Progress in Neurobiology Vol. 35, pp. 451 to 468, 1990 Printed in Great Britain.All rights reserved

0301-0082/90/$0.00+ 0.50 © 1990PergamonPress pie

NEUROPHYSIOLOGICAL ACTIONS OF 5-HYDROXYTRYPTAMINE IN THE VERTEBRATE NERVOUS SYSTEM R. ANWYL Department of Physiology, Trinity College, Dublin 2, Ireland

(Received 31 May 1990)

CONTENTS I. Introduction 2. 5-HT induced increase of an "agonist" inward rectifying K conductance and hyperpolarization 2. I. Voltage and current recording, and ionic mechanism 2.2. G protein involvement 2.3. 5-HT mediated IPSP 2.4. Receptor pharmacology 3. 5-HT induced increase in the Na/K inward rectifying conductance (Gh) 3.1. Voltage and current recording, and ionic mechanism 3.2. Receptor pharmacology and intracellular messengers 4. 5-HT induced increase in other slow Na/K conductances 5. Reduction of the inward rectifying K conductance (GtR) 6. Reduction of the "M" K conductance (GM) 7. Reduction of the slow Ca activated K conductance (GAap) 8. 5-HT induced slow depolarization 9. Effects of 5-HT on the calcium channel 9.1. Facilitation 9.2. Inhibition 10. 5-HT receptors expressed in Xenopus oocytes 10.1. Voltage and current recordings, and ionic mechanism 10.2. Receptor pharmacology and intracellular messengers I 1. 5-HT3 receptors 11.1. Voltage and current recordings 11.2. Ionic mechanism 11.3. Single channel currents 11.4. Desensitization 11.5. Receptor pharmacology 12. Modulation of other transmitter receptors by 5-HT 13. Effect of 5-HT on neuronal excitability 14. Conclusion References Note added in proof

1. INTRODUCTION The first neurophysiological studies of the action of 5-hydroxytryptamine (5-HT) in the central nervous system were made in the 1960s, when the effect of iontophoretieally applied 5-HT was studied on single unit recordings in in vivo preparations (Krnjevic and Phillis, 1963; Roberts and Straughan, 1967). These initial results demonstrated that 5-HT caused both excitation and inhibition of neuronal firing in the spinal cord and cortex, and similar results were later found in most areas of the CNS (Bloom et al., 1972). Such mixed responses to 5-HT were initially difficult to interpret, but later studies using putative 5-HT

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antagonists such as methysergide and cinanserin, which blocked the excitatory, but not the inhibitory, effects of 5-HT, indicated that there were at least two types of 5-HT receptors in the CNS (Haigler and Aghajanian, 1977). Since that time, a large number of studies have revealed that 5-HT has many different neurophysiological actions in the nervous system. The present review describes these actions of 5-HT, concentrating on neurophysiologieal studies of 5-HT action involving the use of current, voltage and patch clamp recording techniques, as it is only through the use of such techniques that the large diversity of neurophysiological action of serotonin in the nervous system has been recognised.

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R. ANWYL 2. 5-HT INDUCED INCREASE OF AN "AGONIST" INWARD RECTIFYING K CONDUCTANCE AND HYPERPOLARIZATION

2.1. VOLTAGEAND CURRENT RECORDING, AND IONIC MECHANISM

5-HT was found to cause an increase in an inwardly rectifying "agonist" K conductance and an associated hyperpolarization or outward current at the resting potential of neurons in the following regions of the nervous system: human and guinea-pig cortical pyramidal neurons, including the anterior temporal, occipital and frontal areas in the former (McCormick and Williamson, 1989); rat piriform cortical pyramidal neurons (Sheldon and Aghajanian, 1990); rat hippocampal pyramidal and dentate cells (Segal, 1980; Jahnsen, 1980; Andrade and Nicoll, 1987a; Colino and Halliwell, 1987; Baskys et al., 1987, 1989; Ropert, 1988; Yakel et al., 1988; Beck, 1989; Segal et al., 1989); dorsal raphe nucleus (Aghajanian and Lakoski, 1984; Yoshimura and Higashi, 1985; Sprouse and Aghajanian, 1987, 1988; Innis et al., 1988; Williams et al., 1988), rat lateral septal nucleus (Joels and Gallagher, 1988; Joels et al., 1986, 1987); rat cerebellar Purkinje cells (Hicks et al., 1989); rat striatum (Yakel et al., 1988); guinea-pig prepositus hypoglossi nucleus (Bobker and Williams, 1990); cat and rat spinal motoneurons (Phillis et al., 1968; Engberg et al., 1976; White and Fung, 1989); rat lumber dorsal root ganglion (Abramets et al., 1989); rat superior cervical ganglion (Ireland, 1987; Ireland and Jordan, 1987); rat sympathetic ganglion (Watanabe and Koketsu, 1973; Newberry and Gilbert, 1989); and rat myenteric plexus (Galligan et al., 1988). The threshold concentration for the hyperpolarization was generally 300nM to I#M. In the hippocampus, the ECso was 2-3#M, and the maximum hyperpolarization recorded was 15 mV, generated by 30#M 5-HT (Andrade and Nicoll, 1987a; Beck, 1989). Blockade of 5-HT uptake with fluoxetine plus desipramine resulted in only a small shift to the left of the dose-response curve (Andrade and Nicoll, 1987a). In the prepositus hypoglossi nucleus, 5-HT was somewhat less potent, with the ECso 8.6#~t, and the maximum response, with 100#M 5-HT, averaging 18mV (Bobker and Williams, 1990). In this nucleus, the uptake blocker cocaine produced a large, 64-fold leftward shift of the 5-HT concentration effect curve, reducing the ECs0 to 135 nM (Bobker and Williams, 1990). The hyperpolarization was associated with an increase in input conductance, Gtr~, of up to 30%, including when the membrane potential (EM) was manually clamped at the resting potential. The current-voltage ( I - V ) curve determined under voltage clamp was approximately linear near the reversal potential and at hyperpolarized potentials, but showed inward rectification upon depolarization. Thus the current increased by a smaller amount upon depolarization than hyperpolarization, i.e. the slope conductance decreased, and at potentials more depolarized than - 55 mV the current actually decreased, the slope conductance becoming negative (Andrade

and Nicoll, 1987a; Colino and Halliwell, 1987; Yakel et al., 1988; Williams et al., 1988). In a detailed study of the conductance increase elicited by the 5-HT agonist 5-carboxyamidotryptamine, Gs.cr, Gs.cr was found to increase with hyperpolarization from - 5 0 mV to -120inV. Thus, in 2.5 rnM [K]0, G5-cr was 2.2 nS at - 5 0 mV and increased to 4.0 nS at - 120 mV. High K increased the value of Gs.cr at a given membrane potential, GS.CTbeing 2.2 nS, 3.3 nS and 5.0 nS in 2.5, 6.5 and 10.5 mM external K respectively at - 6 0 inV. High K also increased the dependency of G5-cT on membrane potential (Williams et al., 1988). The reversal or null potential, ES.aT, always had a value identical with Eg (from - 8 5 to -113 mV in control media containing 3-4ram [K]0), whether determined using either current clamp techniques (Ropert, 1988; Baskys et al., 1989; Segal, 1980; Aghajanian, 1984; Joels et al., 1988) or voltage clamp techniques (Andrade and Nicol, 1987a; Colino and Halliwell, 1987; Yakel et al., 1988; Williams et al., 1988; McCormick and Williamson, 1989; Bobker and Williams, 1990). Altering [K]0 between 2.5-10.5 naxt shifted Es.nr to values identical to those predicted by the Nernst equation for a conductance change solely to K ions (Andrade and Nicoll, 1987a; Baskys et al., 1987; Segal, 1988; Colino and Halliwell, 1987; Joels and Gallagher, 1988; Bobker and Williams, 1990). For example, Es.n~ was - 102, - 7 8 and - 6 7 mV in 2.5, 6.5 and 10.5 n~t [K]0 respectively (Williams et aL, 1988). Cs and Ba (1-2mM) both strongly inhibited the 5-HT response (Andrade and Nicoll, 1987a; Colino and Halliwell, 1987; Ropert, 1988)! In a lower concentration of Ba (100 #M), a 5-HT induced inward current could be evoked, but the inward rectification of the current was blocked (Williams et all' 1988). The 5-HT response was not inhibited by the more selective K conductance inhibitors TEA, 4-AP or 8-Br-cAMP, thus eliminating the K currents Ic and I t , IA and IAHp respectively as being increased by 5-HT (Andrade and Nicoll, 1987a; Ropert, 1988; Segal, 1980). Moreover, the inwardly rectifying current I h (Io) does not appear to be involved in the hyperpolarization, since the 5-HT agonist 5-CT did not increase Ih, and moreover, the 5-CT induced inward rectifying current activated much more rapidly than I h (Williams et al., 1988). No change in chloride permeability was initiated by 5-HT, as loading the cells with C1 (thus reversing the chloride gradient across the cell) or replacing external Cl with proprionate, did not alter Es-H~. Ca, either from an intracellular or extracellular source, was not involved in the 5-HT hyperpolarization since the hyperpolarization was not altered by very low external Ca, addition of external Cd or Co, or injection of EGTA intracellularly (Andrade and Nicoll, 1987a; Colino and Halliwell, 1987; Ropert, 1988; Yakel et a/., 1988). Thus the conductance increase and hyperpolarization evoked by 5-HT is mediated by the opening of highly selective K channels showing inward rectification. Such inward rectification will result in the 5-HT exerting a much greater action at hyperpolarized than at depolarized potentials. These "agonist" inwardly rectifying K channels are cormnonly linked to transmitter receptors, including GABA a, A~ adenosine, #

5-HT IN TH~V~T~tATE NERVOUSSYSTEM and 6 opioid, somatostatin, M2 musearinic and ~2 adrenoceptor (North and Williams, 1985; Pfatfmger et al., 1985; Andrade and Nicoll, 1986; Zgombick et al., 1989). There is also often convergence of such transmitter action on the agonist inward rectifying channel in single neurons. For example, in the hippocampus, activation of 5-HT, adenosine and GABAs receptors activate this K channel, and thus maximal activation of the outward current by an adenosine agonist or baclofen reduced the outward current induced by 5-HT (Andrade et al., 1986; Zgombick et al., 1989). The "agonist" and, "non-agonist" activated inwardly rectifying K conductances have many properties in common (Mihara et al., 1987). However, it has been shown that the agonist (opioid) inwardly rectifying single channels were opened in the absence of background "non-transmitter" inwardly rectifying channels (Miyake et al., 1989), and the potential at which the conductance was half-maximal was - 50 mV for the agonist conductance, but - 115 mV for the "non-agonist" conductance (Williams et al., 1988). Moreover, the agonist induced rectification did not show a time-dependent inactivation, unlike the non-agonist rectification (Inoue et al., 1988). Thus the inwardly rectifying agonist and non-agonist channels are probably distinct. Repeated applications of 5-HT (1-2 rain each application) did not result in any desensitization of the hyperpolarizations (Andrade and Nicoll, 1987a). Moreover, continuous application of 5-HT, even at a high concentration producing maximal hyperpolarization (36/~M) caused a sustained hyperpolarization with no desensitization (Ropert, 1988). 2.2. G PROTEININVOLVEMENT Intraventricular injection of pertussis toxin (PT) for 3 days eliminated or greatly reduced the 5-HT hyperpolarization in the hippocampus (Andrade and Nicoll, 1987a). PT ribosylates and inactivates certain G proteins, and therefore it can be concluded that the 5-HT hyperpolarization is mediated via a G protein. Further support for this conclusion comes from experiments in which the 5-HT hyperpolarization was greatly reduced or abolished by intracellular injection of guanosine 5-O-thiodiphosphate (GDP//S), a non-hydrolysable GDP analogue which binds to G proteins and inhibits GTP binding. Moreover, guanosine 5-O-3-thiotriphosphate (GTPTS), a nonhydrolysable GTP analogue, mimicked the action of 5-HT by causing sustained hyperpolarization and thereby blocking the action of 5-HT (Andrade and Nicoll, 1987a; Innis et al., 1988). Following the formation of a whole cell patch clamp with low resistance electrodes, the outward current elicited by 5-HT decreased with time, and was abolished in 5 rain due to a rapid exchange between the cytoplasm and the patch pipette. However, the inclusion of GTP in the patch pipette prevented the loss of the 5-HT responses (Yakel et al., 1988). It is therefore most likely that 5-HT opens the K channels directly via a G protein, i.e. without the intervention of an intracellular messenger. Such direct G protein linking to the inwardly rectifying K channel has also been found for muscarinic, JPN 35/6~D

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adenosine and somatostatin receptors (pfaffinger, 1985; Kurachi et al., 1986; Yatani et al., 1987). 2.3. 5-HT MEDL~rV_DIPSP A 5-HT mediated ipsp has been recorded from rat dorsal raphe nucleus neurons and also guinea-pig prepositus hypoglossi nucleus neurons. Thus focal electrical stimulation applied to these nuclei evoked a slow ipsp, and an outward current in voltage clamped cells (Yoshimura and Higashi, 1985; Pan et al., 1989; Bobker and Williams, 1990). The slow ipsps had a latency to onset of 47-53ms, an amplitude of 3-30 mV, a rise time of 114-199 ms, a half-decay time of 529 ms, and a total duration of 1-4 s. The slow ipsp was associated with an increase in Gin, and was abolished by blocking transmitter release in a high Mg, low Ca or a high Mg and Co medium (Yoshimura and Higashi, 1985; Pan et al., 1989; Bobker and Williams, 1990). The reversal potential of the serotonergic ipsp was - 9 6 mV in the dorsal raphe, and -111 mV in the prepositus hypoglossi, similar to that of ES.HT in these cells. Moreover, the reversal potential of the slow ipsp was altered in different K concentrations between 2.5-10.5 rnM according to the Nernst equation for a channel selectively permeable to K (Pan et al., 1988; Bobker and Williams, 1990). The I - V curve of the slow ipsp showed inward rectification (Pan et al., 1989). The uptake blockers fluoxatine and cocaine prolonged the ipsp several fold (Pan et al., 1989; Bobker and Williams, 1990). Thus the slow ipsp had very similar properties to the 5-HT evoked hyperpolarizing response in these cells, and is very likely to be mediated by 5-HT acting on the same receptors. 2.4. RECEPTORPHARMACOLOGY The tryptamine analogues 5-methyoxy-N,Ndimethyltryptamine (5-MoODMT) and 5-methoxytryptamine (5-MOOT) both caused a concentration dependent hyperpolarization and outward current, with the order of potency 5-HT > 5-MeODMT > 5MoOT in the hippocampus (Andrade and Nicoll, 1987a; Wu et al., 1989; Aghajanian and Lakoski, 1984) and superior cervical ganglia (Ireland and Jordan, 1987). The relative potencies were found to be very similar for those at the 5-HT1^ binding site. 5-CT also caused a concentration-dependent hypcrpolarization and outward current. In the dorsal raphe and hippocampus, the threshold concentration for 5-CT was 3-6 riM, and the maximum response was at 0.1-0.3/~M (Williams et al., 1988; Beck, 1989), while in the superior cervical ganglion and prepositus hypoglossi nucleus, the threshold was about 1 rL~, and the ECs0 was 100rim (Ireland, 1987; Bobker and Williams, 1990). Thus 5-CT is 10-100 times more potent than 5-HT at this 5-HT receptor. 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT) has been found to be a full agonist in the dorsal raphe and septal nucleus, but a weak partial agonist in the hippocampus, superior cervical ganglia and neocortex. Thus in the hippocampus, superior cervical ganglion, and cortex, 8-OH-DPAT evoked a

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maximum hyperpolarization of only about 20-35% of that of 5-HT, even using high concentrations of the agonist (Ireland and Jordan, 1988; Andrade and Nicoll, 1987a,b; Ropert, 1988; Yakel, 1988; Segal et al., 1989; McCormick and Williamson, 1989), and it antagonized the hyperpolarizing action of 5-HT (see below). However, in the lateral septal nucleus and dorsal raphe nucleus, 8-OH-DPAT produced a similar maximum conductance change and hyperpolarization to 5-HT. Moreover, it was tenfold more potent than 5-HT in the lateral septal nucleus (Joels et al., 1987) and 1000-10,000 fold more potent than 5-HT in the dorsal raphe nucleus (Williams et al., 1988). In the prepositus hypoglossi nucleus, 8-OHDPAT was a potent partial agonist, producing a hyperpolarization with a maximum amplitude 81% of the maximum 5-HT response, but with an ECs0 of 16 riM, 500 times lower than that of 5-HT (Bobker and Williams, 1990). Buspirone, gepirone and ipsapirone were partial agonists in the hippocampus, evoking a maximum hyperpolarization 20-35% of that of 5-HT (Andrade and Nicoll, 1987). In the hippocampus, 8-OH-DPAT and buspirone antagonized the action of 5-HT (Colino and Halliwell, 1986; Andrade and Nicoll, 1987a,b). For example, 30/ZM 8-OH-DPAT, or 30/tM buspirone, concentrations which caused a maximum response of about 3 mV, reduced the hyperpolarizing action of 20-30/tM 5-HT from 10-15mV to 3-7mV (Andrade and Nicoll, 1987a,b). Ipsapirone (200raM) also completely blocked the 5-HT induced outward current (Colino and Halliwell, 1987). In the superior cervical ganglia, 8-OH-DPAT and ipsapirone, at concentrations below those causing a hyperpolarization, strongly antagonized the effect of 5-HT (Ireland and Jordan, 1987). Spiperone has been found to be an effective antagonist of the 5-HT evoked hyperpolarization. In the hippocampus, 1-3/IM spiperone caused complete inhibition of the 5-HT response, although over 1 hr was required for equilibrium to be reached (Andrade and Nicoll, 1987a; Beck, 1989). In the prepositus hypoglossi nucleus, spiperone antagonized the 5-HT hyperpolarization with a Ka of 10 nM (Bobker and Williams, 1990). In the superior cervical ganglia, spiperone antagonized the 5-HT hyperpolarization at concentrations of 0.1-10/~M, with a pKa of 7.4 (Ireland and Jordan, 1987). Methiothepin, cyproheptadine and mianserin were also antagonists of the 5-HT hyperpolarization, with an IC50 of about 30/~ M, 60VM and 150 #M respectively in the hippocampus (Andrade and Nicoll, 1987a). Metoclopramide blocked the 5-HT induced inward current at high concentrations (50% block at 200#M) in striatal neurons (Yakel et al., 1988), while propranolol and methysergide both reduced the 5-HT response in hippocampal neurons (Segal, 1980; Colino and Halliwell, 1987), although methysergide hyperpolarized these neurons and evoked an outward current at high concentrations of 200#M (Yakel et al., 1988). In the dorsal raphe nucleus, methysergide and lysergic acid diethylamide (LSD) (above 200 nvl) elicited a hyperpolarization (Aghajanian and Lakoski, 1984). Thus the available neurophysiological evidence indicates that the 5-HT induced increase in G~N and

hyperpolarization is mediated by 5-HT acting on the 5-HTIA subtype. Pharmacological evidence was also in favour of the serotonergic slow ipsp in the dorsal raphe and prepositus hypoglossi nucleus being mediated by 5-HT acting on 5-HT1A receptors, as spiperone antagonized the slow ipsp, with a threshold concentration of 10 nM and an ECs0 of 30-100 riM, similar to the antagonism against selective 5-HTIA agonists (Pan et al., 1989; Bobker and Williams, 1990).

3. 5-HT INDUCED INCREASE IN THE

Na]K INWARD RECTIFYING CONDUCTANCE (Gh) 3.1. VOLTAGEAND CURRENT RECORDING,AND IONIC MECHANISM 5-HT caused a slow increase in GiN and an accompanying depolarization due to an enhancement of the Na/K inward rectifying conductance, Gh, in neurons of the rat brainstem prepositus hypoglossi nucleus (Bobker and Williams, 1989), guinea-pig dorsal lateral and medial geniculate nuclei (Pape and McCormick, 1989) and rat spinal motoneurons (Takahashi and Berger, 1990). In spinal motoneurons, the threshold concentration of 5-HT was 1-10 riM, and the ECs0 was 120 nM (Takahashi and Berger, 1990). In the hypoglossi and geniculate nuclei, 5-HT was less potent, with the threshold about 1 #M and the maximal effect elicited with 100/~M 5,HT (Bobker and Williams, 1989; Pape and McCormick, 1989). 5-HT produced a steady-state inward current under voltage clamp at - 8 0 to - 9 0 m V in the prepositus hypoglossi nucleus, geniculate nuclei and in spinal motonenrons, with 10-30 aM 5-HT producing a current of about 100 pA at this voltage (Bobker and Williams, 1989; Pape and McCormick, 1989; Takahashi and Berger, 1990). This 5-HT induced current was inwardly rectifying, and was suggested to be an enhancement of the hyperpolarization activated cationic current I h (also termed IQ, If and IAp), (Bobker and Williams, 1989; Pape and McCormick, 1989). In the prepositus and hypoglossi nuclei, Ih was observed as a slowly activating inward current produced by hyperpolarizing voltage steps from a holding potential of about - 5 5 mV. The amplitude and rate of activation of I h increased with membrane hyperpolarization, the time constant of activation being 2.0s at - 7 0 m V and 0.2s at -100mV. No reversal of the current was observed, but the extrapolated reversal potential was - 32 mV, indicating that it is generated by an increase in conductance to both Na and K (Bobker and Williams, 1989; Pape and McCormick, 1989). 5-HT produced an inwardly rectifying current in these cells that appeared to be an increase in the amplitude of the current I~ (Bobker and Williams, 1989; Pape and McCormick, 1989). The activation potential of Ih also appeared to be shifted to more positive potentials by 5-HT, although I o was probably also enhanced without such a shift. As 5-HT produced an inward current even at potentials more negative than - 90 mV, at which potential lh is fully activated, 5-HT can probably activate a

5-HT IN Ttm VEgr~P.AT~N~vous SYSTEM population of Ih channels that hyperpolarization alone cannot activate. Like lh, the 5-HT increase in lh is carried by both Na and K ions. Thus reducing [Na]0 (from 153n~ to 26rim), or increasing [Kh (from 7.5 rr~ to 2.5 mM) reduced the 5-HT induced current, with a shift along the voltage axis corresponding to 21 and 25 mV per tenfold change in [Na]0 and [K]0, respectively. External perfusion of Cs (2 mM), but not Ba (0.1-1.0 raM) blocked the 5-HT current and Ih (Bobker and Williams, 1989; Pape and McCormick, 1989). In spinal motoneurons, 5-HT also produced an inwardly rectifying current which had very similar properties to the Na/K inward rectifier activated by hyperpolarization in the same cells i.e. similar I - V curves, reversal potentials (about - 3 0 mV in 20 mu K), reduction in low Na, potentiation in high potassium and blockage by Cs, but not Ba (Takahashi and Berger, 1990). However, the 5-HT induced current did differ in some respects from the hyperpolarization activated Na/K inward rectifier, being less sensitive to the blocking effects of Cs, being less potentiated by high K, and being much less suppressed by CI free media (Takahashi and Berger, 1990). Thus 5-HT may activate a Na/K inward rectifier in spinal motoneurons, and perhaps even in the hypoglossi and geniculate nuclei, which is similar to, but distinct from, the hyperpolarization activated Na/K inward rectifier.

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4. 5-HT INDUCED INCREASE IN OTHER SLOW Na/K CONDUCTANCES

5-HT evoked an increase in GIN accompanied by a slow depolarization in 50% of bullfrog dorsal root ganglion A cells (Morita and Katayuma, 1987). ES-HT was --65 mV (20 mV more depolarized than EK), and the I - V curve was linear from - 5 0 to - 9 0 mV. The 5-HT depolarization was reduced by 40% in Na free media. Raising external K from the control 2 n ~ to 10rr~ shifted ES-Hr to - 5 3 m V , while zero Ca, Co and Mn all abolished the 5-HT response. This 5-HT response could be due to an increase in Ca and K conductance, as suggested by the authors (Morita and Katayama, 1987), or more likely, to a Ca-sensitive Na/K current, with a predominant conductance increase to K ions.

5. REDUCTION OF THE INWARD RECTIFYING K CONDUCTANCE (Gin)

5-HT has been found to reduce the inward K rectifier conductance, GxR, in the rat nucleus accumhens (North and Uchimara, 1989). This resulted in a concentration-dependent depolarization with a threshold of about 1/ZMand an ECs0 of 10/~M (North and Uchimara, 1989). The depolarization was usually relatively small with a maximum depolarization of 5-10 mV and it had a slow time to peak, attained only after several seconds (North and Uchimara, 1989). 3.2. RECEPTOR PHARMACOLOGYAND INTRACELLULAR The amplitude of the response to 5-HT increased upon membrane depolarization and decreased upon MESSENGERS membrane hyperpolarization. The reversal potential The action of 5-HT in enhancing I h in the preposi- was - 1 0 5 mV in 2.5 rn~ external K, and it altered tus hypoglossi nucleus and geniculate nucleus was with the membrane potential in agreement with the mimicked by the non-selective 5-HTI agonist, 5-CT Nernst equation for a conductance decrease to K. (0.3-10#M), but not by 8-OH-DPAT, ipsapirone, 5-HT reduced an inwardly rectifying current pro1-(m-trifluoromethylpbenyl) piperazine (TFMPP) or duced by hyperpolarizing voltage steps from holding 2-methyl-5-HT. However, in rat spinal cord neurons, potentials of - 60 mV, with G~Rbeing decreased from 8-OH-DPAT (10 ~M) was an agonist (Takahashi and 34nS in control to 26nS in the presence of 100/~M Berger, 1990). The effect of 5-HT in the hypoglossi 5-HT. The slope conductance of the 5-HT induced and geniculate nucleus, and in spinal motoneurons, current was about 5 times greater at - 120 mV than was blocked by spiperone (1-5/~M), but not by at - 5 0 mV (North and Uchimara, 1989). Following mianserin, ritanserin, ketanserin, LM 21-009, block of the K inward rectifier with 100/~M Ba, 5-HT ICS 205-930 or GR 38032. Thus the results indicate did not produce a response (North and Uchimara, that the receptor is of the 5HTI subtype in all three 1989). However, whereas sufficient concentrations of locations, and that in the spinal motoneurons, Ba blocked all the K inward rectifier channels, 5-HT although not in the prepositus and geniculate nuclei, only blocked a subset of them (Uchimara and North, it is the 5-HTtA subtype. Forskolin (1-25#M), 3- 1990). The receptor subtype generating this response isobutyl- l-methylxanthine (IBMX) (100-300 ~ M) and 8-bromo-cAMP (500 ~M-1 raM) mimicked the effects was identified as 5-HT2. Thus ketanserin, mianserin of 5-HT, producing a slow inward current and aug- and spiperone reversibly blocked the depolarization, menting lh, and therefore this action of 5-HT may be with a KD of 3 riM, 45 nM and l0 riM, respectively. mediated by an increase in the intracellular concen- There was no effect of the 5-HTI agonists 5-CT, tration of cAMP (Bobker and Williams, 1989; Pape 8-OH-DPAT, m-chlorophenylpiperazine (mCPP) and McCormick, 1989). or the 5-HT3 antagonist ICS 205-930 (North and No desensitization of the 5-HT induced response Uchimara, 1989). was observed with continuous application of 5-HT Muscarinic receptor agonists and phorbol esters (Bobker and Williams, 1989; Pape and McCormick, also produced a depolarization and inward current in 1989; Takahashi and Berger, 1990). However, the nucleus accumbens by blocking the same K repeated application of 5-HT did bring about a inward rectifier channels as 5-HT; such action reprogressive decline of the 5-HT responses, which it suited in an occlusion of the 5-HT depolarization was suggested may be induced by a run-down of and inward current. These experiments also suggest intracellular messengers (Takahashi and Berger, the possibility that protein kinase C activation is an intermediate step in the pathway from receptor 1990).

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rat (Andrade and Nicoll, 1987a; Colino and HaUiwell, 1987; Segal et al., 1989; Baskys et aL, 1990); pyramidal neurons of the guinea-pig and human (McCormick and Williamson, 1989); spinal motoneurons in the cat, rat, turtle and lamprey (Houns6. REDUCTION OF THE "M" gaard and Kiehn, 1985, 1989; Hounsgaard et at., K CONDUCTANCE ((~M) 1989; Van Dongen et aL, 1986; Wallen et al., 1989; 5-HT has been shown to inhibit the M conductance White and Fung, 1989) and guinea-pig myenteric in voltage clamped pyramidal neurons in human and neurons (Wood and Mayer, 1979). IAHPis a long-lasting outward current with a duration greater than 1 s guinea-pig cortex (McCormick and Williamson, 1989), rat hippocampus (Colino and Halliwell, 1987), which is generated by an increase in a Ca-dependent K conductance following the influx of Ca during one, rat dorsal root ganglion neurons (Abramets et al., 1989) and Xenopus oocytes following injection of rat or more effectively, a train, of spikes. The increase in brain mRNA (Parker et al., 1990). GM is a voltage GINwhich occurs during the slow AHP was decreased dependent K conductance that is relatively slowly by 5-HT, from a 39% increase in control to a 14% activated at potentials depolarized to about - 70 inV. increase in 5-HT (Wallen et al., 1989). The 5-HT GM does not inactivate with time and, as it increases block of the slow AHP or lahp occurred without a steeply with depolarization, it can be a significant reduction of the amplitude, time course or threshold component of the total membrane conductance be- voltage of the Ca spike (observed in the presence of tween about - 7 0 to - 3 0 mV. 5-HT caused a large TTX and TEA), demonstrating that the block of the inward current when the membrane potential was slow AHP is not generated by an inhibition of the clamped at potentials depolarized to about - 6 0 mV influx of Ca (Andrade and Nicoll, 1987a; Colino and due to suppression of I M(Colino and Halliwell, 1987; Halliwell, 1987; Baskys et al., 1989; Hounsgaard and McCormick and Williamson, 1989; Parker et al., Kiehn, 1989; Wallen et al., 1989). Thus the reduction 1990). Moreover, the slow inward current associated of the slow AHP by 5-HT is probably caused by a with closure of the M channels produced by hyperpo- direct block of the Ca-dependent K channel. larizing command potentials from - 4 5 mV to about In most neurons studied, the resting Ca activated - 60 mV was greatly reduced by 5-HT, and depolar- K conductance is small or insignificant, and therefore izing steps from - 6 0 mV produced a slow inward the 5-HT block of this conductance did not apprecicurrent due to 5-HT inhibition of outward IM (Colino atively alter the resting potential. However, in neurand Halliwell, 1987; McCormick and Williamson, ons in which the Ca activated K conductance has a 1989). The action of 5-HT in suppressing IM persisted high resting value, such as guinea-pig small and large when Ca influx was prevented, such as in low Ca, or intestine type 2 myenteric neurons, 5-HT also in the presence of EGTA, or Mn or Cd (Colino and blocked this resting Ca activated K conductance, Halliwell, 1987; McCormick and Williamson, 1989; generating a substantial depolarization up to 30 mV Parker et al., 1990). However, this action of 5-HT was amplitude, with a 1-5-fold decrease in the resting blocked by Ba (2 mM), which is known to suppress/M conductance, and large slow epsps were observed (Colino and Halliwelt, 1987; Parker et al., 1990). (Wood and Mayer, 1979; Grafe et al., 1980; Tamura The 5-HT receptor subtype responsible for the and Wood, 1989). inhibition of IM has not been identified in the cortex The 5-HT block of the slow AHP appeared to be of hippocampus. However, the presence of a slow very similar to that in hippocampal pyramidal depolarization in the cortex mediated by 5-HT2 recep- neurons produced by activation of adenylate cyclase tors (see below) raises the possibility that these and consequent cAMP production in these cells. receptors mediate the IM inhibition. The block of IM However, such a mechanism generating the 5-HT in rat dorsal root ganglion neurons has been reported block of the slow after-hyperpolarization was exas being evoked by 5-HT2 receptor activation cluded because increasing intracellular cAMP levels (Abramets et al., 1989). In Xenopus oocytes, it was in the hippocampus by an inhibition of phosphodisuggested that the receptors mediating IM inhibition esterase activity with IBMX did not potentiate the were 5-HTlc-like as they had a high affinity for 5-HT, late AHP blocking effects of 5-HT (Andrade and with a threshold effect of 5-HT of 1 nM and an EC~0 Nicoll, 1987a). Moreover, increasing intraceUular of 30 riM, and the responses were blocked by mian- cAMP concentration in lamprey spinal motoneurons, serin (Parker et al., 1990). Alternatively, the receptors either by direct intracellular injection of cAMP, may be of the high affinity 5-HT2 subtype. or extracellular application of 8-bromo-cAMP or Convergence of transmitter action on the M forskolin, did not alter the late AHP, or the 5-HT channels has been observed, with acetyleholine (via reduction in the late AHP (Wallen et al., 1989). An muscarinic receptors) and 5-HT both blocking I M alternative internal messenger which could mediate in cortical pyramidal neurons (McCormick and these effects of 5-HT is protein kinase C (PKC), since Williamson, 1989). the PKC activator phorbol ester has been found to block the slow ahp in the hippocampus (Baraban et al., 1985). However, in lamprey spinal motoneurons, the PKC activator phorbol-12,13-di-butyrate, or 7. REDUCTION OF THE SLOW Ca arachidonie acid, did not alter the AHP (Wallen et ACTIVATED K CONDUCTANCE (G~mr) al., 1989). PT also did not affect the action of 5-HT 5-HT has been found to block the stow, afterhyper- in these spinal motoneurons (Wallen et al., 1989). polarizing Ca activated K conductance, G ~ , in CA 1 5-MeODMT and 5-MeOT both mimicked the hippocampal pyramidal and granule neurons of the agonist action of 5-HT on the slow AHP in the activation to G~R channel closure (Uchimara and North, 1990).

5-HT IN THE VERTI~IU,TE NERVOUS SYSTEM

hippocampus, but 8-OH-DPAT and TFMPP were inactive (Andrade and Nicholl, 1987a; Colino and Halliwell, 1987). Spiperone, ketanserin, mianserin, methysergide, and ICS 205-930 failed to antagonize the decrease in the late AHP mediated by 5-HT (Andrade and Nicoll, 1987a; Colino and HaUiwell, 1987). Thus, the 5-HT mediated block of the late AHP does not seem to be mediated by any known 5-HT receptor subtype.

8. 5-HT INDUCED SLOW DEPOLARIZATION A 5-HT induced slow depolarization has been recorded in the following regions of the nervous system: the guinea-pig somatosensory cortex (Davies et al., 1987); rat piriform cortex (Sheldon and Aghajanian, 1990); rat hippocampus (Andrade and Nicoll, 1987a; Colino and Halliwell, 1987; Asaaf et al., 1981); rat brainstem facial motoneurons (VanderMaelen and Aghajanian, 1982; Aghajanian et al., 1988); rat nucleus accumbens (North and Uchimara, 1989); rat nucleus tractus solitarius (Jacquin et al., 1989); rat and cat spinal cord motoneurons (Engberg et aL, 1976; White and Fung, 1989); bullfrog, rat and rabbit dorsal root ganglion (Holz et al., 1985; Todorovic and Anderson, 1990); guinea pig small and large intestine myenteric plexus (Wood and Mayer, 1979; Galligan et al., 1988; Mawe et al., 1986; Tamura and Wood, 1989); rat and guinea-pig submucous plexus (Suprenant and Crist, 1988; Galligan et al., 1988; Hirst and Silinsky, 1975; Johnston et al., 1980); guinea-pig coeliac ganglion (Wallis and Dun, 1988; Dun et al., 1984); and rat and cat superior cervical ganglion (De Groat and Lalley, 1989; Haefely, 1974; Newberry and Gilbert, 1989). The 5-HT induced conductance change generating the slow depolarization has been shown to be generated by several mechanisms. These include a block of GXR, GM and Gx¢cA), and also an enhancement of Gh The receptor subtypes 5-HT2 and 5-HTlp, and possibly further subtypes, have been shown to be responsible for the slow depolarization. The slow depolarization in neurons of the nucleus accumbens was generated by a block of Girt, and caused by an activation of 5-HT2 receptors (North and Uchimara, 1989) (see above). In the cortex, the slow depolarization was generated by a reduction of GM (McCormick and Williamson, 1989). Although the receptor subtype was not identified in this study, a 5-HT induced slow depolarization generated by a decrease in GtN occurring in pyramidal cells of the somatosensory and piriform cortex was shown to be caused by activation of the 5-HT2 subtype of receptor, as it was blocked by ritanserin and cinanserin (Davies et al., 1987; Sheldon and Aghajanian, 1990). A 5-HT induced slow depolarization accompanied by a decrease in G,r~ and located in facial motoneurons has been identified as generated by activation of 5-HT2 as it was blocked by 5-HT2 antagonists (Aghajanian et al., 1988). 5-HT: receptor antagonists have also been shown to block the 5-HT induced facilitation of facial motoneuron activation (Rasmussen and Aghajanian, 1988), and to block a slow depolarizing epsp in the prepositus hypogiossi nucleus (Bobker and Williams, 1990). In spinal and facial

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motoneurons, methysergide is an antagonist of the 5-HT slow depolarization, and unit studies in these areas have shown that excitatory 5-HT action was blocked by methysergide, metergoline, eyproheptadine and cinanserin (White et al., 1983; Aghajanian et al., 1989). These classical 5-HT antagonists do block both 5-HT, and 5-HT2 receptors, but have a greater affinity for the latter (McCall and Aghajanian, 1980; Barasi and Roberts, 1974; White and Neumann, 1983; Aghajanian et aL, 1988). The slow 5-HT induced depolarization accompanied by a conductance decrease in rat dorsal root ganglion neurons is blocked by ketanserin, with an ICs0 of 8 riM, and also by spiperone and methiothepin at concentrations of 100 riM-1/ZM, demonstrating that the receptors are of the 5-HT2 subtype (Todorovic and Anderson, 1990). This conclusion was supported in further studies in the rat dorsal root ganglia (Abramets et al., 1989). These 5-HT2 receptors in the dorsal root ganglia have a relatively high affinity for 5-HT, and may therefore be the high affinity type of the receptor (Todorovic and Anderson, 1990). Unit studies by 5-HT excitation in the medullary brainstem have also shown antagonism by the 5-HT2 antagonist ketanserin, as well as methysergide (Davies et al., 1988). In submucosal neurons, the slow 5-HT induced depolarization, and inward current under voltage clamp, was shown to be caused by a reduction in a K conductance. The inward current showed strong inward rectification, with usually no reversal to an outward current occurring (Suprenant and Crist, 1988). The threshold concentration of 5-HT was 2-10 riM, with the ECs0 0.1/~M, some l0 times lower than the action of 5-HT acting on 5-HT3 receptors on the same neurons (Suprenant and Crisp, 1988). The 5-HT induced slow depolarization and GIN decrease in guinea-pig myenteric and submucosal neurons has been classified as a 5-HTIp receptor (Mawe et al., 1986; Branchek et ai., 1988), This receptor has a high affinity for 5-HT, and is not inhibited by agents that bind to 5"HT1A.B.c.D,or 5-HT2, or 5-HT3 subtypes. 5and 6-hydroxyindalpine {5- and 6-hydroxy[(indoyl3)-2-ethyl]-4-piperidine} (5-OHIP and 6-OHIP) were agonists at this receptor, producing a slow depolarization and GIN decrease (Mawe, 1986). The action of 5-HT and 5- and 6-OHIP, and also the slow epsps, were antagonized by a dipeptide of 5-hydroxytryptophan, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) (Takaki et al., 1985; Mawe et ai., 1986). It has been confirmed that this 5-HT response in submucosal neurons was not of any well categorized subtype, as no antagonism occurred with ketanserin, mianserin, methysergide, spiperone, cyproheptadine, pirenzapine or ICS 205-930 (Suprenant and Crist, 1988)~ 2-Methyl 5-HT was an effective agonist at this presumed 5-HT,p receptor, with an ECs0 of 0.4/~M in submucosal neurons, similar to that at 5-HT3 receptors (Suprenant and Crist, 1988). 8-OH-DPAT, TFMPP and 5-CT were without effect (Suprenant and Crist, 1988; Galligan et aL, 1988). In pyramidal neurons of the hippocampus, the depolarization was generated by a reduction of Gx, including GM and also possibly of a leak conductance (Asaaf et al., 1981; Andrade and Nicoll, 1987a; Colino and Halliwell, 1987). The depolarization or

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R. ANWYL

inward current in the hippocampus was blocked by Cs or Ba, but it was not altered by reversing the CI gradient, or by pertussis toxin treatment (Andrade and Nicoll, 1987a). The receptor was not of a well known subtype, as spiperone, methysergide, pirenzapine, ICS 205-930, ketanserin and mianserin were ineffective as antagonists. 5-MeODMT (30/LM) and 5-MeOT (100~tM) were agonists but 8-OH-DPAT, TFMPP and 5-CT had no action (Andrade and Nicholl, 1987a; Colino and Halliwell, 1987). The slow depolarization in the prepositus hypoglossi and also geniculate nuclei was generated by an enhancement of Gh, with the receptor subtype unknown (see above) (Bobker and Williams, 1989; Pape and McCormick, 1989). In myenteric type 2 neurons, the depolarization was evoked by block of the resting Ca activated K conductance (Grafe et al., 1980). About half of guinea-pig coeliac neurons were found to respond with a slow depolarization and a decrease in G~Nin response to 5-HT, and also a slow 5-HT mediated epsp (Dun and Ma, 1984; Dun et al., 1984). The 5-HT depolarization and slow epsp was enhanced by membrane hyperpolarization from - 5 0 to - 8 0 mV, and had an extrapolated reversal potential of about - 3 5 mV (Dun and Ma, 1984; Dun et al., 1984). 5-HT is likely to be decreasing a K conductance and simultaneously increasing a Na/K conductance in these cells. The 5-HT mediated depolarization and epsp in the remaining coeliac ganglion cells were found to be generated solely by a GK decrease. Extracellular depolarization in response to 5-HT has also been recorded from neonatal rat hemisected spinal motoneurons, with an EDs0 of 20/~M in control conditions, and an EDs0 of 3.6/~M following blockade of 5-HT uptake with citalopram (Connell and Wallis, 1988). Extensive pharmacological studies of this depolarization evoked by 5-HT have shown that the response cannot be equated with any of the well known 5-HT l , 5-HT2, or 5-HT3 binding sites. Spiperone (10-9-10-7M, PA2 8.2), mesulcrgine (10-s-10-TM, pA 2 8.9) and cyproheptadine (10 -910-8 M, pA2 8.9) were potent antagonists. Metergoline and methysergide were moderately potent antagonists, with a pA2 of 7.4 and 7.5 respectively. Ketanserin was a weak antagonist, with a pA2 of 6.5, while ritanserin, cyanopindolol, quipazine, yohimbine, RU-24969, DPAT, MDL 72222, ICS 205-930 and methiothepin had no antagonistic action. 5-CT, ~Me-HT and 5-Me-OT were full agonists, and tryptamine was a partial agonist. The order of potency was 5-HT > g-Me-5-HT > 5-CT > 5MeOT > > tryptamine (Connell and Wallis, 1988). In neurons of the nucleus tractus solitarius, a novel interaction between 5-HT and Sub P has been observed. Both of these agents caused a sustained depolarization and an inward current accompanied by a decrease in GiN if applied separately and, if applied simultaneously, the resulting depolarization was additive, or greater than additive. However, a 5 min conditioning application of Sub P altered the subsequent 5-HT action to a hyperpolarizing one (i.e. 5-HT reduced the amplitude of the Sub P sustained depolarization). It was suggested that the prolonged application of Sub P may be producing an internal

messenger which converted the usual depolarizing 5-HT action to one of inhibiting the Sub P depolarization (Jacquin et al., 1989). Such an effect is likely to be of physiological importance since Sub P and 5-HT are commonly colocalized in neurons (Wessendorf and Elde, 1987). Stimulation of interganglionic fibre tracts of Auerbach's plexus produced a slow epsp which had identical physiological properties to the exogenous 5-HT produced slow depolarization. Thus, a short train of stimuli produced an epsp of 4-22 mV amplitude, a rise time of several seconds and a total duration of 18-455 s. Cell input resistance was increased, and spike discharge enhanced. Postspike afterhyperpolarization was also reduced (Wood and Mayer, 1979). Thus it is likely that this slow epsp is caused by release of 5-HT. Stimulation of the nucleus raphe obscurus (1 min at 20Hz) depolarized rat lumber spinal motoneurons and increased the population spike amplitude, an effect mimicked by 5-HT (Roberts et al., 1988).

9. EFFECTS OF 5-HT ON THE CALCIUM CHANNEL 9.1. FACILITATION 5-HT has been found to facilitate Ca action potentials in several regions of the central and peripheral nervous system. Iontophoretic application of 5-HT to distal dendrites of rat pars compacta neurons of the substantia nigra produced an average 24% increase in the amplitude of a high threshold Ca spike (evoked by depolarizing stimuli of 10-40 mV in the presence of TTX) recorded from the pars compacta cell bodies (Nedergaard et al., 1988). The spike facilitation was blocked by the 5-HT antagonist cinanserin (Nedergaard et al., 1988). The enhancement of the Ca action potentials in these neurons could be generated either by an increase in the inward Ca current or, alternatively, by a decrease in a K conductance. An attempt to eliminate the latter possibility was made by applying the 5-HT in the presence of three K channel blockers, TEA, 4-AP and Ba. These agents did not alter the 5-HT action, supporting the theory that 5-HT is directly enhancing the Ca current (Nedergaard et al., 1988). 5-HT also increased the amplitude and duration of Ca spikes evoked in a TTX/TEA media in dentate granule cells (Baskys et al., 1989), while in turtle spinal motoneurons, 5-HT (10/~M), enlarged the amplitude, and lowered the threshold of, a Ca mediated depolarizing plateau evoked in the presence of TTX and BAY K8644 (Hounsgaard and Kiehn, 1989). The duration of the Ca component of action potentials elicited in bullfrog dorsal root ganglion cells (in the presence of TEA) was increased 17-82% by relatively high concentrations of 8-OHDPAT (10--50/~M), buspirone (40-100/~M) and pamino-phenylethyl-trifluoromethyl phenyl-piperazine (PAPP) (40-100 pM). This action was sugges~ to be mediated by 5-HT~Areceptors (Marzelec et al., 1988; Marz¢lec and Anderson, 1988). In nucleus tractus solitarius neurons, 5-HT inhibited Ca spike duration when applied alone. However, following a 5 rain

5-HT IN THEV ~ a ~ conditioning dose of Sub P, 5-HT increased spike duration (Jacquin et al., 1989). 9.2. Ih,mm~oN 5-HT caused a dose-dependent decrease of the duration of the Ca component of the mixed Na/Ca action potentials in embryonic chick dorsal root ganglion neurons maintained /n vitro (Dunlap and Fischhach, 1978), in bullfrog dorsal root ganglion cells (Holz et al., 1986) and in rat nucleus tractus solitarius neurons/n vitro (Jac,quin et aL, 1989). Thus 10/~M 5-HT evoked a 33-47% decrease in the duration of spikes evoked in a media containing TEA, and a 40% decrease in the duration of spikes additionally broadened in 4-AP or Ba in bullfrog dorsal root ganglion cells (Holz et al., 1986; Scroggs and Anderson, 1989). About one-half of bullfrog dorsal root ganglion cells responding to 10/~,l 5-HT also showed a slow depolarization and Gm decrease. However, 1 ~M 5-HT decreased the duration of TEA broadened spikes without altering the membrane potential or conductance (Holz et al., 1986). In TTX, 5-HT reduced the maximum rate of rise, the amplitude and the duration of the Ca spike in chick and bullfrog dorsal root ganglion neurons (Dunlap and Fischbach, 1978, 1981; Holz et al., 1986). Moreover, 5-HT was found to decrease the amplitude of/ca in voltage clamped chick dorsal root ganglion neurons in the presence of TTX and TEA. The TTX resistant tail current recorded at EK was also decreased by 5-HT (Dunlap and Fischbach, 1981). The component of the Ca spike reduced by 5-HT was dihydropyridine insensitive, as it was not altered by nifedipine or BAY K8644. However, it was sensitive to a~conotoxin, indicating that the channel inhibited may be of the "N" type (as for other agonists, see later) (Scroggs and Anderson, 1989). 5-HT has also recently been shown to reduce the influx of a high threshold long lasting inter-current through Ca channels in rat spinal cord inter-neurons, with 10 #M 5-HT inhibiting the current by 36% (Sah, 1990). 5-CT was a potent agonist in inhibiting the duration of the TEA Ca spike in frog dorsal root ganglia, with an ECs0 of 19rim (compared to 210rL~ for 5-HT). In the same studies, 5-methoxytryptamine and ~t-methyl 5-HT had an ECs0 of 1.7 and 3.7#M respectively (Scroggs and Anderson, 1990). Methysergide, metergoline and cinanserin (1-100/~M), but not 8-OH-DPAT, buspirone or PAPP, had a weak agonist action (Hoiz et al., 1986). Potent antagonism was observed with methiothepin (2/~M), spiperone (2-10/~M), spiroxatrine (2-7.5/~M), and low, but not high (which caused facilitation), concentrations of 8-OH-DPAT (0.1-2.5 #M), buspirone (up to I0 #M) and PAPP (up to 10/~M). These antagonists reduced the maximum responses to 5-HT as well as shifting the dose-response curves to the right, suggesting some non-competitive action (Holz et al., 1986). The receptor is probably of the 5-HTI subtype (Scroggs and Anderson, 1990). It will be of interest to discover if 5-HT1 receptors are linked to Ca channels and to K channels in these neurons, as is the situation for a 2 receptors in submucous plexus neurons (North et aL, 1988). No desensitization of the effects of 5-HT were observed following its prolonged or repeated appli-

N~VOUSSYSTEM

459

cation in frog dorsal root ganglion cells (Holz et al., 1986). Several other neurotransmitters have been found to act in an apparently identical manner to 5-HT in reducing inward Ca current through voltagedependent Ca channels. These include noradrenaiine, GABA, adenosine, enkephalin, dynorphin A and somatostatin (Mudge et aL, 1979; Gross and McDonald, 1987; Lipscombe et al., 1989). Dynorphin A and noradrenaline (acting via 0t receptors) selectively reduce the N-type Ca current, with a decrease in the mean open time, but not the amplitude, of the single channel current (Gross and McDonald, 1987; Lipscombe et al., 1989). Thus 5-HT may also decrease an N-type Ca channel. It was suggested that the physiological effects of transmitters such as 5HT, which decrease the Ca current, is to decrease the transmitter release in sensory nerve terminals (Dunlap and Fischbach, 1981). However, an effect on accommodation of spike firing may also be important (see below). It has recently been shown that specific opioid agonists have a dual action on individual neurons, prolonging the action potential at very low concentrations (1-10 rL~) and shortening the action potential at higher concentration (10/~M) (Crain and Shen, 1990). It would be interesting to know whether 5-HT has a similar dual action on individual neurons such as dorsal root ganglion cells.

10. 5-HT RECEPTORS EXPRESSED IN

XENOPUS OOCYTES 10.1. VOLTAGEANDCURRENTRECORDINGS,AND IONIC MECHANISM

The neurophysiological properties of mammalian central nervous system 5-HT receptors have been studied in Xenopus oocytes following their expression in Xenopus iaevis oocytes injected with rat, mouse or human poly mRNA. The main response to 5-HT in the oocytes is an oscillatory current which is inward at the normal resting potential of - 70 mV. It can consist of either a series of oscillatory currents which gradually increase in amplitude before declining (Gunderson et al., 1984a,b; Takahashi et al., 1987), or alternatively, an initial large transient phase followed by a delayed long lasting component upon which may be superimposed current fluctuations (Lubbert et al., 1987). The amplitude of the initial peak reflected the amount of mRNA injected. In oocytes injected with 10rig poly(A) RNA, the responses were half-maximal at 5 x 10.8 M and maximal at 10 -+ M 5-HT (Gunderson et aL, 1984; Lubbert et al., 1987). Depolarization of the oocyte membrane decreased the 5-HT induced oscillatory response, with Es-HT being - 2 0 to - 4 0 m V , equivalent to Ea in these oocytes (Gunderson et al., 1984a,b; Takahashi et al., 1987). At more depolarized levels to this reversal potential, the current became inward. Hyperpolarizing from the membrane potential decreased the amplitude of the response due to outward rectification. This rectification could also be seen if a hyperpolarizing voltage step was applied at the peak of the 5-HT

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R. ANWYL

response, for an initial increase in the outward current was quickly followed by a decline over several hundred ms (Gunderson et al., 1984a,b). Reducing external C1 to one third of normal resulted in a 20 mV shift of Es.ar to more positive values (Takahashi et al., 1987). Reducing Na0 to one half of normal did not alter ES.HT(Takahashi et al., 1987). Thus Cl is the ion-carrying species. The smooth current response was found to be composed of two currents, one carried by C1 ions, and reversing at Eo, and a further current which could be seen by clamping the potential at 0 mV. 5-HT then elicited an outward current which declined over several seconds to leave a smaller maintained inward current which was associated with a decrease of K conductance, probably GM (Gunderson et al., 1987; see above). 5-HT evoked single channel currents were evoked when 5-HT was applied from a source even outside the patch clamp electrode. The single channel currents were mainly of a population that reversed at - 2 9 m V , had a duration of l l6ms, and had a slope conductance of 3 pS between 0 to + 60 mV. A second population of channels were found in one third of patches which had a conductance of 22 pS and an ES-HT of --3 mV (Takahashi et al., 1987). If oocytes are injected with smaller amounts of RNA ( < 10 ng), the response to a second application of low concentrations of 5-HT (5 x 10 -s M or lower), 2 min following the first, was facilitated by as much as 200%. However, a reduced response resulted following a second application of a higher concentration of 5-HT (10-7-10 -5 M) 2 min after the first (Lubbert et al., 1987). 10.2. RECEPTOR PHARMACOLOGY AND INTRACELLULAR MESSENGERS

The finding that oocyte membrane CI channels are activated by 5-HT applied outside the tightly sealed recording patch clamp electrode suggests that intracellular messengers mediate the 5-HT response (Takahashi et al., 1987). The 5-HT responses are abolished by PT and GDPfls, mimicked by GTP~S (Dascal et al., 1986; Nomura et al., 1987) and they cause an elevation of intracellular Ca, measured using fura 2, both in oocytes and in transformed mammalian fibroblast ST 3T3 cells (cells into which cloned D N A encoding the 5HTlc receptor has been introduced) (Julius et al., 1988). 5-HT also induces inositol 1,4,5-trisphosphate (IP3) production (Nomura et al., 1987). The oscillatory current was only activated after a long latency, with a minimum of 2 s delay following even iontophoretic application of 5-HT, indicative of intracellular mediators (Gunderson et al., 1984a,b). Injection of the Ca chelating agent EGTA into the oocytes to reduce the intracellular Ca to very low levels abolished the oscillatory current (Parker et al., 1985), Inhibiting the influx of Ca from the external media by addition of La, Mn or reducing external Ca to very low levels did not block the oscillatory response (Parker et al., 1985). It would appear 5-HT receptor activation leads to production of IP3 via a G protein, liberation of Ca from an intracellular store, and the subsequent acti-

vation of membrane CI channels due to the rise in intracellular Ca. The 5-HT receptor mediating the oscillatory response has been characterized as the 5-HTlc receptor. Thus 5-HT binds with high affinity to the receptor (EC~0 10-50riM), indicative of a 5-HTt receptor. Moreover, ritanserin, ketanserin and mianserin were very potent antagonists, with apparent K~ values of 30, 35 and 40 nM respectively (Lubbert et al., 1987; Tohda et al., 1989). The antidepressant imipramine was also a potent antagonist, with a K~ of 60 nM (Tohda et al., 1989). Mesulergine and cyproheptadine were moderately potent antagonists, with a K~ of 250 and 300 nM, respectively. Spiperone and yohimbine were very weak antagonists (Lubbert et al., 1987; Sakai, 1986). 5-HT~c receptors have been localized by in situ hybridization and RNA blot analysis to numerous regions of the CNS, including the choroid plexus, lateral habenula, basal ganglia, hypothalamus, hippocampus, pens, medulla and spinal cord (Julius et al., 1988).

11. 5-HT3 RECEPTORS 11.1. VOLTAGE AND CURRENT RECORDINGS

The main characteristics of the neurophysiological response of 5-HT3 receptors to 5-HT application are a rapid depolarization, or inward current, under current clamp or voltage clamp conditions respectively, both accompanied by a large increase in membrane conductance. Such responses have been recorded from cultured mouse hippocampal pyramidal and striatal neurons (Yakel and Jackson, 1988; Yakel et al., 1988), rat D R G neurons (Bevan and Robertson, 1987; Todorovic and Anderson, 1990), rabbit SCG (Wallis and Woodward, 1975; Wallis and North, 1978; Round and Wallis, 1986), rabbit nodose ganglia (Higashi, 1977; Higashi and Nishi, 1982), guinea-pig coeliac ganglion cells (Wallis and Wood, ward, 1974; WaUis and Dun, 1988), guinea, pig submucous plexus (Suprenant and Crist, 1988; Derkach et al., 1989), and cultured neuroblastoma cells NIE115, NG108-15 and NCB-20 (McDermott et al., 1979; Peters and Usherwood, 1984; Guharay et at., 1985; Neijt et al., 1986, 1988, 1989; Peters et al., 1988; Peters and Lambert, 1989; Lambert et al., 1989). The depolarization or inward current evoked by the activation of 5 - H T 3 receptors has a more rapid time course than that caused by activation of other 5-HT receptors. Thus, iontophoretically induced 5HT3 potentials recorded from submucous plexus neurons have a duration similar to nicotinic fast epsps in the same neurons, 30-50 ns (Nield, 1981; Suprenant and Crist, 1988). The 5,HT response was dose dependent when applied by bath perfusion or iontophoresis. The threshold dose of 5-HT has commonly been found to be 0.5-10/aM in autonomic ganglionic neurons (Higashi and Nishi, 1982; Derkach et al., 1989) and 0.1-1.0/aM in N E I - I I 5 cells (Guharay et al., 1985; Neijt et al., 1988), with an ECs0 of 1.8-2,0/aM and a maximal response generated by 10/aM in the latter cells (Guharay et al., 1985). However, more sensitive responses to 5-HT have been recorded. In

5-HT IN THEV ~ t A T E NERVOUSSYSTEM neurons of the submucous plexus, the threshold was 50-100 nM and the maximum response was 10-20 ~M 5-HT (Suprenant and Crist, 1988), while in dorsal root ganglion cells, the ECs0 was 67 nM (Todorovic and Anderson, 1990). The Hill slope of the concentration-response curve was found to have a value of 2.5 or 3.0, indicating that at least 2, and probably 3, molecules of 5-HT are required for activation of a single receptor (Higashi and Nishi, 1982; Peters and Usherwood, 1984). The activation of 5-HT3 receptors always generated a large depolarization (up to 50 mV), the amplitude of which is only limited by the reversal potential of the response (Neijt et al., 1988). Repetitive spike firing was generated by the depolarization (Higashi and Nishi, 1982).

461

Es-~3 which resulted from alterations in the concentrations of Na and K ions were not found to be in full agreement with the Goldman equation. In particular, the shift in E , ~ 3in low external Na or in low internal K was less than that predicted from the Goldman equation (Lambert et aL, 1989). The deviation from the Goldman equation may be due to the substitute cations for Na and K being slightly permeable through the 5-HT3 channel. The amplitude of the depolarization or current generated by activation of the 5-HT3 receptor has been found to be very dependent on the concentration of divalent ions in the bathing media. Ganglionic extracellular depolarization in response to 5-HT was increased by 20% in either Ca or Mg free media (Nash and Wallis, 1981). Moreover, the 5-HT induced depolarization or inward current was increased 11.2. IONIC MECHANmM severalfold when Ca or Mg was reduced from 10 rnM The I-V relationship of the potential or current to 0.1 raM, and the increase was larger if Ca and Mg induced by activation of the 5-HT3 receptor was were reduced simultaneously (Peters and Usherwood, found to be linear between -110 and + 50 m V in 1984; Peters et aL, 1988). Ca and Mg are unlikely to autonomic ganglion cells (Higashi and Nishi, 1982; be major current carriers through the channels since Dcrkach et ,,L, 1989; Suprcnant and Crist, 1988; altering their concentration had no effect on ER. It Wallis and Dun, 1988) and neuroblastoma cells is more likely that Ca and Mg are influencing (Guharay et aI., 1985; Neijt et aI., 1989), although the alfmity of the receptors for 5-HT, or affecting the inward rectificationhas been observed in some stud- receptor-channei coupling. An alteration of the ies on cultured neuroblastoma and hippocampal cells desensitization rate of the receptor being responsible (Yakel et al., 1988; Peters et al., 1988; Lambert et al., for the effects of Ca and Mg was shown to be unlikely 1989). The reversal potential of the 5-HT3 response from the experiments of Peters and Usherwood was between - 1 0 to + 2 0 m V in all cells studied. (1984) in which reduction of Ca from 1.8 to 0.18 mM Thus ES-HT3measured + 7 mV (Higashi and Nishi, had no consistent effect on desensitization rate. 1982), - 2 2 m V (Wallis and Dun, 1988) and +3.3mV (Derkach et ai., 1989) in autonomic 11.3. SrNOLECHANNELCURRENTS ganglion cells; - 1 0 m V (Christian et al., 1978), The first single channel recordings of 5-HT3 acti0 mV (Guharay et al., 1985; Hales et aL, 1988), + 17 to + 2 0 m V (Neijt et al., 1988, 1989), - 8 . 6 m V vated receptors were made by Guharay et al. (1985) (Peters and Usherwood, 1984), +10 to + 2 0 m V from neuroblastoma NIE-115 cells. At - 4 0 m V , (Neijt et aL, 1986), and - 2 to - 3 mV (Lambert et single channel currents recorded in the cell attached aL, 1989) in neuroblastoma cells, and - 7 to - 15 mV or outside-out mode averaged 6 pA in amplitude, had in cultured striatal and hippocampal neurons (Yakel a single channel conductance of 140 pS and a reversal et al., 1988). potential of 0 inV. Two channel open states occurred Replacement of external Na with choline or Tris in intact cells and in inside-out patches, with open was found to abolish the 5-HT induced response time constants of 240-260 ~s and 3.7-4.3 ms. Out(Wallis and Woodward, 1975; Higashi and Nishi, side-out patches had only 1 open state time constant 1982; Suprenant and Crist, 1988; Wallis and Wood- of 1.1 ms. The channel closed times also had 2 states ward, 1988). Reducing external Na by 50*/, and 86*/, in intact and inside-out patches, with closed time caused a negative shift in E~Hr3 of 15 mV and 36 mV constants of 338-350 ~s and 52-78 ms. Outside-out respectively (Lambert et al., 1989; Derkach et aL, patches had only one closure time constant of 34 ms. 1989). Increasing external K from 4.5 to 20ram Channel activity patterns consisted of brief openings shifted ES-HT3positively by 14 mV (Derkach et al., and brief closings, or brief openings and longer 1989), while reducing internal K by 86*/, shifted closures. The unitary currents reversed at 0 mV Es.HT3 positively by 24 mV (Lambert et al., 1989). An (Guraray et aL, 1985). Single channel currents have also been recorded external or internal media deficient in CI did not alter ES.HT3 (Lambert et al., 1989; Derkach et al., 1989), from submucous plexus neurons. Two discrete and varying Ca and Mg between 0.1-3.0 mM did not amplitude unitary currents are evoked by 5-HT, with alter ER (Peters et al., 1988). These results demon- single channel conductances of 15 and 9 pS. The strate that activation of the 5-HT3 receptor opens 15 pS channels had two open'states, with open time non-selective cation channels that conduct Na and K constants of 0.1 and 5.0 ms. Reversal of the large ions. The ratio of the increase in conductance to Na conductance channels occurred at + 13 mV (Derkach and K (AGN,/AGK) was calculated as 2.3 (Higashi et al., 1989). In a further study of 5-HT3 receptors on and Nishi, 1982), while the ratio of the increase in neuroblastoma NIE-115 and NCB-20 cells, discrete permeability to Na to K (APNJAPK), derived from single channel events were not observed in outsidethe Goldman-Hodgkin-Katz equation, has been cal- out patches. Fluctuation analysis of the increase in culated as 1.1 ('Yakel and Jackson, 1988; Peters and noise accompanying the inward currents in outsideLambert, 1989), 0.92 (Lambert et al., 1989), 2.4 out patches indicated the single channel current (Neijt, 1989) and 2.3 (Derkach, 1989). The changes in amplitude to be 22 fA at - 6 0 mV, and the single

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462

channel elementary conductance to be 360 iS at - 6 0 mV (Lambert et al., 1989). 11.4. DESENSITIZATION 5HT3 receptor responses undergo rapid desensitization. Thus, the responses are transient in response to continuous application of 5-HT (Guharay et al., 1985; Neijt et al., 1986, 1989; Yakel et aL, 1988), and repetitive application of pulses of 5-HT rapidly results in a decrease in the responses (Wallis and North, 1978; McDermot et aL, 1979; Wallis and Dun, 1988; Yakel et al., 1988). The rate of onset of desensitization was dose dependent (Neijt et al., 1988, 1989). In experiments applying 5-HT by perfusion to neuroblastoma cells and measuring 5-HT induced inward current, the lowest concentrations of 5-HT found to induce desensitization of the 5-HT induced inward current were 100-200 riM, a concentration 5-10 times lower than the threshold for eliciting a response (Neijt, 1988). Moreover, the concentration of 5-HT which caused half-maximal desensitization, 110 riM, was 10 times smaller than that causing half-maximal activation of the receptors (1.8/ti) (Neijt et al., 1988). The time constant of desensitization induced by 0.7, 1.0, 1.5, 2.0 and 3 #M (and higher concentrations) was 155s, 38s, 16s, 8s and 6.5s, respectively, at - 7 0 m V (Neijt, 1989). In experiments applying 5-HT by pressure ejection from a pipette, desensitization was faster, with a half time at - 7 0 mV of 1.3s and 2.5s in neuroblastoma and hippocampal pyramidal cells, respectively (Yakel and Jackson, 1988). The rate of this fast desensitization was voltage dependent. Thus, desensitization half-time increased from 16s at - 4 0 mV to 2s at - 100 mV (Yakel et al., 1988). However, these measurements may need further clarification since the desensitization was biphasic, with both a fast and a slow rate (Yakel et al., 1988). The finding of a slow desensitization process in these studies may explain the absence of an effect of voltage on desensitization when 5-HT was applied by perfusion (Neijt et al., 1989), if the fast, but not the slow, desensitization process is voltage dependent. Desensitization of the 5-HT 3 receptor is a reversible process. The time constant of recovery from desensitization was 18s in neuroblastoma cells, a value not affected by the length of period of desensitization or the membrane potential (Neijt, 1989). 11.5. RECEPTORPHARMACOLOGY 2-Methyl-5-HT has been found to be an agonist at the 5-HT3 receptor, although it has been found to be less potent than 5-HT (Bevan and Robertson, 1987; Niejt, 1988; Yakel and Jackson, 1988; Derkach et al., 1989). In hippocampal and neuroblastoma cells, 50 # M 2-methyl-5-HT activated a response 8% of that of the same concentration of 5-HT, and the time course of the 2-methyl-5-HT inward current was slower than that of 5-HT (Yakel and Jackson, 1988). In submucosal neurons, the threshold concentration for 2-methyl-5-HT was 200°500 nM, as compared to 50-100nu for 5-HT, and the maximum responses were similar for both agents (Suprenant and Crist, 1988). 5-Fluorotryptamine and tryptamine were shown to be full agonists in neuroblastoma cells,

both agents giving parallel dose-response curves and a similar maximum response to 5-HT. The order of potency was 5-HT > 5-fluorotryptamine > tryptamine (Peters and Usherwood, 1984). Dopamine is a partial agonist at the 5-HT3 receptor, giving a shallower dose-response curve and a smaller maximum response than 5-HT in neuroblastoma cells (Peters and Usherwood, 1984). The potency of dopamine was 300-1000 times lower than 5-HT in neuroblastoma cells (Neijt et al., 1986). Dopamine was also an agonist at 5-HT3 receptors on submucosal neurons (Vanner and Surprenant, 1990). ICS 205-930, MDL 72222 and GR 38032F have been shown in many studies to be specific antagonists at the 5-HT 3 receptor, and tubocurarine, quipazine and metoclopramide are also effective antagonists. One and 5/~u MDL 7222 reduced the 5-HT response by 33 and 50% respectively in coeliac neurons, with a plCs0 of 5.8 (WaUis and Dun, 1988). 1/~M ICS 205-930 antagonized 5-HT responses on coeliac neurons by 64% (Wallis and Dun, 1988). The threshold of the ICS 205-930 antagonism in submucous plexus neurons was 5-10 h i , and complete block occurred with 600 riM-2 #M (Suprenant and Crist, 1988). The IC50 of ICS 205-930 and GR 38032F on the submucosal neurons was 12rim and 100riM respectively, with the inhibition being voltage independent (Vanher and Suprenant, 1990). In neuroblastoma cells, ICS 205-930 caused a complete block at 1 riM, and the IC50 was 0.17 nM (Neijt et al., 1986, 1988). In coeliac ganglion cells, I/tM quipazine depressed the 5-HT response by 65%, while 1, 10, 50 and 100/~M curare reduced the response by 7, 30, 50 and 75% respectively, with plCs0 measuring 4.4 (Wallis and Dun, 1988). In nodose ganglion cells, 1.5 #M curare shifted the 5-HT concentration-response curves to the right in a parallel manner, suggesting a competitive inhibition at these low concentrations, while concentrations above 10 #M reduced the maximum 5-HT response, implying non-competitive antagonism, such as channel blockade, at higher concentrations (Higashi and Nishi, 1982). Tubocurare has also been shown to block the 5-HT3 induced fast depolarization in neuroblastoma, hippocampal and submucosal plexus cells, with an IC50 of 0.8 riM, 1.5 nM and 20 #M respectively (Yakel and Jackson, 1988; Vanner and Suprenant, 1990). Metoclopramide had an IC50 of 440 and 180 nM in hippocampal and neuroblastoma cells respectively (Yakel and Jackson, 1988), while GR 38032F almost completely blocked responses in neuroblastoma cells at 1 nM (Hales et al., 1988).

12. MODULATION OF O T H E R TRANSMITTER RECEPTORS BY 5-HT

In Purkinje cells of the rat cerebellum, 5-HT markedly suppressed the inward currents evoked by L-glutamate or quisqualate (by 43% and 77% respectively). The N-methyl-D-aspartate (NMDA) evoked inward currents were much less reduced (29%) by 5-HT. These effects of 5-HT usually occurred without any change of the resting membrane current or conductance (Hicks et al., 1989). In contrast, in the cat and rat cortex, 5-HT was found to facilitate the excitatory amino acid induced

5-HT IN ~

VERTImt~TEN.~vous SYSTEM

depolarization, even at concentrations which do not affect the passive membrane properties (Nedegaard et al., 1986, 1987; Reynolds et al., 1988). In layer V of the rat cortex, the depolarization or inward current induced by NMDA was increased in amplitude and duration by 5-HT, although qulsqualate responses were not altered (Reynolds et al., 1988). Epsps with an NMDA component were also enhanced by 5-HT and, like the enhancement of the NMDA responses, the action of 5-HT was only very slowly reversible (Reynolds et al., 1988). In a further study in the rat cortex, both glutamate and quisqualate depolarization was enhanced by 5-HT and this action of 5-HT was blocked by cinanserin, but not by methysergide (Nedergaard et al., 1986, 1987). 5-HT also inhibited the acetylcholine inward current and the nicotinic fast epsp in bullfrog sympathetic ganglia. The threshold dose for inhibition was about 5~M and 50% inhibition occurred with 50-100~M 5-HT. The antagonism was competitive and it was concluded that 5-HT acts by reducing the binding of acetylcholine to the nicotinic acetylcholine receptor (Akasu and Koketsu, 1986).

463

frequency of firing (Hounsgaard and Kiehn, 1989). The rate of accommodation may also be altered by the effect of 5-HT on the duration of the Ca spike. Thus a lengthening, or a shortening, of the Ca spike duration would be expected to increase or decrease, respectively, accommodation by enhancing or diminishing the Ca mediated AHP. A second consequence of the 5-HT induced suppression of GM and G ~ is an enhancement of the responsiveness of the neuron to excitatory synaptic input, with the increased fast epsps generating spikes more effectively. High amplitude and frequency activated inputs in particular will be enhanced, with the neurons generating prolonged bursts of spikes in response to barrages of epsps. Weak and low frequency inputs, however, will be relatively ineffective. The inward rectifying current Ih iS a current that produces an increase in conductance and depolarization when the membrane is hyperpolarized. Thus, it is a current that is particularly important in cells with pacemaker activity. The dorsal lateral and medial geniculate neurons were shown to have spontaneous rhythmic burst firing arising as rebound responses from rhythmic ipsps or afterhyperpolarizations. 5HT reduced the rhythmic burst firing, promoting a state of increased responsiveness to excitatory input 13. EFFECT OF 5-HT ON NEURONAL (Pape and McCormick, 1989). This occurred because EXCITABILITY 5-HT (by its conductance increase) reduced and 5-HT has a wide diversity of action on spike firing shortened the afterhyperpolarization and ipsps, and due to the large number of actions at the membrane thus reduced the rebound Ca spike which generated level of 5-HT. The simplest action of 5-HT is the the burst firing. The hyperpolarization produced by activation of direct initiation of action potentials by the fast and large depolarizing epsps, generated by activation of 5-HTIA receptors has been shown, as expected, to 5-HT3 receptors. A major consequence of the 5-HT inhibit spontaneous spike firing, for example in the induced suppression of K conductance, such as G~.~r hippocampal CAI (Colino and Halliwell, 1987). The and GM, is a block of accommodation. Accommo- population spike was also always reduced by 5-HT dation is the decrease in the train spike frequency, in CA1, the inhibition being most pronounced with and often cessation of spikes, which occurs during a low stimulation currents, and being less pronounced prolonged depolarizing current stimulus. 5,HT was with higher stimulation currents (Segal, 1980). This found to cause a block of accommodation in the demonstrates that there is a large increase in conduchippocampal pyramidal cells, in guinea-pig and hu- tance in the CA I cell body layer induced by 5-HT. man cortical pyramidal cells, and in cat, turtle, and The inhibitory effect of 5-HTIA receptor activation on lamprey spinal motoneurons (Andrade and Nicoll, the excitatory synaptic input though has been shown 1987a; Colino and Halliwell, 1987; McCormick and to be much less than expected. Thus 5-HT was found Williamson, 1989; Hounsgaard and Kiehn, 1989; to reduce the intracellular epsp in CA1 of the hippoWallen et al., 1989) and in myenteric neurons of the campus in only 6 out of 14 pyramidal cells, with an guinea-pig (Wood and Mayer, 1979). For example, in increase in 1 cell and no change in 7 cells (Segal, pyramidal neurons of the hippocampus and cortex, in 1980). In the lateral septal nucleus, 10 ~M 5-HT did which accommodation has a rapid onset and a large not alter the fast epsp amplitude in any cells, and the amplitude, a prolonged depolarizing current pulse fast ipsp was only weakly inhibited, by an average of only produces a maximum of 6--8 spikes in control 33% in 6 out of 9 cells (Joels and Gallagher, 1988). conditions. However, in the presence of, or a few The lack of, or relatively small, inhibitory effect of minutes following the washout of, 10-30/ZM 5-HT, 5-HT on the fast epsp may be due to an insufficient many more (13-20) spikes were evoked (Andrade and GiN increase in the synaptic area evoked by 5-HT. The slow ipsp in the septal nucleus was strongly Nicoll, 1987a; McCormick and Williamson, 1989). In the spinal motoneurons of cat and turtle, the late ahp inhibited by 5-HT, being reduced by an average of is smaller than in the CA I hippocampal pyramidal 80% (the inhibition still occurred when the memcells, and a prolonged depolarizing stimulus pro- brane potential was adjusted to E[psp (Joels and duced a train of spikes in which firing continued Gallagher, 1988)). The slow ipsp is also blocked by throughout the stimulus, and which displayed a fast 5-HT in the hippocampus (Segal, 1990). It is and a slow phase of accommodation (Hounsgaard suggested that the inhibitory effect of 5-HT on the and Kiehn, 1989). 5-HT, and 5-HT precursors, due to slow ipsp may be due to competition for identical K their block of the late afierhyperpolarization, and channels by 5-HT and the transmitter of the slow subsequent increase in G~N and plateau potential ipsp, which is likely to be 7-amino-butyric acid depolarization, caused an initial acceleration of spike (GABA) acting on GABA B receptors. 5-HT, acting firing which was followed by a jump to a higher via 5-HT~^ receptors, and GABA, acting via GABAB

464

R. ANWYL

receptors, are known to open an identical K channel population (Andrade et aL, 1986), and therefore if the number of such channels is limited, then the opening of a large percentage of these channels by 5-HT will result in a lack of such available channels for opening by GABA. Thus it is possible that a major role of 5-HTLA receptor activation at the synaptic level is to reduce the amplitude of the slow ipsp. 14. C O N C L U S I O N The neurophysiological recordings of membrane potential and membrane current generated b y a p p l i cation of 5-HT which have been described in this review have demonstrated the great variety o f neurophysiological actions of 5-HT in the nervous system. 5-HT can thus evoke a simple large fast depolarization, generated by an increase in conductance to N a and K (5-HT3 receptor); activate an "agonist" inward rectifying K conductance which generates a hyperpolarization; enhance the hyperpolarizing activated N a / K inward rectifying conductance, Gh; reduce the hyperpolarizing activated K inward rectifying conductance, Gin; reduce the depolarization activated M conductance, GM; reduce the slow Ca activated K conductance, G ~ p ; enhance a little studied N a / K conductances; facilitate, or inhibit, a Ca conductance; and evoke a Ca activated conductance. A specific receptor subtype is not always linked to the same conductance change. F o r example, the 5-HTm receptor subtype increases a N a / K inward rectifier in motoneurons, decreases or increases the duration of the Ca spike in dorsal root ganglia, and very widely increases a K inward rectifier. The 5-HT2 receptor would also appear to reduce at least two conductances, G M and Gm. REFERENCES AegAMETS, I. I., KOmSAgOV, I. Y. and SAMOVL1CH,I. M. (1989) Serotonin-induced responses of rat dorsal root ganglion neurons. Their metabolic and ionic dependence. Neurofiziologiya 21, 86-93. AGHAJANIAN,G. K. and LAKOSKI,J. M. (1984) Hyperpolarization of serotonergic neurons by serotonin and LSD: studies in brain slices showing increased K conductance. Brain Res. 30'3, 181-185. AGHAJAN1AN,G. K., SVROUSE, J. S. and P.O~MUSSEN,K. (1988) Electrophysiology of central serotonin ~ t o r subtypes. In: The Serotonin Receptors, Ed. E. SANDERS-BusH.The Humana Press. AKASU,T. and KOKETSU,K. (1986) 5-Hydroxytryptamine decreases the sensitivity of nicotinic acetylcholine receptor in bull-frog sympathetic ganglion cells. J. Physiol. ~ , 93-109. AVa~RAVE,R. and NICOLL,R. A. (1987a) P h a r m a c o l o g y distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro~ J. Physiol. 394, 99-124. ANDRADE,R. and NICOLL,R. A. (1987b) Novel amtiolytics discriminate between postsynaptic serotonin receptor mediating different physiological responses on single neurons of the rat hippocampus. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 336, 5-10. ANDRADE,R., MALENKA,R. C. and NICOLL,R. A. (1986) A G protein couples serotonin and GABAn receptors to the same channels in hippocampus. Science 7,34, 1261-1265.

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5-HT IN Tim VERTEBRAlZN~vous

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N O T E ADDED IN P R O O F The 5-HT induced hyperpolarization in rat hippocampal CAI neurons recorded with K acetate filled microelectrodes was greatly reduced when the electrode was filled with the fast Ca chelator Bapta. Thus it was postulated that the inward rectifying Gg activated by 5-HT is dependent on a transient rise in Cai, with the Ca released from intracellular stores (Segal, 1990). The outward current induced by 5-HT in rat CA3 hippocampal neurons was inhibited by kainic and quisqualic acid (Rovira et al., 1990). A hyperpolarization of frog motoneurons was produced by low (0.01-1.0/~M) concentrations of 5-HT, 8-OH-DPAT and ipsapirone, and this hyperpolarization was blocked by spiroxatrine and spiperone, suggesting a 5-HTIA receptor (Holohean et al., 1990). Evidence for two types of 5-HT induced hyperpolarization, one long-lasting mediated via 5-HT~A receptors and mimicked by 8-OH-DPAT, and

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R. ANWYL

another short lasting and mediated by a different receptor type, has been put forward in rat hippocampal neurons (Gurevich et al., 1990). Studies employing the technique of rat brain mRNA injection into oocytes showed that 5-HT2 receptor activation decreased the outward K current of the RBK1 delayed rectifier (North, cited by Henderson, 1990). High concentrations of 5-HT (I0-100 t~M) generated a slow long-lasting depolarization of frog motoneurons, an effect mimicked by the 5-HTtc/5-HT2 agonist ~-methyl-5-HT. The depolarization was blocked by the 5 - H T J 5 - H T 2 antagonists ketanserin, mianserin and methysergide (Holohean et al., 1990). The 5-HT induced depolarization in rat facial motoneurons was only partially abolished by ketanserin, suggesting that 5-HT2 receptors and another receptor subtype is responsible for the depolarization (Larkman, cited by Henderson, 1990). The 5-HT2/5-HTjc agonist 1-[2,5dimethoxy-4-methyiphenyl]-2-aminopropane (IX)M) induced a slow depolarization and an increase in evoked spikes in the rat facial nucleus (Rasmussen and Aghajanian, 1990). 5-HT was shown to reduce both the rate of activation and amplitude of a high threshold N-like Ca current with a transient and sustained component (activated by depolarization from - 100 mV to - I0 mV) in serotonergic neurons of the dorsal raphe (Pennington and Kelly, 1990). 5-Carboxyamidotryptamine, 5-HT, ~-methyl 5-HT, and 5methoxytryptamine possessed full agonist activity in shortening the Ca dependent plateau of spikes recorded from frog sensory neurons, with ECs0s of 19 riM, 210nm, 3.7/ZM and 1.7 l z i respectively. This suggests a 5-HT:like receptor (Scroggs and Anderson, 1990). In rat spinal motoneurons, 5-HT enhanced a low voltage activated Ca current (Berger and Takahashi, 1990). Further studies have verified that the single channel conductance of the 5-HT 3 receptor is extremely variable in different cell types. Peters, and Yakel and Jackson, reported single channel conductances of t7 pS, and 9pS and 3pS from rabbit nodose ganglia, and differentiated and undiffer-

entiated NGI08-15 neuroblastoma ceils respectively (cited by Henderson, 1990). Peters also reported the otubocurarine was approximately 100-fold more potent as an antagonist of 5-HT in NIE-115 neuroblastoma cells than in rabbit nodose ganglion cells (cited by Henderson, 1990).

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