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The effects of 5-hydroxytryptamine (5-HT) in the central nervous system are mediated through seven classes of receptors (Hoyer et al., 1994). At least four classes of 5-HT receptors (5-HT1–4) are known to modify neurone excitability and/or neurotransmitter release (Segal, 1976; 1980; Aghajanian & Lakoski, 1984; Segal et al., 1989; Andrade & Nicoll, 1987; Araneda & Andrade, 1991; Ropert & Guy, 1991; Beck et al., 1992; Torres et al., 1994). Although neurones may not express all known 5-HT receptors, 5-HT released by 5-hydroxytrypta-minergic terminals projecting to the hippocampus from the raphe nuclei is likely to change synaptic responses in this region (Segal, 1975; Segal & Weinstock, 1983; Segal et al., 1989; Klancnik & Phillips, 1991). In the rat hippocampus, 5-HT induces hyperpolarization of CA1 pyramidal cells through 5-HT1A receptor stimulation (Colino & Halliwell, 1987; Ropert, 1988; Beck et al., 1992; Passani et al., 1994; Corradetti et al., 1996). This inhibitory effect masks the concomitant block of a potassium conductance (Colino & Halliwell, 1987), probably exerted by 5-HT4 receptor stimulation, which leads to cell membrane depolarization and to increased excitability of pyramidal cells once the 5-HT1A receptor-mediated response is blocked (Andrade & Nicoll, 1987; Torres et al., 1994).
The increase in potassium conductance and consequent hyperpolarization produced by activation of 5-HT1A receptors strongly decrease the excitability of pyramidal cells and may participate in the blocking of long-term potentiation (LTP), as observed in CA1 and CA3 regions of the rat hippocampus (Corradetti et al., 1992; Villani & Johnston, 1993).
It has been suggested that in the CA1 region 5-HT decreases excitatory postsynaptic potentials (e.p.s.ps) by eliciting a 5-HT1A receptor-mediated increase in potassium conductance (see Anwyl, 1990 for a review) and/or presynaptic mechanism(s) involving a decrease in calcium conductance (Schmitz et al., 1995a). Indeed, several 5-HT1A receptor agonists such as 5-HT, 5-carboxamidotryptamine (5-CT), (8-hydroxy-2 di n-propylamino) tetralin (8-OH-DPAT), buspirone, and ipsapir-one (Jahnsen, 1980; Segal, 1980; Peroutka et al., 1987; Rowan & Anwyl, 1987; Ropert, 1988; Sakai & Tanaka, 1993; Schmitz et al., 1995a) reduce the electrically evoked e.p.s.p. in this brain region. However, all the compounds tested were not selective enough to insure that the observed decrease in e.p.s.p. was not due to stimulation of other 5-HT receptors. For instance, 8-OH-DPAT and 5-CT, in addition to their agonist action on 5-HT1A receptors, exert 5-HT1B/D receptor-mediated effects and bind to 5-HT6–7 receptors (Hoyer et al., 1994). Furthermore, 8-OH-DPAT may act as a partial antagonist (Colino & Halliwell, 1987; Beck et al., 1992) and decrease fibre volley through still non-elucidated mechanisms (Peroutka et al., 1987; Hiner et al., 1988). The antagonists available to us until now were either non-selective or they acted as partial or full agonists in the raphe nucleus (Greuel & Glaser, 1992), a phenomenon ascribed to the presence of a reserve of receptors in the 5-hydroxytryptaminergic cells of the raphe (Fletcher et al., 1993). In spite of these drawbacks, the evidence obtained with non-selective 5-HT receptor antagonists such as methysergide (Segal, 1980; Klancnik et al., 1991) spiperone (Beck et al., 1985; Rowan & Anwyl, 1987), methiothepin (Corradetti et al., 1992), NAN-190 (Sakai & Tanaka, 1993) converge to indicate a role of 5-HT1A receptors in the inhibitory modulation of e.p.s.ps by 5-HT.
WAY 100635 is a new antagonist devoid of intrinsic activity in several biochemical and behavioural tests (Fletcher et al., 1996) having a selective and competitive high-affinity binding for the 5-HT1A receptors. In vitro and in vivo autoradiographic studies with 3H-labelled WAY 100635 confirmed the regional distribution and receptor density differences previously revealed by binding of agonists (Laporte et al., 1994; Gozlan et al., 1995; Khawaja et al., 1995). In electrophysiological recordings in vitro, WAY 100635 acted as a full antagonist of 5-HT1A receptor-mediated responses in the CA1 region of the hippocampus and the dorsal raphe nuclei (Fletcher et al., 1996; Mundey et al., 1996; Corradetti et al., 1996).
The aim of the present work was to study the overall effect of the selective 5-HT1A antagonist WAY 100635 on neuro-transmission and to re-evaluate the role of these receptors in the modulation of excitatory neurotransmission in the CA1 region of the hippocampus.
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The major results of our investigation were that the 5-HT1A selective antagonist WAY 100635 blocked the 5-HT1A receptor-mediated responses and the decrease in excitatory neurotransmission elicited by 5-HT, whereas it did not affect either neurone excitability or synaptic transmission in the CA1 region of the rat hippocampus. We used 10 nM WAY 100635, a concentration that afforded full antagonism of 5-HT1A responses in CA1 pyramidal cells without affecting the selectivity of the drug (Fletcher et al., 1996; Corradetti et al., 31996). At this concentration, WAY 100635 did not change either the amplitude or the duration of e.p.s.ps evoked by the stimulation of the stratum radiatum. The antagonist did not modify any of the parameters of cell excitability including r.m.p., Rin, or action potential amplitude. In particular, it did not change either the action potential frequency adaptation or the slow AHP which follows the repetitive cell discharge caused by a long (400 ms) cell depolarization. In addition, the presence of WAY 100635 unmasked a depolarization elicited by 5-HT, that was blocked by the selective 5-HT4 antagonist RS 23597–190 hydrochloride, confirming that this response is indeed mediated by 5-HT4 receptors (Torres et al., 1995).
This work confirms and extends to excitatory synaptic potentials previous results which investigated the selectivity of WAY 100635 on 5-HT1A receptors in CA1 pyramidal neurons and 5-hydroxytryptaminergic cells of the dorsal raphe nuclei (Gozlan et al., 1995; Fletcher et al., 1996; Corradetti et al., 1996). The fact that WAY 100635 antagonized the decrease in e.p.s.p. amplitude elicited by 5-HT and the 5-HT1A agonist 5-CT demonstrates that activation of 5-HT1A receptors strongly inhibits excitatory neurotransmission in the CA1 region of rat hippocampus.
Previous studies showed that 5-HT1A antagonists (methy-sergide, spiperone, and NAN 190) which block 5-HT1A receptor-mediated hyperpolarization, also antagonize the reduction of excitatory synaptic transmission caused by 5-HT in the CA1 region of the hippocampus (Beck et al., 1985; Klancnik & Phillips, 1991; Sakai & Tanaka, 1993). Accordingly, 5-HT1A agonists (8-OH-DPAT, ipsapirone, buspirone) mimicked the effects of 5-HT on the e.p.s.ps (Rowan & Anwyl, 1987; Hiner et al., 1988; Sakai & Tanaka, 1993). However, these early results were obtained with non-selective agonists and antagonists and extracellular recordings. Our results are the first conclusive evidence that activation of 5-HT1A receptors modulate synaptic transmission in this brain region. Indeed, we used WAY 100635 which selectively binds to 5-HT1A receptors, both in vitro (Gozlan et al., 1995; Forster et al., 1995) and in vivo (Laporte et al., 1994; Fletcher et al., 1993), and blocks 5-HT1A receptor-mediated responses in the CA1 region, without affecting pyramidal cell excitability, or the responses due to activation of GABAB and 5-HT4 receptors (Corradetti et al., 1996 and present results).
As previously found by Segal (1980), we observed that the action of 5-HT on e.p.s.ps in control aCSF was small (-15%) in the presence of the GABAA receptor antagonist, though, 5-HT reduced the amplitude of the e.p.s.p. by 45%. This may be explained by the fact that two opposing factors contribute to the determination of the amplitude of the early e.p.s.ps: glutamatergic excitation and GABAergic inhibition via activation of GABAA receptors (Dingledine & Gjerstad, 1979). 5-HT decreases not only the excitatory but also the inhibitory neurotransmission that impinges upon pyramidal cells, through, presumably, 5-HT1A receptors (Schmitz et al., 1995b). Therefore, the modulatory effect of 5-HT on excitatory neurotransmission is counterbalanced by the reduced inhibitory effect of GABAergic interneurones. This apparently led to an overall larger reduction of the e.p.s.p. amplitude when GABAA neurotransmission was blocked, than when it was not.
5-HT decreased both the AMPA and NMDA components of the e.p.s.ps. Using extracellular recordings of dendritic potentials, Staubli & Otaky (1994) showed that 5-HT decreases the NMDA component of excitatory neurotransmission during conditioning trains aimed at inducing long term potentiation (LTP). This effect may contribute to the block of LTP exerted by 5-HT in the rat hippocampus (see also: Corradetti et al., 1992; Villani & Johnston, 1993). Our results directly demonstrate that 5-HT decreases the NMDA component of e.p.s.ps through a 5-HT1A receptor-mediated action. The fact that both components of e.p.s.ps were affected by 5-HT may suggest that 5-HT decreases glutamate release from presynaptic terminals. However, the reduction of the e.p.s.p. amplitude is more probably due to a postsynaptic mechanism, where the synaptic currents are shunted through the potassium conductance activated by the 5-HT1A receptors. At the moment, the location of the 5-HT1A receptors involved in the modulation of e.p.s.ps remains to be elucidated. Biochemical data suggest that 5-HT1A receptors have a postsynaptic location in the hippocampus (Pompeiano et al., 1992; Mengod et al., 1996), although Schmitz et al., (1995a) observed that the 5-HT1A agonist 8-OH-DPAT decreases e.p.s. currents recorded in CA1 pyramidal cells and suggested that 5-HT exerts its effects partially through presynaptic 5-HT1A receptors. In brain regions other than the hippocampus the release of neurotransmitters is inhibited by presynaptic 5-HT1B/D and 5-HT2 receptors (Maura et al., 1991; Tanaka & North, 1993; Maura & Raiteri, 1996). So far, though, there is no direct evidence of heterosynaptic 5-HT1B/D receptors modulating the release of excitatory aminoacids in the hippocampus. In addition, our experiments conducted with CGS 12066A, a 5-HT1B agonist (Neale et al., 1987), showed that the inhibitory action of 5-HT on synaptic potentials was not elicited by the interaction with 5-HT1B receptors. This finding is in agreement with results obtained in the CA1 region of the hippocampus, with CP 93129, another 5-HT1B agonist (Dr H.W.G.M. Boddeke, personal communication). Regardless of the location of the effectors, though, our results clearly demonstrate that the effects of 5-HT on e.p.s.ps are exerted through 5-HT1A receptors and that WAY 100635 blocks them. The CA1 region of the hippocampus appears to be involved in several functions of the CNS, including memory processing, anxiety and mood control. 5-HT released by terminals originating in the dorsal raphe nuclei regulates CA1 pyramidal cell discharge through activation of several types of receptors. It is conceivable that at least part of the anxiolytic and antidepressant effects observed with 5-HT1A agonists in vivo (reviewed by De Vry, 1995) result from inhibition of CA1 pyramidal cells.
In conclusion, our results demonstrate that WAY 100635 per se is devoid of any noticeable effects on e.p.s.ps, while it selectively blocks the decrease in neurotransmission produced by 5-HT. This latter finding, obtained by using the most potent and selective 5-HT1A receptor antagonist currently available, also demonstrates that 5-HT inhibits e.p.s.ps through stimulation of 5-HT1A receptors. WAY 100635 has been recently used in healthy people to delineate the regional distribution of 5-HT1A receptors (Pike et al., 1995; Osman et al., 1996) and the knowledge of the functional consequences of the blockade of 5-HT1A receptors may become necessary in order to interpret these investigations. The lack of aspecific effects of WAY 100635 on synaptic transmission further supports the potential usefulness of this compound in studies on central 5-HT1A receptor location and function in vivo.