Electrophysiological evidence that spiperone is an antagonist of 5-HT1A receptors in the dorsal raphe nucleus

European Journal of Pharmacology, 149 ( 1988) 9-15

9

Elsevier EJP 50236

Electrophysiological evidence that spiperone is an antagonist of 5-HT1A receptors in the dorsal raphe nucleus J a n e t T. L u m a n d M o n t f o r d F. Piercey * CNS Research, The Upjohn Company, Kalamazoo. MI 49001. U.S.A.

Received 15 October 1987, revised MS received 29 December 1987, accepted 9 February 1988 The neuroleptic spiperone, which binds to 5-HT~A, 5-HT2 and dopamine iDA) receptors, was studied for its effects on serotonin (5-HT) and DA neurons in dorsal raphe nucleus and substantia nigra pars compacta, respectively. We found that 1 mg/kg i.v. spiperone, but not LY53837 (a 5-HT2 antagonist), antagonized the inhibition induced by 5-HTIA agonists 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and buspirone in the dorsal raphe nucleus. Lower spiperone doses blocked DA receptors in substantia nigra pars compacta, but did not affect 5-HT neurons. Doses of 8-OH-DPAT completely silencing dorsal raphe neurons were ineffective in substantia nigra pars compacta. However, buspirone antagonized DA receptors in substantia nigra pars compacta with doses similar to those depressing dorsal raphe neurons. It is concluded that spiperone is an antagonist of 5-HT~.,, receptors in the dorsal raphe nucleus. Spiperone; Buspirone: 8-OH-DPAT: Dorsal raphe: Serotonin (5-HT); (Electrophysiology)

1. Introduction

Although classical pharmacological approaches originally suggested that there were only two subtypes of serotonin (5-HT) receptors ( G a d d u m and Picarelli, 1957), recent binding studies have suggested that there may be as many as seven 5-HT receptor subtypes (Richardson and Engel, 1986). 5-HT 2 receptors were differentiated from 5-HT~ receptors by the high affinity of the former for spiperone and the latter for 5-HT (Leysen, 1981; Peroutka and Snyder, 1979). Subsequent studies demonstrated that 5-HT~ receptors can be further subdivided into multiple subtypes, with 5-HT~A receptors being differentiated from 5-HT~r~ and 5-HTlc receptors by its greater affinity for spiperone (Nelson et al., 1981; Pedigo et al., 1981; Peroutka, 1985). Interest in the physiological and pharmacological role for the 5-HTlA receptor sub-

* I'o whom all correspondence should be addressed: CNS Diseases Research 7251-209-4, The Upjohn Company, Kalamazoo, MI 49001, U.S.A.

type has grown with the discovery that 8hydroxy-dipropyl aminotetralin (8-OH-DPAT) is a relatively selective 5-HT~A agonist (Middlemiss and Fozard, 1983; Hall et al., 1985; H a m o n et al., 1984; Hjorth et al., 1982). It has further been demonstrated that the novel anxiolytic buspirone binds to the 5-HT~A receptor (Glaser and Traber, 1983; Gozlan et al., 1983; Riblet et al., 1984) and that both 8 - O H - D P A T (Sinton et al., 1986) and buspirone (McMiilen et al., 1984; VanderMaelen and Wilkerman, 1984; VanderMaelen et al., 1986) inhibit firing rates of serotonergic neurons in the dorsal raphe nucleus. The presence of 5-HT~A binding sites on the raphe neuron (Ennis, 1981; Pazos and Palacios, 1985; Verge et al., 1985; 1986) has strengthened the argument that these receptor subtypes function as 5-HT somatodendritic autoreceptors. Although the neuroleptic spiperone is widely known as a dopamine (DA) receptor antagonist (Creese et al., 1978; Laduron el al., 1978), little is known of its properties at 5-HT~A receptor sites. In this study, we used single cell activity to examine the role of spiperone in 5-HT and DA receptor sites. We conclude that spiperone

0014-2999/88/$03.50 :(" 1988 Elsevier Science Publishers B.V. (Biomedical Division)

10 antagonizes the effects of 5-HTjA agonists on serotonergic neurons in dorsal raphe nucleus via a blockade of 5-HT]A receptors.

2. Materials and methods

2.1. Preparation of animals" Male Sprague-Dawley rats weighing between 250 and 350 g were anesthetized with chloral hydrate (400 m g / k g i.p.). Supplemental doses between 50 and 100 m g / k g were administrated as needed to maintain anesthesia. The femoral artery and vein were cannulated to measure blood pressure and to permit i.v. administration of drugs, respectively. Body temperature was maintained at 37 o C using a deitaphase isothermal pad (Braintree Scientific). Animals were placed in David Kopf stereotaxic apparatus for electrophysiological measurements.

lambda suture. The electrode, positioned at the midline and at a 34 ° angle to the vertical plane, was hydraulically advanced through this hole to reach the dorsal raphe nucleus (ant. 0.5-1.7 mm, lat. 0 ram, vert. 3.5-4.2 mm), according to Paxinos and Watson (1982). Serotonergic neurons were identified by large positive-negative biphasic action potentials with slow and regular firing rates (approximately 0.8-2.5 spikes/s) according to the criteria of Aghajanian ct al. (1970). For substantia nigra pars compacta nucleus, the electrode was hydraulically lowered vertically 7.0-8.0 mm below cortical surface (post. 2.8-3.2 mm, lat. 2.0-2.2 ram) according to Pelligrino et al. (1979). Dopaminergic neurons were identified by their long duration action potential ( > 2.5 ms), shape, and firing pattern ( < 12 spikes/s) according to traditional criteria (Bunney and Aghajanian, 1975" Vogelsang and Piercey, 1985).

2.4. Drugs 2.2. Extracellular recording Single barrel Omega Dot tubing (Glass Company of America) with an internal glass fiber were pulled by a Narashige microelectrode puller. The tips were broken back to 1 /~m under microscopic control and filled with a filtered 2 M NaCI solution saturated with pontamine sky blue dye (Gurr). The atlases of Pelligrino et al. (1979) and Paxinos and Watson (1982) were used for electrode placement in substantia nigra pars compacta and dorsal raphe nucleus, respectively. Unit activity was passed through a high input impedance amplifier and displayed on an oscilloscope. The unit discharge was led into a window discriminator whose threshold was set so that it was triggered by the individual spikes of the unit. An integrated ratemcter measured each 10 s interval of the analog output from this discriminator and plotted it on a paper chart. Blood pressure was monitored throughout the experiment.

2.3. Electrode placement For dorsal raphe nucleus, a small burr hole was made at the midline and 2-3 m m posterior to the

Drugs were obtained from the following sources: 8 - O H - D P A T (Research Biochemicals, Inc., Natick, MA) buspirone (Bristol-Myers, Evansville, IN), spiperone (Janssen Pharmaceuticals, Beerse) and LY 53837 (Lilly Pharmaceutical, Indianapolis, IN). All drug solutions were made in distilled water. Each injection contained no more than 0.15 ml of a given concentration, followed by a 0.2-0.4 ml of physiological saline to clean the cannula of any residual drug.

2.5. Histology At the termination of each recording session, the location of the unit was identified by passing 10 /~A cathodic current through the recording electrode for 10-20 min to deposit a discrete blue dye at the recording site. The brain was then removed, fixed in 10% formalin, sectioned at 20 /~m, mounted and stained in cresyl violet. For each experiment, the pontamine sky blue spot was verified to be within the dorsal raphe or substantia nigra pars compacta nucleus.

11-

2.6. Statistical analysis All drug effects were measured as percent alterations in firing rate by comparison with control rates prior to treatment. Potencies were expressed as EDs0s, the doses required to cause half maximal effects. The average potencies of the individual experiments were expressed as means + S.E.M. Population potencies were measured by interpolation of population dose-response curves, where the individual points were obtained by averaging the responses to each dose of drug. The Student's t-test was used for measuring statistical significance. 3. Results

3.1. Effects of ~piperone dopaminergic cells

on serotonergic and

All six cells identified as dopaminergic were depressed by amphetamine. Figure 1 illustrates

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that spiperone reversed this depression with an EDs0 of 62 ~ g / k g (n = 6). The inset for this curve shows a substantia nigra pars compacta neuron, first being depressed by amphetamine to 20% of control, after which spiperone dose dependently reversed this effect. In contrast, five cells recorded in the dorsal raphe nucleus revealed that spiperone up to 1 m g / k g had only minimal effects on the spontaneous firing rates of 5-HT neurons in dorsal raphe nucleus (fig. 1).

3.2. ~ffects of 8-OH-DPA T on serotonergic and dopaminergic neurons The spontaneous firing of all eight cells identified as serotonergic was potently depressed by the selective 5-HT1A agonist 8-OH-DPAT in a doserelated manner. Dorsal raphe neuronal firing was completely suppressed at a cumulative dose of 10 # g / k g or less. The calculated individual mean EDs0 (average of each experiment) of 1.6 + 0.6 ~ g / k g (n = 7) was similar to the population EDso (obtained from the dose-response curves of all experiments) of 1.5/.tg/kg (fig. 2). However, in all five cells studied, 8-OH-DPAT did not depress or stimulate DA neurons at doses up to 100 times (1000/~g/kg cumulative dose) of that required to completely suppress all 5-HT neurons (fig. 2). In addition, for all six DA cells studied, 8-OH-DPAT could only reverse the amphetamine-depressed substantia nigra pars compacta neuron at very high doses (individual EDso = 612 _+ 289 ~g/kg, n = 6, fig. 2).

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Fig. 1. Population dose-response illustrating the effects of spiperone in substantia nigra pars compacta and dorsal raphe neurons. Abscissa represents cumulative i.v. spiperone doses. Ordinate represents percent reversal of amphetamine depression in substantia nigra pars compacta (SNPC, open circles, n = 6) or percent depression in dorsal raphe nucleus (DRN, closed circles, n = 5) as measured by changes in control firing rates. Insets show representative chart recordings for individual experiments in substantia nigra pars compacta and dorsal raphe nuclei. Doses shown in inset are non-cumulative and ordinate shows absolute firing rates.

3.3. Effects of buspirone on serotonergic and dopaminergic neurons Like 8-OH-DPAT, buspirone depressed all six serotonergic cells in a potent dose-dependent manner. The individual mean EDs0 of 15.4 + 5.5 ~ g / k g (n = 6) was very similar to the population EDs0 of 13 ~tg/kg (fig. 3). In the substantia nigra pars compacta (five cells recorded), buspirone reversed amphetamine's effects with an individual EDs~~of 9.9 + 3.3 ~ g / k g (n = 5) and a population mean EDs0 of 8 ~ g / k g (fig. 3 inset). This reversal effect was strong enough to overshoot beyond control firing rates, a frequently observed phe-

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Fig. 2. I)opulation dose-response illustrating the effects of 8-OH-I)PAT in substantia nigra pars compacta and dorsal raphe neurons. Abscissa represents cumulative i.v. 8-OH-DPAT doses. Ordinate represents percent reversal of amphetamine depression in substantia nigra pars compacta (SNPC, triangles, (n = 6), percent depression in substantia nigra pars compacta firing rates (SNPC. closed circles, n = 5) or percent depression in dorsal raphe nucleus (DRN, open circles, n = 7) as measured by changes in control firing rates. Inset shows the effects of 8-OH-DPAT, I,Y53837, and spiperone on firing rates of a single dorsal raphe neuron. Abscissa and ordinate of inset are as described in fig. 1.

n o m e n o n with n e u r o l e p t i c s (Bunney a n d A g h a j a n i a n , 1975). W h e n the neuroleptic h a l o p e r i d o l was given following b u s p i r o n e , cell firing increased even higher. Buspirone, when given without a m p h e t a m i n e , s t i m u l a t e d rather than depressed s u b s t a n t i a nigra pars c o m p a c t a n e u r o n s ( d a t a not shown). It was o b s e r v e d that b u s p i r o n e ' s d o s c - r e s p o n s e curve for reversing a m p h e t a m i n e i n d u c e d d e p r e s s i o n of s u b s t a n t i a nigra p a r s c o m p a c t a n e u r o n s was essentially c o n g r u e n t with that for depressing dorsal raphe nucleus n e u r o n s (see fig. 3).

3.4. Antagonism of 5-HTIA agonists by spiperone T h e ability of the two 5-HTtA agonists to depress 5 - H T cells was readily reversed b y t r e a t m e n t with 1 m g / k g s p i p e r o n e (figs. 3 a n d 4, response of all 10 cells recorded). Low doses of s p i p e r o n e elicited less reversal, a n d higher s p i p e r o n e doses either p r o d u c e d no further effect or, ironically, d e p r e s s e d d o r s a l r a p h e n e u r o n a l firing. In s o m e of the 5 - H T cells studied, s p i p e r o n e actually in-

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Fig. 3. Population dose-response illustrating the effects of buspirone in substantia nigra pars compacta and dorsal raphe neurons. Abscissa represents cumulative i.v. doses of buspirone. Ordinate represents percent reversal of amphetamine depression in substantia nigra pars compacta (SNPC, closed circles, n = 5), or percent depression in dorsal raphe nucleus (DRN, open circles, n = 6) by buspirone as measured by changes in control firing rates. Insets show representative chart recordings for individual experiments in substantia nigra pars compacta and dorsal raphe nuclei, where abscissa and ordinate arc described in fig. 1.

creased firing rates a b o v e control. The mean reversal with 1.0 m g / k g s p i p e r o n e was 152 + 35% (n = 4) after 8 - O H - D P A T and 100 + 26% (n = 6) after buspirone. T h e overshoot seen after s p i p e r o n e was reminiscent of the o v e r s h o o t measured when using neuroleptics to reverse a m p h e t a m i n e ' s depression in the s u b s t a n t i a nigra pars c o m p a c t a . However, the effects in dorsal r a p h e were statistically insignificant, the size of the percentage increase being e x a g g e r a t e d by the very slow p r e - d r u g firing rates of this neuron. F u r t h e r studies into the effect of s p i p e r o n e p r e t r e a t m e n t on 8 - O H - D P A T were also carried out. G i v i n g 1 m g / k g s p i p e r o n e prior to 8 - O H D P A T , the EDs0 was 6.1 + 1.3 /~g/kg, n = 5 (fig. 4). This value was significantly different from that

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Fig. 4. Population dose-response illustrating the antagonism of 8-OH-DPAT by spiperone pretreatment in dorsal raphe nucleus. Abscissa represents cumulative i.v. 8-OH-DPAT doses. Ordinate represents percent depression of control dorsal raphe firing rates by 8-OH-DPAT either without (open circles, n = 7) or with (closed circles, n = 5) 1 mg/kg i.v. spiperone given 2-3 min prior to the first 8-OH-DPAT injection. Complete 8-OH-DPAT dose-response curves were obtained in each animal. without spiperone pretreatment (1.6 _+ 0.6 p,g/kg, n = 7, P < 0.01). Although spiperone readily reversed the depression of dorsal raphe neuronal firing caused by 5-HT~A agonists, the selective 5-HT 2 antagonist LY53837 had no effect in any of these experiments (fig. 2 inset). LY53837 was tested for reversal of 8 - O H - D P A T five times, and buspirone once.

4. Discussion

The antagonism of 5-HT1A agonist effects could be observed both as a reversal of already suppressed activity or by shifting the agonist dose-response curve following spiperone pretreatment. Although these two modes of evaluation are not quantitatively comparable (due to different agonist doses, different pharmacokinetic patterns, and the pretreatment design making overshoots impossible to observe), the results of the two protocols are qualitatively identical. To neurophysiologists evaluating new compounds which depress dorsal raphe neurons, the reversal protocol is qualitatively more useful to quickly assess the role of 5-HT1A agonists. The reversal protocol is initiated when a dose is found causing complete or near complete neuron inhibition. Since these doses vary

somewhat from animal to animal, the procedure is not strictly quantitative. The pretreatment protocol is quantitatively more meaningful because it measures the effects of a single dose of spiperone against the entire 5-HT~A agonist dose-response curve. The present data are in agreement with previous results that both 8 - O H - D P A T and buspirone depress dorsal raphe cell firing (Fallon et al., 1983; Sinton, 1986; VanderMaelen et al., 1986; VanderMaelen and Wilkerman, 1984). The fact that spiperone (Seeman, 1981) but not LY53837, a selective 5-HT 2 antagonist (Cohen et al., 1983) was able to antagonize this depression supports the deduction that spiperone's action is by a mechanism other than 5 - H % antagonism. Our results also revealed that in the chloral hydrate anesthetized rat, there is little interaction between dopaminergic and serotonergic neurons in the substantia nigra pars compacta and dorsal raphe nucleus, respectively. This conclusion is supported by the fact that 8-OH-DPAT, at doses which completely silenced dorsal raphe neuron, was ineffective in substantia nigra pars compacta. Similarly, though both buspirone and spiperone are DA antagonists, they show opposite effects in the dorsal raphe nucleus. However, our observations in the anesthetized rat do not rule out the possibility that important interactions between dorsal raphe and substantia nigra pars compacta may occur in other conditions. In agreement with Goodwin and Green (1985) and Hjorth el al. (1982), our data also demonstrated that 8-OH-DPAT, a 5-HT1A agonist, was at least 350-fold more sensitive in dorsal raphe than in substantia nigra pars compacta. In addition, buspirone was found to be equipotent both as a 5-HT~A agonist and as a DA antagonist. It is therefore clear that DA receptor antagonism does not prevent the depression of 5-HT neurons by 5-HTIA agonists. Consistent with this deduction, spiperone was inactive as a 5-HT~A antagonist in the dose range where it antagonized DA: only higher doses interacted with 5-HT1A sites in the dorsal raphe nucleus. Since spipemne's antidopaminergic actions cannot readily explain the reversal of 5-HTIA agonists, the most reasonable conclusion is that spiperone acts as a 5-HT~A

14

antagonist in dorsal raphe nucleus. This reasoning is consistent with the fact that spiperone binds to 5-HT~A receptor sites (Nelson el al., 1981; Pedigo et al., 1981: Peroutka, 1985). In turn, this deduction reinforces the conclusion that the actions of 8-OH-DPAT and buspirone are mediated by a direct stimulation of 5-HT~A autoreceptors located on cell bodies of dorsal raphe nucleus (Gozlan et al., 1983: Pazos and Palacios, 1985: Verge et al., 1985: VanderMaelen et al., 1986). Our results also showed that both spiperone and buspirone possess DA antagonistic properties. The presence of neuroleptic-like properties in the anxiolytic buspirone may be cause for concern. Remarkably, there have been few reported cases of extrapyramidal side effects in clinical trials of this drug (Sussman, 1986). Possibly, 5-HTIA agonist activity suppresses extrapyramidal syndromes. Regardless, the depression of 5-HT cells through 5-HT~,~ receptors has been suggested as the most likely mechanism for its novel anxiolytic effects (VanderMaelen et al., 1986). In conclusion, we have demonstrated that systemic spiperone can be used as a 5-HT~,x antagonist in the dorsal raphe nucleus. Sprouse and Aghajanian (1986) have demonstrated that microiontophoretic (-)propranolol also blocks 51-tl'~,x receptors. Nonetheless. the presence of concomitant D A , . 5-HT,, and tt2-antagonist properties require extreme caution in using these agents. The identification of a more selective 5-HT~, x antagonist is desirable.

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