DH~E and atropine could entirely block the action of ACh, and also by the fact that DH~E could block the action of acetyl-/3-methylcholine. We therefore feel bound to conclude that there is a species difference between cat and rat Renshaw cell ACh receptors. We have no explanation for this difference except to point out that purely nicotine or muscarinic actions by ACh seem to be the exception rather than the rule in the cat central nervous system; purely nicotinic responses have been seen only with cat Renshaw cells, and muscarinic responses in the neocortex and hippocampus (Krnjevic" and Phillis, 1963; Crawford and Curtis, 1966; Biscoe and Straughan, 1966). In other sites ACh receptors have been judged to be intermediate, on the bases of the time course of ACh excitation, the responses to cholinomimetics and the effects of antagonists. However, the interpretation of such results is somewhat compromised by the almost universal lack of tests of the interactions of nicotinic and muscarinic agonists and antagonists on the same cell; for instance, apparently only Legge et al. (1966) have compared directly the actions of atropine and DH~E, in this case on cells in the pyriform cortex. We have attempted to perform such comprehensive tests on rat Renshaw cells, and these do clearly indicate that the ACh receptors at this site cannot be categorised in the traditional manner. It is perhaps relevant to note that atropine does have curare-like effects at the neuromuscular junction. Abdon (1940) showed some effects similar to curare. Beranek and Vyskogil (1967) subsequently demonstrated that atropine reduced the magnitude of end plate potentials (e.p.p.s) in rat diaphragm;it was also shown to shorten the time course and decrease the amplitude of e.p.p.s (Beranek and Vysko~il, 1968; Kordas, 1968) and elementary depolarizations

(Katz and Miledi, 1973) in frog muscle. In addition, according to Hubbard and Wilson (1970), Potapova (1969) found that atropine shifted the equilibrium potential for the e.p.p, towards the sodium equilibrium potential. Hubbard and Wilson (1970) subsequently showed that there were pre- and post-synaptic effects of atropine at the neuromuscular junction although Katz and Miledi (1973) demonstrated qualitative as well as quantitative differences between atropine and curare. The above work has established that atropine has effects at the neuromuscular junction in rat and frog even though in all cases atropine is at least 100 times less effective than curare. Our findings suggest that it may well be the case that at recurrent collateral synapses on Renshaw cells of rats, this action of atropine is relatively much more marked than in the cat. Lastly, these experiments cast further doubt upon the validity of the classic distinction between muscarinic and nicotinic actions of acetylcheline as applied to the central nervous system. Acknowledgements We are grateful to Merck, Sharpe & Dohme for a gift of dihydro-/3-erythroidine, to Miss E. Thornton, Mr. K. Caddy and to Mrs. A. Crouch for technical assistance, and to the Animal Health and Wellcome Trusts for grants towards recurrent costs.

References Abdon, N.O., 1940, On the influence of atropine on some nicotine-hke actions of acetylcholine, Acta Physiol. Scand. 1, 153. Beranek, R. and F. Vysko~il, 1967, The action of tubocurarine and atropine on the normal and denervated rat diaphragm, J. Physiol. 188, 53, Beranek, R. and F. Vysko6il, 1968, The effect of atropine on the frog sartorius neuromuscular junction, J. Physiol. 195, 493. Biscoe, T.J., A.W. Duggan and D. Lodge, 1973, Actions of pancuronium on Renshaw cells of the rat spinal cord, Comp. Gen. Pharmacol. 4, 179. Biscoe, T.J. and D.W. Straughan, 1966, Micro-electrophoretic studies of neurones in the cat hippocampus, J. Physiol. 183, 341. Crawford, J.M. and D.R. Curtis, 1966, Pharmacological studies on fefine Betz cells, J. Physiol. 186, 121. Curtis, D.R., 1964, Microelectrophoresis, in: Physical Techniques in Biological Research, Vol. 5, ed. W.L. Nastuk (Academic Press, New York) p. 144.

P.M. Headley et al., Rat Renshaw ceU ACh receptors Curtis, D.R. and R.M. Eccles, 1958, The excitation of Renshaw cells by pharmacological agents applied electrophoretically, J. Physiol. 141,435. Curtis, D.R. and J.W. Phillis, 1960, The action of procaine and atropine on spinal neurones, J. Physiol. 153, 17. Curtis, D.R., J.W. Phillis and J.C. Watkins, 1961, Cholinergic and non-cholinergic transmission in the mammalian spinal cord, J. Physiol. 158, 296. Curtis, D.R. and R.W. Ryall, 1966a, The excitation of Renshaw ceils by cholinomimetics, Exptl. Brain Res. 2, 49. Curtis, D.R. and R.W. Ryall, 1966b, The acetylcholine receptors of Renshaw cells, Exptl. Brain Res. 2, 66. Curtis, D.R. and R.W. Ryall, 1966c, The synaptic excitation of Renshaw ceils, Exptl. Brain Res. 2, 81. Duggan, A.W., P.M. Headley and D. Lodge, Acetylcholine sensitive cells in the caudal medulla of the rat: distribution, pharmacology and effects of pentobarbitone, Brit. J. Pharmacol. (in press). Eccles, J.C., 1969, Historical Introduction, Federation Proc. 28, 90. Eccles, J.C., R.M. Eccles and P. Fatt, 1956, Pharmacological investigations on a central synapse operated by acetylcholine, J. Physiol. 131,154. Eccles, J.C., P. Fatt and K. Koketsu, 1954, Cholinergic and

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inhibitory synapses in a pathway from motor-axon collaterals to motoneurones, J. Physiol. 126,524. Hubbard, J.l. and D.F. Wilson, 1970, Rbduction of the quantum content of endplate potentials by atropine, Experientia 26, 1234. Katz, B. and R. Miledi, 1973, The effect of atropinc on acetylcholine action at the neuromuscular junction, Proc. R. Soc. Lond. B. 184, 221. Kordas, M., 1968, The effect of atropine and curarine on the time course of the end-plate potential in frog sartorius muscle, Intern. J. Neuropharmacol. 7, 523. Krnjevi6, K. and J.W. Phillis, 1963, Pharmacological properties of acetylcholine-sensitive cells in the cerebral cortex, J. Physiol. 166,328. Kuno, M. and P. Rudomin, 1966, The release of acetylcholine from the spinal cord of the cat by antidromic stimulation of motor nerves, J. Physiol. 187, 177. Legge, K.F., M. Randi6 and D.W. Straughan, 1966, The pharmacology of neurones in the pyriform cortex, Brit. J. Pharmacol. Chemotherap. 26, 87. Potopova, T.V., 1969, Potentsialy ravnovesiia toka kontsevoi plastinki myshtsy liagushki pri deistvii prozerina i atropina, Biofizika 14, 757. Quastel, D.M.J. and D.R. Curtis, 1965, A central action of hemichollnium, Nature, London 208, 192.