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Modulation of the firing activity of rat serotonin and noradrenaline neurons by (±)pindolol
Modulation of the Firing Activity of Rat Serotonin and Noradrenaline Neurons by (6)Pindolol Nasser Haddjeri, Claude de Montigny, and Pierre Blier Background: (6)Pindolol is a b-adrenergic/5-HT1A receptor antagonist used in combination with certain antidepressant drugs to accelerate the onset of the antidepressive response. Methods: The aim of the present study was to assess, using an in vivo electrophysiologic paradigm, the effect of (6)pindolol on the spontaneous firing activity of rat dorsal raphe serotonin (5-HT) and locus coeruleus noradrenaline (NA) neurons. Results: (6)Pindolol did not modify the firing activity of dorsal raphe 5-HT neurons at low doses (10 and 200 mg/kg, IV), but it prevented the suppressant effect of the 5-HT autoreceptor agonist lysergic acid diethylamide (LSD, 10 mg/kg, IV) but not that of the 5-HT1A receptor 8-hydroxy-N,N-dipropyl-aminotetralin (8-OHDPAT, 5 mg/ kg, IV). At a higher dose (500 mg/kg, IV), (6)pindolol decreased 5-HT neuronal firing and this effect was reversed by the selective 5-HT1A receptor antagonist WAY 100635 (100 mg/kg, IV), suggesting that it could act as a partial 5-HT1A autoreceptor agonist. In the locus coeruleus, the high dose of (6)pindolol decreased the firing activity of NA neurons and this effect was reversed by the 5-HT2A receptor antagonist MDL 100907 (200 mg/kg, IV). Finally, both a lesion of NA neurons and the administration of MDL 100907 prevented the suppressant effect of (6)pindolol on the firing of 5-HT neurons. Conclusions: It is suggested that, at low doses, (6)pindolol acts as a somatodendritic 5-HT1A autoreceptor antagonist whereas at a higher dose, it decreases the tonic excitatory input from NA neurons to 5-HT neurons. Biol Psychiatry 1999;45:1163–1169 © 1999 Society of Biological Psychiatry Key Words: (6)Pindolol, dorsal raphe nucleus, locus coeruleus, LSD, WAY 100635, MDL 100907
Introduction
A
lthough the physiopathology of major depression has not been elucidated, there is a growing body of evidence that supports the implication of the serotonin
From the Neurobiological Psychiatry Unit, McGill University, Montre´al, Que´bec, Canada. Address reprint requests to Nasser Haddjeri, PhD, Neurobiological Psychiatry Unit, McGill University, 1033 Pine Avenue West, Montre´al, Que´bec, Canada H3A 1A1. Received July 29, 1998; revised October 28, 1998; accepted November 2, 1998.
© 1999 Society of Biological Psychiatry
(5-HT) system in the therapeutic effect of antidepressant treatments (Blier and de Montigny 1994; Maes and Meltzer 1995). Various classes of antidepressant treatments enhance 5-HT neurotransmission with a time course that is consistent with their delayed therapeutic effect. This would be mediated via different mechanisms such as postsynaptic sensitization to 5-HT, desensitization of the somatodendritic and/or terminal 5-HT autoreceptors, or a desensitization of a2-adrenergic heteroreceptors located on 5-HT terminals (see for review Blier and de Montigny 1994). A new strategy to potentiate the antidepressant response of medications acting on the presynaptic component consists in combining the arylalkylamine (6)pindolol, which is a b-adrenergic/5-HT1A receptor antagonist, with a selective 5-HT reuptake inhibitor (SSRI), a monoamine oxidase inhibitor (MAOI), or a 5-HT1A receptor agonist from the beginning of a treatment to obtain a more rapid onset of antidepressant action. Although negative results have been reported (Berman et al 1997), there are now six placebo-controlled studies showing that this approach can exert a rapid onset of action, and in some cases, a greater efficacy in major depression (see Blier and Bergeron 1998 for review; Bordet et al 1998; Perez et al 1997; Maes et al 1996; Tome et al 1997; Zanardi et al 1997, 1998). Accordingly, preclinical studies have shown that (6)pindolol can potentiate the effects of SSRIs on extracellular 5-HT concentration by preventing the activation of somatodendritic 5-HT1A autoreceptors, but not those located at the postsynaptic level (Romero et al 1996; Tada et al 1998). However, (2)pindolol possesses a weak partial agonist activity at human 5-HT1A receptors (Newman-Tancredi et al 1998) and it has been shown that its intravenous administration dose-dependently suppresses the firing activity of dorsal raphe 5-HT neurons in freelymoving cats (Fornal et al 1997). Given these apparently discordant observations, the present studies were undertaken to characterize the effects of acute administration (6)pindolol on the firing activity of dorsal raphe (DR) 5-HT and locus coeruleus (LC) noradrenaline (NA) neurons, using in vivo electrophysiologic paradigms in anesthetized rats.
Methods and Materials The experiments were carried out in male Sprague-Dawley rats weighing 250 to 300 g, which were kept under standard labora0006-3223/99/$20.00 PII S0006-3223(98)00354-0
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tory conditions (12:12 light– dark cycle with free access to food and water). They were anesthetized with chloral hydrate (400 mg/kg, IP) and supplemental doses were given to maintain constant anesthesia, monitored by the absence of nociceptive reaction to a tail pinch.
Extracellular Recordings From Dorsal Raphe 5-HT Neurons Unitary extracellular recordings of 5-HT neurons were performed with single-barrelled glass micropipettes preloaded with fibreglass filaments in order to facilitate filling. The tip was broken back to 2 to 4 mm and filled with a 2 mol/L NaCl solution saturated with Fast Green FCF. The burr hole was drilled 1 mm anterior to lambda on midline. Dorsal raphe 5-HT neurons were encountered over a distance of 1 mm starting immediately below the ventral border of the Sylvius aqueduct. These neurons were identified using the following criteria: a slow (0.5 to 2.5 Hz) and regular firing rate and long-duration (0.8 to 1.2 ms) positive action potentials. The effect of IV administration (6)pindolol (10, 200, and 500 mg/kg) on the firing rate of dorsal raphe 5-HT neurons was assessed prior to the IV injection of the 5-HT autoreceptor agonist LSD (10 mg/kg, IV), of the selective 5-HT1A receptor antagonist WAY 100635 (100 mg/kg, IV), prior to the IV injection of the 5-HT1A receptor agonist 8-OH-DPAT (5 mg/kg, IV), and prior to and after the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg, IV). The effect of (6)pindolol on the firing rate of dorsal raphe 5-HT neurons was also determined in 6-hydroxydopamine (6-OHDA) lesioned rats. Lesion of NA neurons was performed under chloral hydrate anesthesia by injecting 6-OHDA intracerebroventricularly (120 mg free base in 20 mL of .9% NaCl and .1% ascorbic acid) 1 h after the injection of the 5-HT reuptake blocker fluoxetine (10 mg/kg, IP) to protect 5-HT neurons. The rats were tested 10 days later. The same volume of vehicle was injected in control rats.
Extracellular Recordings From Locus Coeruleus (LC) NA Neurons LC NA neurons were also recorded with single-barrelled glass micropipettes at the following stereotaxic coordinates: 2.7 mm posterior to lambda and 1.1 to 1.4 mm lateral. Noradrenaline neurons were identified by their regular firing rate (1 to 5 Hz), long-duration (0.8 to 1.2 ms) positive action potentials, and their characteristic burst discharge in response to nociceptive pinch of the contralateral hind paw. The effect of IV administration (6)pindolol (500 mg/kg) on the spontaneous firing activity of LC NA neurons was assessed prior to the IV injection of the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg, IV).
Drugs Drugs used were (6)pindolol, WAY 100635 (Research Biochemicals, Natick, MA, USA); MDL 100907 (Hoechst Marion Roussel, Cincinnati, OH, USA); fluoxetine HCL (Eli Lilly, Indianapolis, IN, USA); and mirtazapine (Organon, Oss, The Netherlands). The concentrations and the doses used for these
compounds were chosen on the basis of previous successful experiments carried out in our and other laboratories.
Results It has been clearly demonstrated that 5-HT neuron firing activity is dependent on the activation of somatodendritic 5-HT1A autoreceptors as well as on the tonic activation by its noradrenegic input (Baraban and Aghajanian, 1980; Blier and de Montigny 1987). Accordingly, and as illustrated in Figure 1A, the IV administration of the 5-HT autoreceptor agonist LSD (10 mg/kg, IV) suppressed the firing activity of DR 5-HT neurons, and this suppressant effect was reversed by the selective 5-HT1A receptor antagonist WAY 100635 (100 mg/kg, IV). (6)Pindolol, at doses of 10 and 200 mg/kg did not modify the firing activity of DR 5-HT neurons (22 6 2% for a dose of 10 mg/kg, n 5 7 and 21 6 3% for a dose of 200 mg/kg, n 5 13) but significantly attenuated the suppressant effect of LSD (Figure 1B, C, and D). Interestingly, (6)pindolol at a dose of 200 mg/kg (n 5 6), did not prevent or reverse the suppressant effect of a dose of 8-OH-DPAT (5 mg/kg, IV, n 5 7), which completely shuts off neuronal firing (Figure 2A and B). On the other hand, 500 mg/kg of (6)pindolol by itself reduced the firing activity of 5-HT neurons by about 50% (Figure 3A and C). As exemplified in Figure 3A, this suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons, which appeared with a short latency (,60 s), was reversed in all cases by the subsequent IV administration of WAY 100635 (100 mg/ kg, n 5 5) but not that of the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg). Finally, MDL 100907 (200 mg/kg, IV) did not modify by itself 5-HT neuronal firing but prevented suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons (prior to: 47 6 14%, n 5 7; after MDL 100907: 7 6 14%, n 5 6, p , .03). It was previously reported that (2)pindolol decreased the firing activity of LC NA neurons when given at a dose of 15 mg/kg, IP (Haddjeri et al 1997). To determine a possible involvement of NA neurons in the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons at the doses mentioned above, the lesioning of NA neurons was performed using the neurotoxin 6-OHDA. As illustrated in Figure 3B and C, the suppressant effect of (6)pindolol on the spontaneous firing activity of DR 5-HT neurons was markedly and significantly attenuated by the 6-OHDA pretreatment. Finally, the effect of (6)pindolol (500 mg/kg) administered systemically was investigated on electrophysiologically identified LC NA neurons. A dose of 500 mg/kg of (6)pindolol reduced the firing activity of NA neurons by 54 6 12% (n 5 8). In contrast to DR 5-HT neurons, this
(6)Pindolol and Rat Dorsal Raphe Nucleus
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Figure 2. Integrated firing rate histograms of dorsal raphe 5-HT neurons showing their responses to 8-OH-DPAT (5 mg/kg, IV), (6)pindolol (200 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (A); to (6)pindolol (200 mg/kg, IV), 8-OH-DPAT (5 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (B); and to MDL 100907 (200 mg/kg, IV), (6)pindolol (200 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (C). Note that the suppressant effect of 8-OHDPAT was antagonized by WAY 100635 but not (6)pindolol (A and B) and that MDL 10097 prevented the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons.
Figure 1. Integrated firing rate histograms of dorsal raphe 5-HT neurons showing their responses to LSD and WAY 100635 (A), to (6)pindolol (10 mg/kg, IV), LSD (30 mg/kg, IV), and WAY 100635 (100 mg/kg, IV); (B) and to (6)pindolol (200 mg/kg, IV), LSD (30 mg/kg, IV), and WAY 100635 (100 mg/kg, IV). (C) Mean results 6 SEM of the suppressant effect of LSD (10 mg/kg, IV) on the firing activity of 5-HT neurons in control rats after the i.v. administration of 10 and 200 mg/kg of (6)pindolol are presented in D. The numbers at the bottom of the columns indicate the number of rats tested. *p , .05 (unpaired Student’s t test).
suppressant effect of (6)pindolol on the firing activity of LC NA neurons was reversed by the IV administration of 200 mg/kg of MDL 100907 (Figure 3D). It is noteworthy that MDL 100907 (200 mg/kg, IV), when administered by itself, did not modify the firing activity of LC NA neurons (n 5 4, data not shown).
Discussion In the present study, the systemic administration of low doses of (6)pindolol (10 and 200 mg/kg, IV) did not
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Figure 3. Integrated firing rate histograms of 5-HT neurons recorded in the DR nucleus showing their responses to (6)pindolol (500 mg/kg, IV) in a control rat (A) and in a 6-OHDA pretreated rat (B). (C) Responsiveness of 5-HT neurons to (6)pindolol (500 mg/kg, IV) in control and 6-OHDA-pretreated rats (means 6 SEM). The numbers at the bottom of the columns indicate the number of neurons tested, *p , .05, using the unpaired Student t test. (D) Integrated firing rate histogram of NA neurons recorded in the LC showing its response to (6)pindolol (500 mg/kg, IV) and MDL 100907 (500 mg/kg, IV) in a control rat.
modify the firing activity of 5-HT neurons but prevented the suppressant effect of LSD, suggesting that (6)pindolol antagonized somatodendritic 5-HT1A autoreceptors in the DR. However, a high dose of (6)pindolol (500 mg/kg, IV) reduced the spontaneous firing activity of both DR 5-HT
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and LC NA neurons. This suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons was reversed by WAY 100635 (100 mg/kg, IV), suggesting that (6)pindolol could act as a partial 5-HT1A autoreceptor; however, this inhibitory action was abolished by a 6-OHDA pretreatment, indicating that the NA system is involved in the suppressant effect of (6)pindolol on the spontaneous firing activity of DR 5-HT neurons. Pindolol interacts with 5-HT1A receptors with nanomolar affinity (Ki 5 8 nmol/L in recombinant human 5-HT1A receptors using (2)pindolol and Ki 5 13 nmol/L in rat hippocampal membranes using (6)pindolol (Hoyer and Schoeffter 1991; Ne´none´ne´ et al 1996; Newman-Tancredi et al 1998); and prevents some biologic responses mediated via 5-HT1A receptor activation (Aulakh et al 1988; Lesch et al 1990; Seletti et al 1995). One peculiar characteristic found with this ligand is its preferential activity at the presynaptic 5-HT1A receptor. In vitro, it has been previously demonstrated that 1 mmol/L (6)pindolol antagonizes the inhibitory effect of gepirone on the firing activity of rat DR 5-HT neurons (Gehlbach and VanderMaelen 1987). In vivo, it has been also shown that a 2-day treatment with (2)pindolol (15 mg/kg/day, SC) prevents the inhibitory effect of LSD on rat DR 5-HT neuronal firing activity as well as the reduction of firing activity observed after a 2-day treatment with the SSRI paroxetine, thus supporting its antagonistic property for DR somatodendritic 5-HT1A autoreceptors. However, using the latter regimen, (2)pindolol did not antagonize postsynaptic 5-HT1A receptors mediating the suppression of firing of dorsal hippocampus CA3 pyramidal neurons recorded extracellularly (Romero et al 1996; Haddjeri et al 1998b), further suggesting the existence of pharmacologically distinct subpopulations of 5-HT1A receptors (Blier et al 1993; Hadrava et al 1994). Nevertheless, in vitro, an antagonistic activity of (6)pindolol for postsynaptic 5-HT1A receptors on CA1–CA3 pyramidal neurons has been reported (Corradetti et al 1998). In the present studies, both LSD and 8-OH-DPAT have been used to activate somatodendritic 5-HT1A autoreceptors in the DR and (6)pindolol prevented only the suppressant effect of LSD on the firing activity of 5-HT neurons. It has been recently shown that (6)pindolol (0.1 to 1 mg/kg, IV) fails to antagonize the suppressant effect of 8-OH-DPAT (10 mg/kg, IV) on the firing activity of DR 5-HT neurons in freely moving cats (Fornal et al 1997). As previously proposed (Blier and de Montigny 1987), the latter discrepancy between the effects of LSD and 8-OH-DPAT could be explained by the existence of a negative feedback loop. In fact, the IV administration of 8-OH-DPAT would activate postsynaptic 5-HT1A receptors located on neurons, possibly in the medial prefrontal cortex, which exert a negative feedback influence on DR 5-HT neurons (Blier and de Montigny 1987; Ceci et al 1994; Hajos et al 1998). Thus, (6)pindolol (200 mg/kg, IV) would be unable to
(6)Pindolol and Rat Dorsal Raphe Nucleus
antagonize the effect of 8-OH-DPAT because (6)pindolol may not antagonize this postsynaptic 5-HT1A receptor as is the case in the dorsal hippocampus. In contrast, both (6)pindolol and WAY 100635 antagonized the suppressant effect of LSD on the firing activity of DR 5-HT neurons because both antagonists are effective on the somatodendritic 5-HT1A autoreceptors. In recent years, numerous studies have documented several interactions between NA and 5-HT systems. It is well established that NA neurons modulate the activity of the 5-HT system and there are several lines of evidence supporting the notion that the 5-HT system also influences brain NA neurons (see Haddjeri et al 1997). Using the potent and selective 5-HT1A receptor antagonist WAY 100635 (Fletcher et al 1996), the involvement of 5-HT1A receptors in the modulation of LC NA neurons by the 5-HT system was recently described. WAY 100635 suppressed the firing activity of NA neurons and this inhibitory effect was abolished by lesioning 5-HT neurons. It has been concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurons was due to an enhancement of the function of 5-HT neurons via a presynaptic 5-HT1A receptor which is, however, not the somatodendritic one since the dose of WAY 100635 used in these experiments did not alter 5-HT neuronal activity. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurons appears to be of the 5-HT2A subtype, as indicated by antagonism of 5-HT2A receptors (Haddjeri et al 1997). Since (2)pindolol (15 mg/kg, IP) attenuated the firing activity of LC NA neurons in the latter study, the possibility that (6)pindolol could act in part via NA neurons had to be taken into account in the present work (Figure 2). In the present study, the high dose of (6)pindolol (500 mg/kg, IV) indeed suppressed the firing activity of both DR 5-HT and LC NA neurons, and the lesion of NA neurons with the neurotoxin 6-OHDA nearly abolished the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons. These results suggest that the suppressant effect of the high dose of (6)pindolol on the firing activity of DR 5-HT neurons could be due, at least in part, to a decrease of the tonic excitatory input from NA neurons. It is worth mentioning that the lesion of NA neurons with the neurotoxin 6-OHDA decreases the firing activity of 5-HT neurons for the first few days, but after 4 to 7 days their normal pattern of firing is restored (Svensson et al 1975). This clearly indicates that 5-HT neurons adapt in the presence of an impaired NA activation. The reason why the suppressant effect of the high dose of (6)pindolol was prevented by the lesion of the NA neurons remains elusive. However, one possibility could be a permissive role of normal LC neuronal firing. As mentioned, it has been shown that the suppressant effect of WAY 100635
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(100 mg/kg, IV) on the firing activity of LC NA neuronal firing was due to an enhancement of the function of 5-HT neurons via a presynaptic 5-HT1A receptor and that the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurons was of the 5-HT2A subtype (Haddjeri et al 1997). Similarly, MDL 100907 could have prevented the suppressant effect of the high dose of (6)pindolol on the firing activity of DR 5-HT neurons by preventing an attenuation of the LC neuronal firing. On the other hand, Fornal et al (1997) recently reported in awake cats that systemic administration of both (6) and (2)pindolol suppressed the firing activity dorsal raphe 5-HT neurons and this effect was reversed by WAY 100635. This result implies that (6)pindolol can act as an agonist at 5-HT1A autoreceptors and this assumption was confirmed (Clifford et al 1998; present studies). This appears inconsistent with a potentiation of the firing activity of 5-HT neurons by (2)pindolol in combination with SSRIs (Romero et al 1996). The present studies, however, put into evidence a biphasic effect of (6)pindolol on the firing activity of DR 5-HT neurons. Although still speculative, the low intravenous doses, which did not affect the neuronal firing activity and prevented 5-HT1A autoreceptor activation, might be similar to the sustained subcutaneous administration of 15 mg/kg/day used by Romero et al (1996). It is possible that low doses of (6)pindolol might exert an enhancing effect on 5-HT neurotransmission by acting on neuronal elements outside the raphe nuclei. The critical role of the concentration of (6)pindolol can be further emphasized by recent studies, using different models in humans (e.g., body temperature and cortisol levels) showing that (6)pindolol at a high dose (30 mg p.o.) behaves as a 5-HT1A receptor agonist per se but prevents the full effect of subsequent administration of an agonist (Seletti et al 1995; Meltzer and Maes 1996; Haddjeri et al 1998a). Furthermore, (2)pindolol, but not the partial 5-HT1A receptor agonist buspirone, potentiates the actions of SSRIs in facilitating 5-HT release (Romero et al 1996; Hjorth and Auerbach 1996; Gobert et al 1997). It is also noteworthy that (6)pindolol can also potentiate the antidepressant response to the 5-HT1A receptor agonist buspirone (Blier et al 1997). In the light of (6)pindolol currently being used in combination with several antidepressants to accelerate the onset of the antidepressive response, further studies, using different concentrations of the compound, are required to determine whether its long-term administration could by itself affect 5-HT and NA neurotransmission. This work was supported by the Medical Research Council of Canada (MRC) grants (to PB, MT11014 and C de M, MA6444). NH was a recipient of a fellowship from the “Fonds de la Recherche en Sante´ du Que´bec” and PB a recipient of a Scientist award from the MRC.
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Introduction
A
lthough the physiopathology of major depression has not been elucidated, there is a growing body of evidence that supports the implication of the serotonin
From the Neurobiological Psychiatry Unit, McGill University, Montre´al, Que´bec, Canada. Address reprint requests to Nasser Haddjeri, PhD, Neurobiological Psychiatry Unit, McGill University, 1033 Pine Avenue West, Montre´al, Que´bec, Canada H3A 1A1. Received July 29, 1998; revised October 28, 1998; accepted November 2, 1998.
© 1999 Society of Biological Psychiatry
(5-HT) system in the therapeutic effect of antidepressant treatments (Blier and de Montigny 1994; Maes and Meltzer 1995). Various classes of antidepressant treatments enhance 5-HT neurotransmission with a time course that is consistent with their delayed therapeutic effect. This would be mediated via different mechanisms such as postsynaptic sensitization to 5-HT, desensitization of the somatodendritic and/or terminal 5-HT autoreceptors, or a desensitization of a2-adrenergic heteroreceptors located on 5-HT terminals (see for review Blier and de Montigny 1994). A new strategy to potentiate the antidepressant response of medications acting on the presynaptic component consists in combining the arylalkylamine (6)pindolol, which is a b-adrenergic/5-HT1A receptor antagonist, with a selective 5-HT reuptake inhibitor (SSRI), a monoamine oxidase inhibitor (MAOI), or a 5-HT1A receptor agonist from the beginning of a treatment to obtain a more rapid onset of antidepressant action. Although negative results have been reported (Berman et al 1997), there are now six placebo-controlled studies showing that this approach can exert a rapid onset of action, and in some cases, a greater efficacy in major depression (see Blier and Bergeron 1998 for review; Bordet et al 1998; Perez et al 1997; Maes et al 1996; Tome et al 1997; Zanardi et al 1997, 1998). Accordingly, preclinical studies have shown that (6)pindolol can potentiate the effects of SSRIs on extracellular 5-HT concentration by preventing the activation of somatodendritic 5-HT1A autoreceptors, but not those located at the postsynaptic level (Romero et al 1996; Tada et al 1998). However, (2)pindolol possesses a weak partial agonist activity at human 5-HT1A receptors (Newman-Tancredi et al 1998) and it has been shown that its intravenous administration dose-dependently suppresses the firing activity of dorsal raphe 5-HT neurons in freelymoving cats (Fornal et al 1997). Given these apparently discordant observations, the present studies were undertaken to characterize the effects of acute administration (6)pindolol on the firing activity of dorsal raphe (DR) 5-HT and locus coeruleus (LC) noradrenaline (NA) neurons, using in vivo electrophysiologic paradigms in anesthetized rats.
Methods and Materials The experiments were carried out in male Sprague-Dawley rats weighing 250 to 300 g, which were kept under standard labora0006-3223/99/$20.00 PII S0006-3223(98)00354-0
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tory conditions (12:12 light– dark cycle with free access to food and water). They were anesthetized with chloral hydrate (400 mg/kg, IP) and supplemental doses were given to maintain constant anesthesia, monitored by the absence of nociceptive reaction to a tail pinch.
Extracellular Recordings From Dorsal Raphe 5-HT Neurons Unitary extracellular recordings of 5-HT neurons were performed with single-barrelled glass micropipettes preloaded with fibreglass filaments in order to facilitate filling. The tip was broken back to 2 to 4 mm and filled with a 2 mol/L NaCl solution saturated with Fast Green FCF. The burr hole was drilled 1 mm anterior to lambda on midline. Dorsal raphe 5-HT neurons were encountered over a distance of 1 mm starting immediately below the ventral border of the Sylvius aqueduct. These neurons were identified using the following criteria: a slow (0.5 to 2.5 Hz) and regular firing rate and long-duration (0.8 to 1.2 ms) positive action potentials. The effect of IV administration (6)pindolol (10, 200, and 500 mg/kg) on the firing rate of dorsal raphe 5-HT neurons was assessed prior to the IV injection of the 5-HT autoreceptor agonist LSD (10 mg/kg, IV), of the selective 5-HT1A receptor antagonist WAY 100635 (100 mg/kg, IV), prior to the IV injection of the 5-HT1A receptor agonist 8-OH-DPAT (5 mg/kg, IV), and prior to and after the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg, IV). The effect of (6)pindolol on the firing rate of dorsal raphe 5-HT neurons was also determined in 6-hydroxydopamine (6-OHDA) lesioned rats. Lesion of NA neurons was performed under chloral hydrate anesthesia by injecting 6-OHDA intracerebroventricularly (120 mg free base in 20 mL of .9% NaCl and .1% ascorbic acid) 1 h after the injection of the 5-HT reuptake blocker fluoxetine (10 mg/kg, IP) to protect 5-HT neurons. The rats were tested 10 days later. The same volume of vehicle was injected in control rats.
Extracellular Recordings From Locus Coeruleus (LC) NA Neurons LC NA neurons were also recorded with single-barrelled glass micropipettes at the following stereotaxic coordinates: 2.7 mm posterior to lambda and 1.1 to 1.4 mm lateral. Noradrenaline neurons were identified by their regular firing rate (1 to 5 Hz), long-duration (0.8 to 1.2 ms) positive action potentials, and their characteristic burst discharge in response to nociceptive pinch of the contralateral hind paw. The effect of IV administration (6)pindolol (500 mg/kg) on the spontaneous firing activity of LC NA neurons was assessed prior to the IV injection of the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg, IV).
Drugs Drugs used were (6)pindolol, WAY 100635 (Research Biochemicals, Natick, MA, USA); MDL 100907 (Hoechst Marion Roussel, Cincinnati, OH, USA); fluoxetine HCL (Eli Lilly, Indianapolis, IN, USA); and mirtazapine (Organon, Oss, The Netherlands). The concentrations and the doses used for these
compounds were chosen on the basis of previous successful experiments carried out in our and other laboratories.
Results It has been clearly demonstrated that 5-HT neuron firing activity is dependent on the activation of somatodendritic 5-HT1A autoreceptors as well as on the tonic activation by its noradrenegic input (Baraban and Aghajanian, 1980; Blier and de Montigny 1987). Accordingly, and as illustrated in Figure 1A, the IV administration of the 5-HT autoreceptor agonist LSD (10 mg/kg, IV) suppressed the firing activity of DR 5-HT neurons, and this suppressant effect was reversed by the selective 5-HT1A receptor antagonist WAY 100635 (100 mg/kg, IV). (6)Pindolol, at doses of 10 and 200 mg/kg did not modify the firing activity of DR 5-HT neurons (22 6 2% for a dose of 10 mg/kg, n 5 7 and 21 6 3% for a dose of 200 mg/kg, n 5 13) but significantly attenuated the suppressant effect of LSD (Figure 1B, C, and D). Interestingly, (6)pindolol at a dose of 200 mg/kg (n 5 6), did not prevent or reverse the suppressant effect of a dose of 8-OH-DPAT (5 mg/kg, IV, n 5 7), which completely shuts off neuronal firing (Figure 2A and B). On the other hand, 500 mg/kg of (6)pindolol by itself reduced the firing activity of 5-HT neurons by about 50% (Figure 3A and C). As exemplified in Figure 3A, this suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons, which appeared with a short latency (,60 s), was reversed in all cases by the subsequent IV administration of WAY 100635 (100 mg/ kg, n 5 5) but not that of the selective 5-HT2A receptor antagonist MDL 100907 (200 mg/kg). Finally, MDL 100907 (200 mg/kg, IV) did not modify by itself 5-HT neuronal firing but prevented suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons (prior to: 47 6 14%, n 5 7; after MDL 100907: 7 6 14%, n 5 6, p , .03). It was previously reported that (2)pindolol decreased the firing activity of LC NA neurons when given at a dose of 15 mg/kg, IP (Haddjeri et al 1997). To determine a possible involvement of NA neurons in the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons at the doses mentioned above, the lesioning of NA neurons was performed using the neurotoxin 6-OHDA. As illustrated in Figure 3B and C, the suppressant effect of (6)pindolol on the spontaneous firing activity of DR 5-HT neurons was markedly and significantly attenuated by the 6-OHDA pretreatment. Finally, the effect of (6)pindolol (500 mg/kg) administered systemically was investigated on electrophysiologically identified LC NA neurons. A dose of 500 mg/kg of (6)pindolol reduced the firing activity of NA neurons by 54 6 12% (n 5 8). In contrast to DR 5-HT neurons, this
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Figure 2. Integrated firing rate histograms of dorsal raphe 5-HT neurons showing their responses to 8-OH-DPAT (5 mg/kg, IV), (6)pindolol (200 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (A); to (6)pindolol (200 mg/kg, IV), 8-OH-DPAT (5 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (B); and to MDL 100907 (200 mg/kg, IV), (6)pindolol (200 mg/kg, IV), and WAY 100635 (100 mg/kg, IV) (C). Note that the suppressant effect of 8-OHDPAT was antagonized by WAY 100635 but not (6)pindolol (A and B) and that MDL 10097 prevented the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons.
Figure 1. Integrated firing rate histograms of dorsal raphe 5-HT neurons showing their responses to LSD and WAY 100635 (A), to (6)pindolol (10 mg/kg, IV), LSD (30 mg/kg, IV), and WAY 100635 (100 mg/kg, IV); (B) and to (6)pindolol (200 mg/kg, IV), LSD (30 mg/kg, IV), and WAY 100635 (100 mg/kg, IV). (C) Mean results 6 SEM of the suppressant effect of LSD (10 mg/kg, IV) on the firing activity of 5-HT neurons in control rats after the i.v. administration of 10 and 200 mg/kg of (6)pindolol are presented in D. The numbers at the bottom of the columns indicate the number of rats tested. *p , .05 (unpaired Student’s t test).
suppressant effect of (6)pindolol on the firing activity of LC NA neurons was reversed by the IV administration of 200 mg/kg of MDL 100907 (Figure 3D). It is noteworthy that MDL 100907 (200 mg/kg, IV), when administered by itself, did not modify the firing activity of LC NA neurons (n 5 4, data not shown).
Discussion In the present study, the systemic administration of low doses of (6)pindolol (10 and 200 mg/kg, IV) did not
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Figure 3. Integrated firing rate histograms of 5-HT neurons recorded in the DR nucleus showing their responses to (6)pindolol (500 mg/kg, IV) in a control rat (A) and in a 6-OHDA pretreated rat (B). (C) Responsiveness of 5-HT neurons to (6)pindolol (500 mg/kg, IV) in control and 6-OHDA-pretreated rats (means 6 SEM). The numbers at the bottom of the columns indicate the number of neurons tested, *p , .05, using the unpaired Student t test. (D) Integrated firing rate histogram of NA neurons recorded in the LC showing its response to (6)pindolol (500 mg/kg, IV) and MDL 100907 (500 mg/kg, IV) in a control rat.
modify the firing activity of 5-HT neurons but prevented the suppressant effect of LSD, suggesting that (6)pindolol antagonized somatodendritic 5-HT1A autoreceptors in the DR. However, a high dose of (6)pindolol (500 mg/kg, IV) reduced the spontaneous firing activity of both DR 5-HT
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and LC NA neurons. This suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons was reversed by WAY 100635 (100 mg/kg, IV), suggesting that (6)pindolol could act as a partial 5-HT1A autoreceptor; however, this inhibitory action was abolished by a 6-OHDA pretreatment, indicating that the NA system is involved in the suppressant effect of (6)pindolol on the spontaneous firing activity of DR 5-HT neurons. Pindolol interacts with 5-HT1A receptors with nanomolar affinity (Ki 5 8 nmol/L in recombinant human 5-HT1A receptors using (2)pindolol and Ki 5 13 nmol/L in rat hippocampal membranes using (6)pindolol (Hoyer and Schoeffter 1991; Ne´none´ne´ et al 1996; Newman-Tancredi et al 1998); and prevents some biologic responses mediated via 5-HT1A receptor activation (Aulakh et al 1988; Lesch et al 1990; Seletti et al 1995). One peculiar characteristic found with this ligand is its preferential activity at the presynaptic 5-HT1A receptor. In vitro, it has been previously demonstrated that 1 mmol/L (6)pindolol antagonizes the inhibitory effect of gepirone on the firing activity of rat DR 5-HT neurons (Gehlbach and VanderMaelen 1987). In vivo, it has been also shown that a 2-day treatment with (2)pindolol (15 mg/kg/day, SC) prevents the inhibitory effect of LSD on rat DR 5-HT neuronal firing activity as well as the reduction of firing activity observed after a 2-day treatment with the SSRI paroxetine, thus supporting its antagonistic property for DR somatodendritic 5-HT1A autoreceptors. However, using the latter regimen, (2)pindolol did not antagonize postsynaptic 5-HT1A receptors mediating the suppression of firing of dorsal hippocampus CA3 pyramidal neurons recorded extracellularly (Romero et al 1996; Haddjeri et al 1998b), further suggesting the existence of pharmacologically distinct subpopulations of 5-HT1A receptors (Blier et al 1993; Hadrava et al 1994). Nevertheless, in vitro, an antagonistic activity of (6)pindolol for postsynaptic 5-HT1A receptors on CA1–CA3 pyramidal neurons has been reported (Corradetti et al 1998). In the present studies, both LSD and 8-OH-DPAT have been used to activate somatodendritic 5-HT1A autoreceptors in the DR and (6)pindolol prevented only the suppressant effect of LSD on the firing activity of 5-HT neurons. It has been recently shown that (6)pindolol (0.1 to 1 mg/kg, IV) fails to antagonize the suppressant effect of 8-OH-DPAT (10 mg/kg, IV) on the firing activity of DR 5-HT neurons in freely moving cats (Fornal et al 1997). As previously proposed (Blier and de Montigny 1987), the latter discrepancy between the effects of LSD and 8-OH-DPAT could be explained by the existence of a negative feedback loop. In fact, the IV administration of 8-OH-DPAT would activate postsynaptic 5-HT1A receptors located on neurons, possibly in the medial prefrontal cortex, which exert a negative feedback influence on DR 5-HT neurons (Blier and de Montigny 1987; Ceci et al 1994; Hajos et al 1998). Thus, (6)pindolol (200 mg/kg, IV) would be unable to
(6)Pindolol and Rat Dorsal Raphe Nucleus
antagonize the effect of 8-OH-DPAT because (6)pindolol may not antagonize this postsynaptic 5-HT1A receptor as is the case in the dorsal hippocampus. In contrast, both (6)pindolol and WAY 100635 antagonized the suppressant effect of LSD on the firing activity of DR 5-HT neurons because both antagonists are effective on the somatodendritic 5-HT1A autoreceptors. In recent years, numerous studies have documented several interactions between NA and 5-HT systems. It is well established that NA neurons modulate the activity of the 5-HT system and there are several lines of evidence supporting the notion that the 5-HT system also influences brain NA neurons (see Haddjeri et al 1997). Using the potent and selective 5-HT1A receptor antagonist WAY 100635 (Fletcher et al 1996), the involvement of 5-HT1A receptors in the modulation of LC NA neurons by the 5-HT system was recently described. WAY 100635 suppressed the firing activity of NA neurons and this inhibitory effect was abolished by lesioning 5-HT neurons. It has been concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurons was due to an enhancement of the function of 5-HT neurons via a presynaptic 5-HT1A receptor which is, however, not the somatodendritic one since the dose of WAY 100635 used in these experiments did not alter 5-HT neuronal activity. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurons appears to be of the 5-HT2A subtype, as indicated by antagonism of 5-HT2A receptors (Haddjeri et al 1997). Since (2)pindolol (15 mg/kg, IP) attenuated the firing activity of LC NA neurons in the latter study, the possibility that (6)pindolol could act in part via NA neurons had to be taken into account in the present work (Figure 2). In the present study, the high dose of (6)pindolol (500 mg/kg, IV) indeed suppressed the firing activity of both DR 5-HT and LC NA neurons, and the lesion of NA neurons with the neurotoxin 6-OHDA nearly abolished the suppressant effect of (6)pindolol on the firing activity of DR 5-HT neurons. These results suggest that the suppressant effect of the high dose of (6)pindolol on the firing activity of DR 5-HT neurons could be due, at least in part, to a decrease of the tonic excitatory input from NA neurons. It is worth mentioning that the lesion of NA neurons with the neurotoxin 6-OHDA decreases the firing activity of 5-HT neurons for the first few days, but after 4 to 7 days their normal pattern of firing is restored (Svensson et al 1975). This clearly indicates that 5-HT neurons adapt in the presence of an impaired NA activation. The reason why the suppressant effect of the high dose of (6)pindolol was prevented by the lesion of the NA neurons remains elusive. However, one possibility could be a permissive role of normal LC neuronal firing. As mentioned, it has been shown that the suppressant effect of WAY 100635
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(100 mg/kg, IV) on the firing activity of LC NA neuronal firing was due to an enhancement of the function of 5-HT neurons via a presynaptic 5-HT1A receptor and that the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurons was of the 5-HT2A subtype (Haddjeri et al 1997). Similarly, MDL 100907 could have prevented the suppressant effect of the high dose of (6)pindolol on the firing activity of DR 5-HT neurons by preventing an attenuation of the LC neuronal firing. On the other hand, Fornal et al (1997) recently reported in awake cats that systemic administration of both (6) and (2)pindolol suppressed the firing activity dorsal raphe 5-HT neurons and this effect was reversed by WAY 100635. This result implies that (6)pindolol can act as an agonist at 5-HT1A autoreceptors and this assumption was confirmed (Clifford et al 1998; present studies). This appears inconsistent with a potentiation of the firing activity of 5-HT neurons by (2)pindolol in combination with SSRIs (Romero et al 1996). The present studies, however, put into evidence a biphasic effect of (6)pindolol on the firing activity of DR 5-HT neurons. Although still speculative, the low intravenous doses, which did not affect the neuronal firing activity and prevented 5-HT1A autoreceptor activation, might be similar to the sustained subcutaneous administration of 15 mg/kg/day used by Romero et al (1996). It is possible that low doses of (6)pindolol might exert an enhancing effect on 5-HT neurotransmission by acting on neuronal elements outside the raphe nuclei. The critical role of the concentration of (6)pindolol can be further emphasized by recent studies, using different models in humans (e.g., body temperature and cortisol levels) showing that (6)pindolol at a high dose (30 mg p.o.) behaves as a 5-HT1A receptor agonist per se but prevents the full effect of subsequent administration of an agonist (Seletti et al 1995; Meltzer and Maes 1996; Haddjeri et al 1998a). Furthermore, (2)pindolol, but not the partial 5-HT1A receptor agonist buspirone, potentiates the actions of SSRIs in facilitating 5-HT release (Romero et al 1996; Hjorth and Auerbach 1996; Gobert et al 1997). It is also noteworthy that (6)pindolol can also potentiate the antidepressant response to the 5-HT1A receptor agonist buspirone (Blier et al 1997). In the light of (6)pindolol currently being used in combination with several antidepressants to accelerate the onset of the antidepressive response, further studies, using different concentrations of the compound, are required to determine whether its long-term administration could by itself affect 5-HT and NA neurotransmission. This work was supported by the Medical Research Council of Canada (MRC) grants (to PB, MT11014 and C de M, MA6444). NH was a recipient of a fellowship from the “Fonds de la Recherche en Sante´ du Que´bec” and PB a recipient of a Scientist award from the MRC.
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