Nicotinic and muscarinic ACh receptors in rhythmically

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1 MS 2517, pp Journal of Physiology (1994), Nicotinic and muscarinic Ch receptors in rhythmically active spinal neurones in the Xenopus laevis embryo Ray Perrins and lan Roberts School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG 1. Intracellular recordings were made from presumed motoneurones in the Xenopus embryo spinal cord, and their response to cholinergic agents was investigated. Nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP; both 1-1 /M) strongly depolarized, and muscarine and oxotremorine (2-2,uM) weakly hyperpolarized, these neurones. Tetrodotoxin (1 um), which blocks action potentials in Xenopus neurones, did not affect either of these responses. 2. The extrapolated reversal potential of the nicotinic depolarization was mv (mean + S.E.M.) suggesting the opening of a mixed conductance. The nicotinic response was antagonized by dihydro-fl-erythroidine, d-tubocurarine and mecamylamine (1-2 4M) but not by a-bungarotoxin (1 /M). 3. The muscarinic response was not reversed when recorded with electrodes filled with potassium chloride but was antagonized by atropine ( 1/uM). 4. cetylcholine (Ch, 1/uM) caused a strong depolarization of the neurones which was blocked by d-tubocurarine and dihydro-/3-erythroidine, suggesting that its effects are mediated predominantly by nicotinic Ch receptors. 5. Ch and nicotinic agonists applied to the spinal cord produced a barrage of IPSPs that were blocked by TTX and strychnine. cetylcholine (Ch) is an important vertebrate neurotransmitter in both the central and peripheral nervous system acting at two main types of receptor, the nicotinic and muscarinic Ch receptors (nchrs and mchrs, respectively). The properties and roles of both receptor types have been extensively studied in the peripheral nervous system but less is known about these factors in the central nervous system, although they have been discovered in a wide variety of sites including motoneurones (Wang, Neuman & Bieger, 1991), the mesencephalon (Lacey, Calabresi & North, 199), the hippocampus (Halliwell, 199) and the cerebral cortex (McCormick & Prince, 1987). The best studied example of the central action of naturally released Ch is in the mammalian spinal cord where recurrent collaterals from motoneurone axons make excitatory cholinergic connections to the inhibitory Renshaw cells (Eccles, Fatt & Koketsu, 1954). However, there is still much debate over the actual function of this connection (Noga, Shefchyk, Jamal & Jordan, 1987). The hatchling Xenopus embryo has a simple spinal cord with only eight classes of anatomically defined neurone (Roberts & Clarke, 1982). The way in which these neurones produce the embryo's swimming behaviour is now sufficiently well understood to form a working model of the spinal network (Roberts, 199; Roberts & Tunstall, 199). The presence on motoneurones of receptors to the neurotransmitters glycine (Soffe, 1987), y-aminobutyric acid (GB; Soffe, 1987), 5-hydroxytryptamine (5-HT; Sillar, Wedderburn & Simmers, 1992) and an excitatory amino acid (Dale & Roberts, 1984) has been established. Since all of these major central neurotransmitter receptors have been found we thought it likely that ChRs might also be present. Previous studies in the developing Xenopus embryo have shown that there are no Ch receptors on the sensory Rohon-Beard neurones (Bixby & Spitzer, 1982), but that some cultured spinal neurones, possibly motoneurones, are depolarized by Ch at later stages of development (Bixby & Spitzer, 1984). The discovery of ChRs on Xenopus spinal neurones would be of interest in two ways. Firstly, the properties of the receptors themselves could be studied in great detail since there now exists a technique for dissociating the spinal neurones, making them available for study using patch-clamp techniques (Dale, 1991). Because some classes of neurone, such as motoneurones and inhibitory commissural interneurones, can be identified after dissociation this would provide a rare opportunity for the study of central ChRs and currents in identified neurones. Secondly, possible functions of these receptors during locomotor behaviour should be more easy to elucidate in the simple Xenopus system than in more complex adult vertebrates. Preliminary work has shown an excitatory effect of Ch

2 222 R. Perrins and. Roberts J. Physiol on the Xenopus spinal cord recorded both extracellularly (Panchin, Perrins & Roberts, 1991) and intracellularly (Perrins & Roberts, 1992). METHODS Recordings were made from hatchling embryos of Xenopus laevis (developmental stage 37/38; Nieuwkoop & Faber, 1956). Experiments were carried out at C in a saline solution of composition (mm): NaCl, 115; KCl, 3; CaCl2, 4; MgCl2, 1; NaHCO3, 2-4; N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes), 1, at ph 7-4. nimals were anaesthetized in a 41% solution of 3-aminobenzoic acid ethyl ester (MS-222) in saline solution and the dorsal fin slit to allow access of the neuromuscular blocker a-bungarotoxin (1 um). The embryos were left in a-bungarotoxin until they no longer swam in response to normally effective stimuli. The embryos were then pinned to a rotatable Sylgard block in a bath continuously perfused with saline and the skin overlying the myotomes was removed with finely etched tungsten microneedles. To make intracellular recordings, rostral myotomes were removed from one side of the embryo and microelectrodes inserted into the spinal cord. Microelectrodes were filled with 3 M potassium acetate except where specified in the results and had DC resistances of MQ. total of 166 neurones (mean resting potential -7 mv, mean input resistance 124 MQ) from 126 animals were impaled using a piezoelectric jolting device (Weevers, 198) or by capacitance overcompensation. Neurones were used if they had a stable resting potential more negative than -5 mv and showed clear rhythmic activity during fictive swimming (Roberts & Kahn, 1982). ll neurones used were in the ventral quarter of the cord where marker studies have demonstrated that almost all the neurones are motoneurones (Roberts & Clarke, 1982; Soffe & Roberts, 1982). Therefore the majority of recordings in this study were from motoneurones, and the term motoneurone, rather than presumed motoneurone, will be used throughout to simplify the text. Drugs were applied to the whole embryo via a multibarrelled microperfusion system consisting of multiple polythene tubes exiting through a common orifice 1,um in diameter. One barrel contained control saline solution which was applied between drug applications to prevent perfusion artifacts. Fictive swimming was evoked by dimming the lights or with a 1 ms electric current pulse applied to the tail skin via a suction electrode. Data were stored on a Racal (Southampton, UK) magnetic FM tape-recorder and printouts made using a Graphtec (Tokyo, Japan) thermal arraycorder or a Gould series 6 plotter. During measurement of the reversal potential for the nicotinic response the microelectrode amplifier bridge was balanced at each new membrane potential before drug application. To achieve this, the capacitance compensation was carefully adjusted until an injected current pulse was as near to rectangular as possible without oscillation. The bridge was then adjusted to null any very rapid change in the voltage response at the start of an injected current pulse until only the slower component response was left. To calculate input resistance (Ri,), current (--2 to - 3 n) was injected into neurones via the recording electrode and the measured steady-state voltage change was divided by the amount of current injected. ll values for changes in membrane potential and Rin are means + S.E.M. Drugs used were: acetylcholine chloride, nicotine hydrogen tartrate, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), muscarine chloride, oxotremorine sesquifumarate, atropine sulphate, glutamate, d-tubocurarine chloride, mecamylamine hydrochloride, a-bungarotoxin, muscimol, kynurenic acid, strychnine sulphate, tetrodotoxin (TTX) (all from Sigma, Poole, UK), dihydro-fl-erythroidine (DH,/E, kindly supplied by Merck, Sharp & Dohme, Hoddlesdon, Hertfordshire, UK) and methyllycaconitine (kindly supplied by Dr Susan Wonnacott, Department of Biochemistry, University of Bath, UK). RESULTS Effects of exogenously applied nicotinic agents Nicotine (1-1 /1M) or DMPP (1,UM) strongly depolarized motoneurones (Table 1, Fig. 1 and B). This was accompanied by a decrease in the input resistance of the neurone (Table 1). The depolarization and drop in Rin in response to 1/SM nicotine were not abolished by TTX (1UM, n =7, Fig. 1). The excitatory amino acid antagonist kynurenic acid (1 mm), had no effect on the depolarization and drop in Rin induced by nicotine (in TTX; n = 7). The nicotinic antagonist d-tubocurarine (1-2 /um) blocked the effects of 1 /tm nicotine (n= 6, Fig. I). The nicotinic antagonists mecamylamine (1-2,UM, n = 9, Fig. 1B) and dihydro-,- erythroidine (1-2 /SM, n = 6) blocked the effects of 1 #UM DMPP. DMPP was used as the standard agonist for testing potential antagonists as the response to nicotine tended to be slower to reverse (compare Fig. I and B). a-bungarotoxin did not change the effect of DMPP (1 /M, n = 4). Methyllycaconitine is a new cholinergic antagonist (lkodon, Pereira, Wonnacott & lbuquerque, 1992) which blocks a-bungarotoxin-sensitive nchrs at nanomolar concentrations and other nicotinic receptors at micromolar concentrations (Wonnacott, lbuquerque & Table 1. The effects of nicotinic agonists and Ch on motoneurone membrane properties (means + S.E.M.) gonist at 1/M Nicotine DMPP Ch Number of neurones Membrane potential change (mv) Input resistance decrease (MQ2) Input resistance decrease (%)

3 Ch receptors on Xenopus spinal neurones J. Physiol Bertrand, 1993). Methyllyeaconitine produced a partial block of the response to DMPP (1 /tm) at relatively high concentrations (1 /am, n = 6). The reversal potential of the depolarization was estimated by changing the membrane potential with current injection and then applying nicotine (1 /LM, n = 6, Fig. 2). The response to nicotine was largest at hyperpolarized potentials and smallest at depolarized potentials. The motoneurones were not depolarized above about -4 mv, because delayed rectifying K+ currents, which activate at these potentials (Soffe, 199), might have affected the amplitude of the ligand-gated response. The extrapolated reversal potentials for individual neurones varied from -31 to +18 mv, the mean for the six neurones tested being mv (Fig. 2B). Effects of exogenously applied muscarinic agents Muscarine (2-2 1UM, n = 13) and oxotremorine (1/M, n = 5) resulted in a small (1-4 mv) hyperpolarization of motoneurones (Fig. 3 and C). The inhibitory effect of 1 pm nicotine muscarine was confirmed by injecting positive current pulses with an amplitude just above the action potential threshold in control. In such cases the neurone fired reliable action potentials in control and wash but not during application of muscarine (n = 1, Fig. 3 and B). When recordings were made with 2 M KCl electrodes the hyperpolarization to muscarine was not reversed (n = 5). The small hyperpolarization to either muscarine or oxotremorine was not blocked by ITX (1 JaM, n = 1, Fig. 3C). tropine (1 nm) blocked the effects of muscarine and oxotremorine (1 M, n = 7, Fig. 3C), but not those of DMPP (1 /M, n = 3). Effects of exogenously applied Ch Ch (1-1 4aM) had effects very similar to those seen for nicotine and DMPP. There was a depolarization and a decrease in Rin (Table 1, Fig. 1C), both retained in the presence of ITX (n = 6, Fig. IC). If applications were made in kynurenic acid (1 mm) there was no effect on the depolarization and drop in Rin induced by Ch (n =6, 1 um d-tubocurarine 1 /SM nicotine B 1/uM DMPP 2 am mecamylamine 1,uM DMPP C 1 /M Ch 1aM DH/?E I~~~~~~~~~~~~~~~~~~~~~~ NI 1 s -2 mv Figure 1. Effects of nicotinic agonists and antagonists on motoneurone membrane properties, the depolarizing response and drop in input resistance (R1, measured from the amplitude of responses to hyperpolarizing current pulses) of a motoneurone to 1/M nicotine which is blocked by 1/tM d-tubocurarine. B, depolarizing response and drop in Rin of a motoneurone to 1 UM DMPP which is blocked by 2 UM mecamylamine. Note that during the first DMPP-induced depolarization there is an increase in frequency of hyperpolarizing IPSPs. C, depolarizing response to 1 JM Ch which is blocked by 1 /JM dihydro-,i-erythroidine. Experiments in and C performed in 1,UM TTX. Resting potentials are -74, -67 and -7 mv for, B and C, respectively.

4 224 J. Physiol R. Perrins and. Roberts Fig. 4). Both dihydro-,-erythroidine (1-2 /M, n =3, Fig. IC) and d-tubocurarine (1-2,UM, n = 6) antagonized the effects of Ch (1,UM). High concentrations of atropine (1 /SM) partially blocked the response to Ch (1 /SM, n = 6). The decrease in Rin produced by both the specific nicotinic agonists and Ch could be due partly to the opening of voltage-sensitive K+ channels at depolarized levels (Soffe, 199), rather than ligand-gated channels opened by the agonists. To test this, positive current was injected until the neurone reached the same level of depolarization as that caused by Ch (1 /UM, n = 5). The drop in Rin caused by current injection was smaller ( MQ or a reduction of 11P % from the control values) than that caused by the agonist ( MQ or %). This indicates that about 7% of the drop in Rin is due to the opening of ligand-gated channels by Ch. Presynaptic effects of agonists In twelve of thirteen cases when TX was absent, DMPP (1 um) produced a barrage of hyperpolarizing inhibitory postsynaptic potentials (IPSPs) in motoneurones (Fig. IB). Ch (1 /um) also produced a barrage of IPSPs in thirtytwo of thirty-five neurones tested (Fig. 5, C and E). Strychnine (1 /1M), a selective glycinergic antagonist in this preparation (Soffe, 1987), had no effect on the Ch-induced depolarization, but completely abolished the barrage of IPSPs (n = 7, Fig. 4). The barrage of IPSPs was also abolished by ITX (1,M, n = 8, Fig. 4). The IPSPs were not blocked in the presence of kynurenic acid (1 mm, n = 8, Fig. 4). The hyperpolarization produced by muscarinic agonists was unaffected by TTX (Fig. 3C) suggesting that this effect is entirely due to inhibition of the impaled cell. Effects of antagonists at non-cholinergic receptors The specificity of the cholinergic antagonists was investigated in this preparation by testing their effects against various other agonists. The responses of the sensory Rohon-Beard neurones to GB are known to be blocked by d-tubocurarine (Bixby & Spitzer, 1982) so such a cross-reaction was tested for in motoneurones. d-tubocurarine (1 /,M) strongly antagonized the hyperpolarizing response of motoneurones to muscimol (3 /um), a GB agonist (n = 4, Fig. 5). Mecamylamine (1,UM) had no effect on this hyperpolarization (n = 3). Since mecamylamine has been shown to block excitatory amino acid receptors in some central systems (Krnjevic & Phillis, 1963; O'Dell & Christensen, 1988), mecamylamine (1/,M) was also tested against the depolarizing response to mv B 22-4 mv E 16- a) E a) X 1- o 8. cn a) 6-3 O 3 s Vm (mv) -4-2 Figure 2. Estimation of the reversal potential of the depolarization to 1,UM nicotine, responses of a motoneurone to nicotine at different membrane potentials (Vm). The responses attenuate at more positive membrane voltages, indicated underneath each trace. B, plot of amplitude of the responses of six motoneurones as a function of membrane potential. Each motoneurone is represented by a different symbol and the mean regression line for all six neurones is shown. The mean reversal potential from six cells was -12 mv.

5 a Ch receptors on Xenopus spinal neurones J. Physiol FM muscarine C B a b b l -I jj4h M",W.L.L LiU11LJ J-" L"id I TM,rpr ql- fft I'pl'rFTTTTrl c.1 2 s 2 mv 4 mv C 1 jim muscarine _-- SQWNW_--- Control 1 #M TTX.1 #M atropine 8 ms Wash Figure 3. Effects of muscarinic agonists and antagonists on motoneurone membrane properties, small hyperpolarization on application of 2 /SM muscarine. The peak level of noise at the resting potential is indicated by the dashed line. Upward deflections are caused by positive current pulses which were just suprathreshold to evoke an action potential in control. In 2 /M muscarine the probability of impulses decreases. The periods marked a, b and c were used to collect the data in B. The resting potential of this neurone was -69 mv. B, mean values of responses to eight pulses: (a) in control, when the neurone spiked on each pulse; (b) during and just after muscarine application, when no spiking occurred; (c) after 1 min of wash, during which the hyperpolarization was abolished and the spikes returned. C, application of 1/uM muscarine, represented by the bar, causes a small hyperpolarization in control. This is unaffected by 1 1SM TTX, blocked by 1 nm atropine, and returns after washing. Scale as for, resting potential -67 mv. Control 1 #M strychnine Wash 1 FM TTX Wash NVV,W 25 mv 1 ms Figure 4. Presynaptic effects of Ch pplication of 1/M Ch in 1 mm kynurenic acid resulted in a depolarization of about 35 mv from a resting potential of -78 mv. portion of the record from the peak of the depolarization is expanded to show clearly the barrage of IPSPs in control. The barrage of IPSPs, but not the depolarization, is blocked by 1 S,M strychnine and 1/SM TTX, with partial wash in both cases.

6 R. Perrins and. Roberts 226 J. Physiol glutamate (1/,M, n =5, Fig. 5B), but no antagonistic effect was observed. The hyperpolarizing response to muscarine (1 /LM) was unaffected by mecamylamine (4/M, n = 3). DISCUSSION Both nicotinic and muscarinic ChRs are present in Xenopus spinal motoneurones When nchrs are activated, the membrane potential of Xenopus motoneurones is depolarized, and the Rin decreases, indicating a membrane conductance increase. The value for the reversal potential of around -12 mv is similar to that found in other studies of nicotinic receptors in the central nervous system (Egan & North, 1986a; McCormick & Prince, 1987). In the peripheral nervous system and the neuromuscular junction the depolarization caused by Ch acting on nicotinic receptors also has a similar reversal potential and is known to increase the membrane conductance non-specifically to cations (Takeuchi & Takeuchi, 196; Gallagher, Griffith & Shinnick-Gallagher, 1982). It therefore seems likely that a similar process is occurring in Xenopus spinal neurones. When mchrs are activated, the membrane is hyperpolarized. Other studies of muscarinic receptors in the central nervous system have revealed that the response can be mediated by increasing K+ conductances (Egan & North, 1986b). The muscarine-induced hyperpolarization in Xenopus motoneurones is not reversed when recorded with KCl electrodes. Recording with these electrodes causes GBergic and glycinergic hyperpolarizations mediated by chloride ions to become depolarizing (Roberts & Kahn, 1982; Soffe, 1987). This makes it most likely that the hyperpolarization is caused by the opening of a K+ conductance. In this investigation, there is convincing evidence for both nicotinic and muscarinic receptors with distinct pharmacological properties. similar situation is found in other neurones within vertebrate autonomic ganglia (Gallagher et al. 1982) and the central nervous system (Egan & North, 1985, 1986a; Phelan & Gallagher, 1992), although the functional relevance of such co-expression remains obscure. s Ch has an effect similar to that of nicotinic, rather than muscarinic, agonists, and because its effect can be fully blocked by specific nicotinic antagonists, it may be assumed that this excitation is mediated by nchrs. Ch appears to be less potent than either nicotine or DMPP (Table 1), which may be due to the presence of natural acetylcholinesterases within the spinal cord. The muscarinic receptors may also be activated by Ch, but this response is overwhelmed by the nicotinic excitation, at least in the short term. lthough we have shown that 1,UM atropine is specific for muscarinic receptors in this preparation, at 1/M it is able to partially block the depolarization evoked by Ch via nchrs. tropine is therefore non-specific at this concentration, which explains its antagonism of the Ch-mediated excitation reported previously (Panchin et al. 1991). The fact that d-tubocurarine is a potent antagonist of nchrs is especially important because until recently it was used as a neuromuscular blocking agent to immobilize Xenopus embryos in almost all neurophysiological experiments. Since d-tubocurarine also blocks GB receptors at the concentrations used to immobilize the embryos (Bixby & Spitzer, 1982; this Muscimol B Control 1 jsm d-tubocurarine (2 min) Wash (4 min) Glutamate 2 mv 2 s Control 1,M mecamylamine (12 min) Wash (4 min) Figure 5. Specificity of nicotinic antagonists, 3/M muscimol (application represented by the filled bars), a GB agonist, causes a hyperpolarization and drop in Ri. of motoneurones. This is strongly antagonized by 1 /SM d-tubocurarine. B, 1 /M mecamylamine does not affect the depolarizing response of motoneurones to 1 /M glutamate (filled bars), an excitatory amino acid agonist. Resting potentials are -7 mv for and -69 mv for B.

7 J. Physiol Ch receptors on Xenopus spinal neurones 227 study), it can no longer be regarded as a suitable agent for immobilization. Responses to Ch and GB can both be studied in embryos immobilized in a-bungarotoxin. ctivation of nchrs excites at least two classes of Xenopus spinal neurone The excitation of motoneurones is shown directly by their depolarization on application of nicotinic agonists in TTX. However the results also show that another class of neurone is also excited above firing threshold. The IPSPs elicited in motoneurones by nicotinic agonists are abolished by TTX, suggesting that they are the result of spiking activity in another type of neurone. They are of similar polarity and time course to those known to be caused by spiking in a discrete type of spinal neurone, the commissural interneurone (Roberts & Clarke, 1982; Dale, 1985). The commissural interneurones are the only glycinergic neurone class in the Xenopus spinal cord (Dale, Ottersen, Roberts & Storm-Mathisen, 1986). Because the agonist-evoked IPSPs are blocked by strychnine, it can be concluded that they are the result of activity in commissural interneurones which have been excited to firing threshold. The IPSPs are not abolished by kynurenic acid so the excitation is not caused by the release of an excitatory amino acid onto the commissural interneurones from other classes of interneurone excited by Ch. Commissural interneurones must therefore possess nchrs. Possible functional roles for cholinergic receptors The discovery of ChRs on Xenopus spinal neurones raises the possibility that they could be activated by naturally released Ch during swimming and experiments are currently underway to investigate this. 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