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Keywords:

  • 5-HT;
  • 5-CT;
  • WAY 100635;
  • RS 23597-190;
  • CGS 12066A;
  • 5-HT1A receptor;
  • 5-HT4 receptor;
  • CA1 hippocampus;
  • synaptic potentials

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
  • The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine1A (5-HT1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.

  • WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (Rin), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.

  • Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔVm=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced Rin (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.

  • 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABAA receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).

  • WAY 100635 fully antagonized the hyperpolarization, the reduction of Rin, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT4 receptor antagonist.

  • Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT4 receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT1A receptors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

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).

On the other hand, 5-HT is known to either inhibit or excite GABAergic interneurones in the CA1 region by acting on 5-HT1A (Van den Hooff & Galvan, 1991; Schmitz et al., 1995b), and/or 5-HT3 receptors (Ropert & Guy, 1991; Passani et al., 1994). These actions may concur to change synaptic excitatory responses in the CA1 region.

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.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Preparation of hippocampal slices

Experiments were carried out with rat hippocampal slices in vitro, as previously described (Corradetti et al., 1996). Male Wistar rats (100–200 g body weight, Charles River, Como, Italy), were killed by decapitation. The hippocampi were rapidly removed and placed on ice-cold oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF) of the following composition (mM): NaCl 124, KCl 3.0, NaH2PO4 1.25, MgSO4 1.4, CaCl2 2, NaHCO3 25, D-glucose 10 (pH 7.4). Hippocampal slices (400 μM thick) were cut with a McIlwain tissue chopper (Gomshall, U.K.) and kept in oxygenated aCSF for at least 1 h at room temperature. A single slice was then placed on a nylon mesh, completely submerged in a small (700 μl) chamber, and superfused with oxygenated aCSF (28–30°C) at a constant flow rate of 2–3 ml min−1. Drugs were administered through a three-way tap and a complete exchange of the chamber volume occurred within 1 min. Antagonists were superfused for at least 20 min before the effects of 5-HT were tested.

Intracellular recording

CA1 pyramidal neurones were recorded in current-clamp mode with 2 M K-methylsulphate- (50–80 Mβ) or 3 M KCl-(35–50 MΩ) filled electrodes. Test pulses (80–110 μs duration; 0.017–0.05 Hz), triggered either by a pulse programmer (Digitimer 4030, Digitimer, Welwyn Garden City, U.K.) or by an IBM-compatible PC, were delivered by stimulus isolation units (DS2, Digitimer) through bipolar nichrome electrodes positioned in the CA1 stratum radiatum. Stimulus-response curves were constructed at the beginning and during the experiment by gradually increasing the stimulus strength. Electrical signals were amplified with an Axoclamp 2A (Axon Instruments, Foster City, U.S.A.) and displayed on an oscilloscope and chart recorder (2800 Gould, Valley View, U.S.A.). Traces were stored on a digital tape (DTR 1200, BioLogic, Claix, France; sampling frequency 48 kHz) and on a computer by use of pClamp software (Axon Instruments) for off-line analysis. Several criteria were used to accept cells for experiments: stable resting membrane potential (r.m.p.) of at least −60 mV and no spontaneous firing of action potentials; no sudden drops in the neuronal input resistance (Rin) indicating cell damage; constant amplitude of the spike (geqslant R: gt-or-equal, slanted 80 mV) obtained by direct activation of the cell. Modifications of synaptic efficacy by drugs were evaluated by measuring the changes in slope and amplitude of the evoked e.p.s.p. In some experiments the e.p.s.ps were evoked both at r.m.p. and during injection of hyperpolarizing current steps to allow simultanous measurement of cell Rin and e.p.s.p. amplitude, maintained below threshold for cell discharge. To study the effects of 5-HT on cell discharge, direct activation of the impaled neurone was obtained by injection of long (400–500 ms, for action potential frequency adaptation) depolarizing current steps through the recording electrode. The slow AHP (sAHP) which follows repetitive firing elicited by injection of depolarizing current (+ 500 pA, 400 ms) through the recording electrode were measured 150 ms after cessation of the depolarizing step. A series of hyperpolarizing current steps (400–500 ms duration, −500/+ 500pA) were injected repeatedly to monitor cell Rin and to construct I/V plots. The effects of 5-HT on the N-methyl-D-aspartate (NMDA) receptor-mediated component of the synaptic potential were studied in the presence of 10 μM NBQX (6-nitro-7-sulpha-moylbenzo(f)quinoxaline hydrochloride), a selective AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-receptor antagonist, and 10 μM bicuculline to block γ-aminobutyric acid (GABA)A receptor-mediated inhibition. The effects of 5-HT on the AMPA receptor-mediated components of e.p.s.ps were studied in the presence of 50 μM D(—)-2-amino-5-phosphonopentanoic acid (D-AP5), a selective NMDA receptor antagonist and 10 μM bicuculline.

Drugs

WAY 100635 ((N-(2-(-4(2-methoxyphenyl)-1-piperazinyl) ethyl)-N-(2-pyridinyl) cyclohexane carboxamide) was kindly provided by Dr Michel Hamon; (—)-bicuculline methiodide was from Sigma (St. Louis, U.S.A.); 5-hydroxytryptamine creatinine sulphate, 5-CT (5-carboxamidotryptamine), picrotoxin and CGS 12066A (7-trifluoromethyl-4-(4-methyl-1-piperazinyl)pirrolo[1,2-α]quinoxaline) were from RBI (Natick, U.S.A.); D-AP5, 6-nitro-7-sulphamoylbenzo(f)quinoxaline hydrochloride (NBQX) and RS 23597–190 hydrochloride (3-(piperidin-1-yl)propyl-4-amino-5-chloro-2-methoxy benzoate) were from Tocris (Bristol, U.K.).

Data analysis

All numerical data are expressed as mean ± s.e.; for statistical analysis, Wilcoxon or Mann-Whitney tests, as appropriate, were employed. A value of P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

CA1 pyramidal cells were recorded from 56 slices taken from 49 rats. Neurones (n= 56) had a r.m.p. of −65.4 ± 1.5 mV, Rin of 46.3 ± 1.7 MΩ, and action potential amplitude greater than 80 mV (88.3 ± 1.9 mV). In most of the experiments, and in particular when neurones were uninhibited, cells were manually clamped at a membrane potential (-72.0 ± 1.6 mV) hyperpolarized relative to their original r.m.p. by injecting a constant negative current of 50–180 pA through the recording electrode. Although this procedure decreased the amplitude of the hyperpolarization produced by 5-HT1A agonists, it allowed better investigation of stimulus-response relationships. When necessary, the current was adjusted during the application of 5-HT1A agonists to keep the membrane potential at the level chosen for a given cell (see e.g. Figures 3a, 4a).

image

Figure 3. WAY 100635 (WAY) selectively antagonizes the membrane hyperpolarization and the decrease in e.p.s.p. amplitude induced by application of 5-HT in a CA1 hippocampal neurone. (a) Chart record of the membrane potential recorded from a CA1 pyramidal cell in which 5-HT (10 μM) was applied alone or in the presence of the 5-HT1A antagonist WAY 100635 (10 nM). The sequence of downward/upward deflections are cell membrane responses to negative/positive current pulses (-500/+ 500 pA, varied by 100 pA, 400 ms, 1/7 s) injected through the recording electrode to construct I/V curves and to monitor action potential frequency adaptation (accommodation). The following downward deflections are electrotonic cell membrane responses to constant current pulses (-300 pA, 400 ms, 1/30 s) to monitor Rin Upward deflections indicated by arrows are e.p.s.ps evoked by electrical stimulation (1.7 V) of stratum radiatum. During 5-HT application the membrane potential was manually clamped at the control value (-69 mV) by injecting +250 pA (+ d.c.) through the recording electrode (left panel). Note that the effects of 5-HT were completely blocked by WAY 100635 (right panel) and that in the presence of the antagonist, 5-HT elicited a small depolarization. Membrane potential was clamped at the control value by injecting negative current (-d.c: −90 pA). KCl-filled electrode, r.m.p.: −69 mV. Rin: 43 MΩ. (b) E.p.s.ps evoked by stimulation (1 V, fast transient) of stratum radiatum. Upper: control (left trace), 10 min after 10 μM 5-HT application (middle trace). Lower panel: WAY 100635 (10 nM) alone (left trace) or in combination with 10 μM 5-HT (middle trace). In the right panel corresponding traces are superimposed. KCl-filled electrode, r.m.p.: −68 mV. Rin: 30 MΩ.

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image

Figure 4. WAY 100635 blocked the decrease in the amplitude of AMPA receptor-mediated e.p.s.p. elicited by 5-HT. The responses were obtained in the presence of bicuculline (10 μM) and D-AP5 (50 μM). (a) Continuous chart record of the resting membrane potential of a CA1 pyramidal cell in which 5-HT (10 μM) was applied alone or in combination with 10 nM WAY 100635. The sequence of downward/upward deflections are cell membrane responses to negative/positive current pulses (-500/+ 500 pA, varied by 100 pA, 400 ms, 1/7 s) injected through the recording electrode to construct //V curves and to monitor action potential frequency adaptation (accommodation). The following downward deflections are electrotonic cell membrane responses to constant current pulses (-300 pA, 400 ms, 1/30 s) to monitor Rin Upward deflections indicated by arrows are stimulus-response curves constructed by gradually increasing the stimulus strength of the electrical stimulation of stratum radiatum. During 5-HT application the membrane potential was clamped at the control value (-66 mV) by injecting (+ d.c; +190 pA) through the recording electrode (left panel). In the right panel membrane potential was clamped at the control value by injecting negative current (-d.c: −30 pA). KCl-filled electrode, r.m.p.: −66 mV. Rin: 60 MΩ. (b) E.p.s.ps evoked by stimulation (17 V, fast transient) of stratum radiatum. Upper panel: control (left trace), 10 min after 10 μM 5-HT application (middle trace). Lower panel: WAY 100635 (10 nM) alone (left trace) or in combination with 10 μM 5-HT (lower, middle trace). In the right panel the corresponding traces are superimposed. KCl-filled electrode, r.m.p.: −66 mV. Rin: 60 MΩ.

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Effects of 5-HT and of 5-CT on e.p.s.ps evoked in control or in bicuculline-containing aCSF

In an initial series of experiments, we tested the effects of 5-HT and 5-CT, a high-affinity 5-HT1A receptor ligand with maximal agonist effects (Beck et al., 1992; Corradetti et al., 1996), on e.p.s.ps recorded intracellularly from CA1 pyramidal cells in control aCSF. Stimulation of the stratum radiatum evoked e.p.s.ps which increased in amplitude in relation to the stimulus strength and eventually reached the threshold for discharging an action potential. As previously shown, super-fusion of 5-HT (30 μM, 5 min application) caused cell membrane hyperpolarization (ΔVm=−6.3 ± 1.1 mV) accompanied by a decrease in Rin (-40.9 ± 5.0%). When the stimulation of the stratum radiatum was repeated in the presence of 5-HT the amplitude of the e.p.s.ps decreased from 6.3 ± 1.0 to 5.2 ± 0.8 mV (− 15.3 ± 3.8%, n= 6; P < 0.02; Figure 1a).

image

Figure 1. Effect of 5-HT and 5-CT on the amplitude of electrically evoked e.p.s.ps in CA1 pyramidal neurones. (a) Traces are averages of five consecutive responses evoked by stimulation (2.5 V, downward transient) of stratum radiatum in control conditions (left) and after 10 min of superfusion with 30 μM 5-HT (middle). Right panel shows the two responses superimposed. KCl-filled electrode. r.m.p.: −71 mV; Rin: 35 MΩ. (b) E.p.s.ps were induced by electrical stimulation (2.7 V, downward transient) of the stratum radiatum in the presence of bicuculline (10 μM). The cell was kept at a Vm of −73 mV by injection of −150 pA to avoid cell firing during synaptic stimulation. Upper panel: left trace: control; middle trace: 300 nM 5-CT (10 min application). Lower panel: left trace: 30 min washout of 5-CT; middle trace: 10 min superfusion of 5-HT (10 μM, 10 min). Note that in the presence of bicuculline, evoked e.p.s.ps were longer in duration than in control. Right panels: corresponding left and right superimposed traces. KCl-filled electrode, r.m.p.: −66 mV; Rin: 55 MΩ.

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Since 5-HT may affect the stimulus-evoked activity of GABAergic interneurones (see Introduction), unless otherwise stated, the following experiments were carried out in the presence of 10 μM bicuculline, to block the GABAA receptor-mediated fast inhibition. As expected, under these conditions the e.p.s.ps evoked were longer in duration than in control aCSF (Figure 1b).

Application of 5-HT (10 μM; N= 6) hyperpolarized pyramidal cells (ΔVm=−4.0 ± 0.9 mV) decreased both the Rin (-24.7 ± 6.8%) and the action potential frequency adaptation measured by injecting 400 ms depolarizing (+ 500 pA) steps (the number of spikes increased from 9.0 ± 1.6 to 13.2 ± 3.4). Also, the amplitude of the slow afterhyperpolarization (sAHP) that develops at the end of the depolarizing step decreased from −3.0 ± 0.7 to −1.5 ± 0.5mV. 5-HT reduced the amplitude of stimulus-evoked e.p.s.ps from 8.8 ± 1.7 to 4.5 ± 1.0mV (P < 0.05). Similar results were obtained with 5-CT (300 nM; n= 4), which hyperpolarized the cells (ΔVm=−5.6 ± 0.7mV; n= 4) and reduced the Rin (-18.1 ± 1.4%). The e.p.s.p. amplitude was reduced from −6.8 ± 1.2 to 3.6 ± 0.9 mV (P < 0.01). The effects of both 5-HT and 5-CT were observed at all stimulus strengths tested but were greater at stimulus strengths close to the threshold for eliciting an action potential (Figure 2).

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Figure 2. 5-HT and 5-CT decreased the amplitude of evoked synaptic potentials in a CA1 pyramidal neurone. Stimulus-response relationships were constructed in control conditions by gradually increasing the stimulus strength. The protocol was repeated after 10 min superfusion of 300 nM 5-CT; after 20 min washout (wash 1), 10 min application of 10 μM 5-HT and 20 min after the second washout (wash 2). Data were obtained from the same cell shown in Figure 1b. Note that the effects of both 5-HT and 5-CT were greater at higher stimulus strengths.

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Since both 5-HT and 5-CT bind to 5-HT1B receptors, which are known to decrease release of neurotransmitters, including glutamate, in the central nervous system (Maura et al., 1991; Maura & Raiteri, 1996), we tested the action of CGS 12066A, endowed with maximal agonist effect on 5-HT1B receptors (Neale et al., 1987), on e.p.s.ps. The compound (10 μM) neither affected cell membrane properties (ΔVm= 0.7 ± 0.8 mV, RinΔ%= 6.3 ± 0.8), nor the amplitude of evoked e.p.s.ps (from 8.7 ± 0.8 in control to 8.2 ± 1.1 mV, n= 3). Similar results were obtained by with 30 μM CGS 12066A (n= 2, data not shown).

Effects of WAY 100635 on neurone excitability and on e.p.s.ps

Superfusion of WAY 100635 (10 nM, 30 min) did not change the excitability of CA1 pyramidal cells: neither the membrane potential (AVm= 0.1 ± 0.1 mV) nor the Rin (from 41.9 ± 5.5 to 41.5 ± 5.5 MΩ; n= 9) were affected. The action potential amplitude (from r.m.p. to peak) was 102.3 ± 2.5 mV in control and 103.9 ± 3.5mV in WAY 100635-treated cells, and the threshold for firing an action potential upon intracellular current injections was 14.3 ± 1.3 mV in control and 14.6 ± 1.6mV in the presence of WAY 100635 (n= 5). In addition, WAY 100635 did not change the action potential frequency adaptation (the number of action potentials before and during application of WAY 100635 were 12.2 ± 2.1 vs 12.3 ± 2.5), nor the amplitude of the sAHP (from −3.6 ± 0.7 to 3.5 ± 1.0mV; n= 6). In the presence of WAY 100635, the e.p.s.p. amplitude did not change (from 7.7 ± 1.0 to 7.3 ± 1.0mV). WAY 100635 completely antagonized the hyperpolarization and the decrease in Rin caused by application of 5-HT (10–30 μM; n= 7; Figure 3). In the presence of WAY 100635, 5-HT elicited a depolarization (ΔVm=+1.7 ± 1.5 mV; n= 7, Figure 3a), probably due to activation of 5-HT4 receptors (Andrade & Nicoll, 1987; Torres et al., 1994), since this effect was completely antagonized by 10–30 μM RS 23597–190 hydrochloride, a selective (Eglen et al., 1993) 5-HT4 antagonist (ΔVm= 0.1 ± 0.1 mV; n= 7).

The presence of 10 nM WAY 100635 abolished the decrease in e.p.s.p. amplitude caused by 5-HT, both in control aCSF (from 6.2 ± 0.7 to 6.1 ± 0.8 mV; n= 4) and in the presence of bicuculline (from 7.8 ± 1.0 to 7.5 ± 1.0 mV; n= 4; Figure 3b). The effects of WAY 100635 persisted after wash (up to 2 hours), confirming the very slow dissociation of the antagonist from the receptor (Corradetti et al., 1996).

Since the effect of 5-HT was greater at higher stimulus strengths (see Figure 2), i.e. when the evoked (disinhibited) e.p.s.p. was likely to involve the activation of NMDA receptors (Collingridge et al., 1988), we carried out a series of experiments on the AMPA and NMDA components of e.p.s.p. isolated pharmacologically. The AMPA component of the e.p.s.p. was isolated by the presence of 50 μM D-AP5, superfused for at least 25 min. 5-HT (10 μM) decreased the amplitude of the e.p.s.ps from 9.2 ± 1.1 to 5.5 ± 0.7 mV (n= 6; Figure 4). This effect was fully antagonized by 10 nM WAY 100635 (n= 6; Figure 4).

In 3 experiments the NMDA component of the e.p.s.p. was isolated by the addition of 10 μM NBQX in the superfusing solution (Figure 5). The demonstration that in these conditions the evoked synaptic response was NMDA-mediated, was two fold. Firstly, when afferent fibres were stimulated, the voltage-dependence of a pure NMDA receptor-mediated response was different from the voltage dependence of an NMDA/AMPA e.p.s.p. (compare a and b in Figure 5). In addition, D-AP5 (50 μM) completely blocked the synaptic response obtained in the presence of NBQX (not shown). In these neurones 5-HT significantly decreased the amplitude of the NMDA component of the e.p.s.p. from 4.9 ± 0.6 to 3.6 ± 0.9 mV at resting membrane potential. This effect was completely antagonized by WAY 100635 (Figure 5b).

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Figure 5. WAY 100635 antagonized the decrease in amplitude of the NMDA receptor-mediated component of e.p.s.ps induced by 5-HT. (a) E.p.s.ps evoked by stimulation (3.3 V) of stratum radiatum in the presence of bicuculline (10 μM). Synaptic potentials were recorded in the CA1 pyramidal neurone at two different membrane potentials: r.m.p. (-70 mV) and during a hyperpolarizing step (-200 pA, 400 ms). Note that superfusion of 10 nM WAY 100635 per se did not affect membrane resistance and e.p.s.p. amplitude, while it completely antagonized the effects induced by 10 μM 5-HT. KCl-filled electrode. r.m.p.: −70 mV. Rin: 40 MΩ. (b) NMDA receptor-mediated e.p.s.ps, isolated by the presence of NBQX (10 μM), were evoked (6 V, fast transient) at r.m.p. (-74 mV) and during a hyperpolarizing step (-500 pA, 400 ms). Note the voltage-dependent amplitude of responses. KCl-filled electrode. Rin: 52 MΩ.

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In a separate series of experiments, we tested the possibility that the first application of 5-HT, acting through one of its several non 5-HT1A receptors, might have changed the responsiveness of pyramidal cells to a second application of the agonist. In the presence of WAY 100635 applied from the beginning of the recording, 5-HT (10–30 μM) neither hyperpolarized pyramidal cells nor affected the amplitude of e.p.s.ps (n= 7) or of their AMPA (n= 3) and NMDA (n= 4) components.

The decrease in e.p.s.p. amplitude elicited by 5-HT and its antagonism by WAY 100635 in all the conditions tested are summarized in Figure 6, where the statistical significance of the changes is also indicated.

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Figure 6. Comparison of the effects of 5-HT, alone or in combination with WAY 100635, on excitatory synaptic potentials evoked under different pharmacological treatments. Effects of 5-HT (30–10 μM, 5–7 min application) in the absence and in the presence of 10 nM WAY 100365 in different experimental protocols. Each column is the mean ± s.e. of the values expressed as the percentage of control. Asterisks indicate a statistically significant difference (P < 0.05) from controls and from the corresponding treatment with WAY 100635.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

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.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was supported by grants from the E.C. (BIO4-CT96–0752) and from the University of Florence.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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