Introduction

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Previous autoradiographic studies in the rat brain have documented that serotonin 5-hydroxytryptame (5-HT)_1A receptors are located predominantly in the hippocampus, the entorhinal cortex, and the septal area as well as in the raphe nuclei (Pazos and Palacios, 1985; Verge et al., 1985; Weissman-Nanopoulos et al., 1985). These 5-HT_1A receptors are located postsynaptically in the hippocampus and other limbic areas, and presynaptically on 5-HT cell bodies or dendrites (somatodendritic autoreceptors) in the raphe nuclei (Pazos and Palacios, 1985; Verge et al., 1985; Weissman-Nanopoulos et al., 1985). Activation of the somatodendritic autoreceptors by both 5-HT and 5-HT_1A agonists results in inhibition of 5-HT neuronal firing and, consequently, a decrease of 5-HT release from axon terminals (Sharp et al., 1989; Glaser and De Vry, 1992).

Although direct activation of postsynaptic 5-HT_1A receptors by 5-HT also inhibits the firing of hippocampus pyramidal neurons, the effects of 5-HT_1A agonists on these neurons is more complex (Sprouse and Aghajanian, 1988; Blier and de Montigny, 1987, 1990). For example, Blier and de Montigney (1987, 1990) demonstrated that 5-HT_1A agonists are more potent when applied to dorsal raphe 5-HT neurons than when applied to CA3 hippocampal pyramidal neurons. They also demonstrated that coadministration of 5-HT_1A agonists antagonizes the suppressant effect of 5-HT on CA3 hippocampal pyramidal neurons, but fails to do so on dorsal raphe 5-HT neurons. In addition, in vitro extracellular recordings have revealed that 5-HT_1A agonists antagonize the hyperpolarizing effect of 5-HT in dorsal hippocampus pyramidal neurons (Andrade and Nicoll, 1987). These diverse activities may be due to regional differences in 5-HT_1A receptor reserve. Meller et al. (1990) reported that a large receptor reserve exists in the raphe nucleus, whereas Yocca et al. (1992) found the 5-HT_1A receptor reserve lacking in the hippocampus. These previous studies in anesthetized animals and brain slices indicate that 5-HT_1A agonists may act as antagonists in the presence of competing with endogenous 5-HT for receptor sites. Therefore, it is desirable to use unanesthetized animals to investigate the effect of 5-HT_1A partial agonists on postsynaptic 5-HT_1A receptors since the activity of serotonergic neurons is reduced during slow-wave sleep and under chloral hydrate anesthesia (Hyem et al., 1984; Jacobs, 1985). Recently, Fornal et al. (1994a, 1996) demonstrated that 5-HT_1A autoreceptor antagonists spiperone and WAY 100635 increase the activity of serotonergic neurons in freely moving cats. They also observed that the stimulatory action of these antagonists was evident during wakefulness (when serotonergic neurons typically display a relatively high level of activity) but not during sleep (when serotonergic neurons display little spontaneous activity). These observations indicate that the effect of 5-HT_1A receptor antagonists are dependent on the behavioral state of the animals. Thus, it is important to investigate the effects of 5-HT_1A agonists on neuronal activity in awake animals. This approach is especially important since the use of anesthesia is known to reduce dramatically the spontaneous firing rate of pyramidal neurons in the hippocampus (Sprause and Aghajanian, 1988; Blier and de Montigny, 1987, 1990).

We also tested the interaction between buspirone and putative 5-HT_1A antagonists 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190) and ()-pindolol to determine whether or not the effect of buspirone on the spontaneous pyramidal cell activity was mediated by 5-HT_1A receptors. Furthermore, since systemic administration of buspirone activates both somatodendritic and postsynaptic 5-HT_1A receptors, it was important to establish whether the effects of 5-HT_1A agonist on spontaneous firing rates of hippocampal pyramidal neurons is mediated through the activation of somatodendritic or postsynaptic receptors. We, therefore, examined the effects of buspirone after administration of a serotonin synthesis inhibitor, parachlorphenylalanine (PCPA) (Koe and Weissman, 1966).

	Materials and Methods

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Animal Preparation. Adult male Wistar rats weighing 260 to 300 g at the time of surgery were housed three per cage in a temperature (23 ± 0.5°C)- and light-controlled room (light on from 7:00 AM to 7:00 PM) and had free access to food and tap water.

Surgical Procedures. Surgical procedures were performed under pentobarbital (60 mg/kg i.p.) anesthesia, with supplementary injections as necessary. Rats were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The microdrive, which consists of an inner stainless steel cannula (22-gauge) that can be fed through an outer guide cannula (19-gauge), was implanted stereotaxically above the right hippocampus. Two tungsten electrodes (tip diameter 25 µm) or 7 Formvar-insulated nichrome wires (tip diameter 32 µm diameter) were used as recording electrodes. Electrodes were lowered through the inner stainless steel cannula so that each tip was positioned 1 mm above the CA1 hippocampal region. Stereotaxic coordinates with the incisor bar at 1.0 mm from the interaural line were 4.0 mm anterior to the  and 2.7 mm lateral to midline. Stainless steel screw electrodes were threaded into the skull above the right frontal cortex for recording the electroencephalogram and above the left frontal cortex for ground. The entire apparatus was anchored to the skull with dental cement. These rats recovered for at least 7 days before the experiment was begun.

Electrophysiological Recordings and Neuronal Identification. Extracellular single-unit potentials from the microelectrodes were amplified (MEG-2100; Nihon Kohden, Tokyo, Japan), filtered (band-pass, 0.3-10 kHz), monitored continuously on a storage oscilloscope (VC-6023; Hitachi, Tokyo, Japan), and stored on magnetic tape using a data recorder (MR 20, tape speed 38 cm/s; Teac). The data obtained were printed out by thermal recorder (RTA 1200, paper speed 10 cm/s; Nihon Kohden). Cortical and hippocampal EEGs were amplified, band-pass filtered, and recorded (EEG-5109; Nihon Kohden) continuously on the polygraph. Head amplifiers (operational amplifier; TL074CN; Texas Instruments, Dallas, TX) were used to eliminate cable movement artifacts. All experimental trials were conducted in a small acrylic chamber (40 × 40 × 40-cm high). The microdrive was advanced through the hippocampus in small steps (~25 µm) until stable single-unit recordings characteristic of pyramidal neurons were encountered. Only recordings that displayed a signal-to-noise ratio of at least 5:1 were used for data collection.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Unit recordings of hippocampal CA1 pyramidal neurons in awake, unrestrained rat. A, a typical thermal recorder tracing of complex-spike cell firing. B, response of pyramidal neurons to s.c. injection of buspirone (1 and 6 mg/kg). Buspirone (Busp.) produced a dose-dependent inhibition of unit activity.

Drug Administration. Single-unit activity was recorded before and after s.c. injections of 5-HT_1A agonist and antagonist compounds. Buspirone, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), ipsapirone, flesinoxan, and NAN-190 were dissolved in 0.9% NaCl. ()-pindolol was dissolved in a minimum amount of acetic acid and brought to volume with 0.9% NaCl.

Data Collection. O'Keefe and Dostrovsky (1971) reported that complex-spike cells in the hippocampus fire at high rates in specific places within an experimental environment. Since this space-specific activity may make precise interpretation of drug response difficult, data were obtained each time the rat was located in the same part of the chamber. Data were also collected when the rat showed no observable body movement with its eyes open. This was necessary because the activity of pyramidal neurons has been shown to vary in association with the behavioral states (Buzaski et al., 1983; Suzuki and Smith, 1985). The percentage of change in firing rates was determined by comparing the discharge rate over a 20-s period during the time of maximum drug effect to the rate during the period immediately preceding the injection. It was often very difficult to obtain stable recordings for periods of greater than 20 s in the same chamber location, especially after injection.

Drugs. The following drugs were used: 8-OH-DPAT HBr (Sigma Chemical Co., St. Louis, MO), buspirone HCl (Sigma), ipsapirone HCl (Sigma), flesinoxan HCl (a gift from Solvay Duphar, Weesp, the Netherlands), NAN-190 HBr (RBI, Natick, MA), and ()-pindolol (a gift from Novartis Pharma, Basel, Switzerland).

Histology. Verification that the recording electrodes were in the CA1 region was obtained by histological means. At the end of the recording session, rats were anesthetized deeply with lethal doses of pentobarbital sodium, and direct anodal current was passed through the recording electrode (20 µA for 30 s) at sites where acceptable units were recorded. Rats were then perfused intracardially with physiological saline followed by 10% Formalin. If nichrome wire was used, 5% potassium ferrocyanide in Formalin was then perfused to produce a Prussian blue reaction. The location of the resulting dye deposition was analyzed histologically.

Statistics. Data are expressed as means ± S.E.M. A one-way repeated-measures analysis of variance (ANOVA) was used to test the effect of 5-HT_1A agonists and 5-HT_1A antagonists per se. The dose of a drug required to produce a 50% suppression in neuronal activity (ED₅₀) was calculated for each 5-HT_1A agonist along with the 95% confidence limit using the dose-response curve and least-squares linear regression analysis. The degree of statistical significance of the difference among ED₅₀ values of 5-HT_1A agonists was evaluated with a one-way ANOVA. A two-way repeated-measures ANOVA was performed to examine interactions of the 5-HT_1A antagonists and PCPA with the effect of buspirone. All post hoc analyses used the Student-Newman-Keuls test. Chi-square test was used to compare the single-spike/complex-spike ratio before and after buspirone treatment. A P < .05 was considered to denote statistical significance.

	Results

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Data were obtained from 126 cells in 50 rats. Mean firing rates of pyramidal neurons during quiet waking (1.24 ± 0.1 Hz) were comparable to rates previously reported during similar behavioral states (Suzuki and Smith, 1985). Measurements before and after saline injection were relatively stable across data points (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Response of hippocampal CA1 pyramidal neurons in awake, unrestrained rat to a s.c. injection of saline. The number of neurons examined is indicated in parentheses. Measurements before and after saline injection were relatively stable across data points. Values are means ± S.E.M.

Effects of 5-HT_1A Agonists on Activity of Hippocampal CA1 Pyramidal Neurons. Subcutaneous administration of 8-OH-DPAT, buspirone, ipsapirone, and flesinoxan produced a dose-dependent inhibition of the spontaneous basal firing rate of the hippocampal CA1 pyramidal neurons in unrestrained, unanesthetized rats (Figs. 1B and 3). The effects of ipsapirone on the spontaneous firing rate appeared to peak at a dose of 1 mg/kg. The intrinsic activity of ipsapirone in suppressing the spontaneous firing was weaker than the activity of the other tested 5-HT_1A agonists. Table 1 summarizes the ED₅₀ of 8-OH-DPAT, buspirone, and flesinoxan and their 95% confidence limits. Because higher ipsapirone concentrations could not be prepared for s.c. injection, the ED₅₀ value for ipsapirone could not be calculated. A statistically significant potency difference in suppressing the spontaneous firing activity was detected between 8-OH-DPAT and flesinoxan (P < .05). The single-spike/complex-spike ratio was not altered after 0.3 mg of buspirone (Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Dose-response curves for the suppression of hippocampal CA1 pyramidal neurons produced by s.c. injection of 8-OH-DPAT, buspirone, ipsapirone, and flesinoxan. Values are means ± S.E.M. The number of neurons tested is given in parentheses. All drugs produced dose-dependent inhibition of unit activity. Statistically significant decreases in firing were observed at doses of 0.1 to 0.3 mg/kg 8-OH-DPAT, 0.1 to 6 mg/kg buspirone, 0.1 to 3 mg/kg ipsapirone, and 0.3 to 3 mg/kg flesinoxan. * P < .05, ** P < .01, *** P < .001 versus respective baseline by one-way repeated-measures ANOVA and Newman-Keuls test.

Effects of NAN-190 and ()-Pindolol Pretreatment on the Neuronal Suppression Produced by Buspirone. To determine whether the suppression of hippocampal CA1 pyramidal neuronal activity produced by buspirone was antagonized by NAN-190 and ()-pindolol, the action of buspirone was examined with these putative 5-HT_1A antagonists. The responses of hippocampal CA1 pyramidal neurons to a challenge dose of buspirone (1 mg/kg) with and without NAN-190 and ()-pindolol are shown in Fig. 4. Each antagonist alone showed no significant effect on basal firing rate of hippocampal CA1 pyramidal neurons (percentages of baseline; NAN-190, 91.6 ± 14.7%; ()pindolol, 1 and 3 mg/kg, 110.6 ± 13.8 and 81.3 ± 17.0%, respectively). In the presence of NAN-190, the suppressant effects of buspirone were abolished completely (P < .01). In contrast, ()-pindolol did not antagonize the suppressant effects of buspirone (Fig. 4).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Interactions of s.c. administration of NAN-190 (1 mg/kg) and ()pindolol (1 and 3 mg/kg) with the effectiveness of buspirone (1 mg/kg s.c.) in suppressing the firing activity. Open column, the percentage of baseline after buspirone alone; , those after buspirone and pretreatment with each antagonist. Values are means ± S.E.M. The number in each column indicates the number of neurons tested. ** P < .01 versus the effectiveness of buspirone alone () by two-way repeated-measures ANOVA and Newman-Keuls test.

Effects of PCPA Pretreatment. Previous experiments in our laboratory showed that PCPA treatment (500 mg/kg/day × 3 day i.p.) depleted 5-HT in the hippocampus to about 4% of the normal level (Kasamo et al., 1994). The average spontaneous firing rate of hippocampal CA1 pyramidal neurons after PCPA pretreatment was 1.65 ± 0.45 Hz (n = 14), and this did not differ significantly from the firing rates in rats without the PCPA pretreatment (1.24 ± 0.1 Hz, n = 126).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of PCPA pretreatment (500 mg/kg/day × 3 days) on the effectiveness of buspirone (0.3 and 1 mg/kg s.c.) in suppressing the firing activity. Columns represent percentages of the baselines after buspirone with () or without (open column) PCPA pretreatment. Values are means ± S.E.M. The number in each column indicate the number of neurons tested.

	Discussion

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Recently, Phillips and LeDoux (1992) demonstrated that lesions in the amygdala interfered with conditioning of fear response to both cue and context, whereas lesions of the hippocampus interfered with conditioning to context only. Similarly, Kim and Fanselow (1992) found that contextual fear was abolished in rats given hippocampal lesions 1 day after fear conditioning. These studies indicate that the hippocampus plays an essential role in associative fear memories evoked by contextual stimuli. Since the density of 5-HT_1A receptors is high in the hippocampus (Pazos and Palacios, 1985; Verge et al., 1985; Weissmann-Nanopoulos et al., 1985), it is reasonable to speculate that 5-HT_1A agonists exert their antianxiety effects via interaction with 5-HT_1A receptors located on hippocampal neurons. In fact, it has been shown that the conditioned fear stress-induced freezing elicited by contextual stimuli is attenuated by ipsapirone (Rittenhouse et al., 1992; Inoue et al., 1996). Since the effect of ipsapirone is not modified in rats with 5-HT neuron lesions, ipsapirone may exert its anxiolytic effect via postsynaptic 5-HT_1A receptors (Inoue et al. 1996). It has also been found that buspirone and S-15535, a benzodioxopiperazine class of 5-HT_1A agonist, exert anxiolytic action in the conditioned fear response (fear-induced ultrasonic vocalization) to contextual stimuli but fail to do so in an elevated plus-maze test (Millan et al., 1997).

As mentioned, previous studies using anesthetized animals or brain slices revealed partial agonistic properties of anxiolytic 5-HT_1A agonists in the hippocampus (Andrade and Nicoll, 1987; Blier and de Montigney, 1987, 1990). Present results demonstrate that systemic administration of the 5-HT_1A agonists 8-OH-DPAT, buspirone, flesinoxan, and ipsapirone dose-dependently inhibited pyramidal cell activity, which indicates that they act as agonists at postsynaptic 5-HT_1A receptors located on CA1 hippocampal pyramidal neurons. The potency order was 8-OH-DPAT > flesinoxan, which is in agreement with previous electrophysiological studies in anesthetized and unanesthetized rats (Kasamo et al., 1994; Hadrava et al., 1995). The dose range of the 5-HT_1A agonists for suppressing pyramidal cell firing was similar to the range for exerting anxiolytic action in rats (Treit, 1991). The intrinsic activity of ipsapirone was weaker than that of the other tested 5-HT_1A agonists, which is in agreement with previous electrophysiological studies (Kasamo et al., 1994). It has been pointed out that azapirone derivatives such as buspirone and ipsapirone produce 1-(2-pyrimidinyl)piperazone, which has potent an alpha-2 adrenoreceptor antagonistic property (Caccia et al., 1986). This has complicated the understanding of previous experiments related to the effects of 5-HT_1A agonists on neuronal activities in various brain regions. In contrast to these azapirone derivatives, flesinoxan does not produce metabolites that possess an alpha-2 adrenoreceptor antagonistic property (Schipper et al., 1991). Thus, it is unlikely that suppression of hippocampal CA1 pyramidal neuronal activity produced by these 5-HT_1A agonists is mediated by an alpha-2 adrenoreceptor blockade.

Although our procedure likely kept the state of the recorded neuron constant across drug conditions, it did not completely control for the place field of the neuron. Because many complex-spike cells are responsive to place field, it is reasonable to assume that some of the neurons we recorded were responsive to the place in which recordings took place. Given the uniformity of the drug effects, it seems that even if this mix of "states" contributed to the results, our present findings would still be interpretable as showing inhibitory effects of 5-HT_1A agonists. However, further research is required to confirm this interpretation.

The present results demonstrating NAN-190 antagonizes the suppressant effects of buspirone on hippocampal CA1 pyramidal neuronal activity are in agreement with previous biochemical and behavioral studies (Hjorth and Sharp, 1990; Claustre et al., 1991). It has also been suggested that NAN-190 is a partial agonist at the presynaptic 5-HT_1A receptor (Greuel and Glaser, 1992). For example, Greuel and Glaser demonstrated that NAN-190 inhibited neuronal activity of dorsal raphe neurons in a concentration-dependent manner, but it also antagonized suppressant effects of 8-OH-DPAT. Furthermore, Fornal et al. (1994b) demonstrated that i.v. administration of NAN-190 produced a dose-dependent inhibition of serotonergic dorsal raphe unit activity in behaving cats. Therefore, it is reasonable to assume that the observed antagonism between buspirone and NAN-190 occurs at postsynaptic 5-HT_1A receptors located on the pyramidal neurons. A previous behavioral study also revealed interaction between ipsapirone and NAN-190 (Przegalinski et al., 1994) in the hippocampus. Since the anticonflict effect of ipsapirone administered intrahippocampally is antagonized by NAN-190 administered intrahippocampally or i.p., it is suggested that the anticonflict effect of ipsapirone stems from stimulation of postsynaptic 5-HT_1A receptors in the hippocampus.

The lack of a clear antagonistic property of ()-pindolol on the buspirone-induced suppression of the hippocampal pyramidal neuronal activity is puzzling because ()-pindolol has been shown to antagonize the postsynaptic 5-HT_1A receptor activation in behavioral models (Millan et al., 1993; Sanchez et al., 1996). However, a recent electrophysiological study demonstrated that ()-pindolol did not alter the responsiveness to microionotophoretic application of 5-HT and 8-OH-DPAT in the CA3 region of the hippocampus (Artigas et al., 1996). Thus our results support the idea that ()-pindolol does not possess an antagonistic property at postsynaptic 5-HT_1A receptors on hippocampal pyramidal neurons.

The serotonergic projection to the hippocampus consists of two types of fibers (Lidov et al., 1980; Kosofsky and Molliver, 1987; Tork, 1990). Freund and Buzaki (1996) have suggested that these two types of serotonergic afferents have different mechanisms of action in the hippocampus. Those originating from dorsal raphe nucleus (Kosofsky and Molliver, 1987) release serotonin at nonsynaptic sites, which may diffuse to different target cells having 5-HT1-2 receptors to exert a slow, tonic, G-protein-mediated action. Those originating mostly from the median raphe nucleus (Kosofsky and Molliver, 1987) always make synaptic contacts. The postsynaptic elements are interneuron types exerting dendritic inhibition. The anatomical evidence suggests that the alterations of 5-HT tone have profound effects on hippocampus electrical activities. However, it seems unlikely that 5-HT_1A receptors located on CA1 pyramidal neurons were stimulated tonically by endogenous 5-HT released from 5-HT nerve terminals, since chemical denervation of 5-HT by PCPA did not produce dramatic changes in the spontaneous firing rate of these neurons. In addition, no significant effects on spontaneous firing rate after NAN-190 injection also support the idea that the 5-HT_1A receptors on pyramidal neurons are not stimulated tonically by endogenous 5-HT because NAN-190 theoretically decreases 5-HT tones by stimulating presynaptic 5-HT_1A autoreceptors and antagonizing endogenous 5-HT at postsynaptic 5-HT_1A receptor sites. However, it is possible that the results might be different if we determine the effects of the 5-HT_1A antagonist during periods of more behavioral activity, when tonic 5-HT activity is expected to be greater.

Present results also revealed that the suppressant effect of buspirone was unaffected by the selective depletion of brain 5-HT with the PCPA pretreatment. Taken together, our results strongly suggest that suppression of the hippocampal CA1 pyramidal neuronal activity by 5-HT_1A agonists is mediated by postsynaptic 5-HT_1A receptors possibly located on pyramidal neurons. Recently, Meller et al. (1990) showed that the postsynaptic 5-HT_1A receptor has little or no receptor reserve so that partial agonists can have antagonistic effects. The 5-HT_1A agonists used in this study are known to act as partial agonists at postsynaptic pyramidal neurons in the hippocampus. Therefore, it has been argued that these 5-HT_1A agonists might be acting as postsynaptic antagonists in the hippocampus especially under conditions where 5-HT neurons are activated. Although the antagonistic actions of 5-HT_1A agonists might be seen in the presence of clear 5-HT stimulation of the pyramidal neurons, the present results do not support the idea that 5-HT_1A agonists act as antagonists at postsynaptic 5-HT_1A receptors, at least under the present experimental conditions. Furthermore, systemically administered 5-HT_1A agonists decrease 5-HT release from axon terminals, it is therefore difficult to assume that 5-HT_1A agonists antagonize endogenous 5-HT at postsynaptic 5-HT_1A receptors (Sharp et al., 1989; Glaser and De Vry, 1992).

In a placebo-controlled panic disorder study, it was suggested that buspirone reduce anxiety levels, especially anticipatory anxiety (Robinson et al., 1989). The hippocampal formation is considered to mediate contextual fear conditioning, which could be thought of as an anticipatory anxiety behavioral model. Thus, inhibition of hippocampal pyramidal neuronal activity via postsynaptic 5-HT_1A receptor activation may contribute to anxiolytic effects of 5-HT_1A agonists. Further elucidation is needed to confirm this hypothesis.