The hypothalamic suprachiasmatic nucleus (SCN) is primarily responsible for the regulation of circadian rhythmicity. Melatonin, the pineal-derived neurohormone, modulates the rhythmic output of the SCN. Property timed exposure to melatonin is able to induce changes in rhythmic function and thereby entrain circadian rhythms of activity. c-fos is an immediate early gene that is transiently expressed in neurons in response to receptor activation. The ventrolateral portion of the SCN (vSCN) is activated in response to phase-shifting stimuli, an event which is marked by an increase in the expression of c-fos.

Recent evidence suggests that a novel serotonin receptor 5-HT 7 localized in the hypothalamus downregulates in response to treatment with the antidepressant fluoxetine ( Sleight et al. 1995 ). This receptor has also been implicated in the regulation of circadian rhythms ( Lovenberg et al. 1993 ). Here, we show that several agents administered in a profile consistent with activity at the 5-HT 7 receptor produce significant functional Fos immunoreactivity in the suprachiasmatic nucleus (SCN), an effect reduced upon chronic exposure. Furthermore, binding studies demonstrate that chronic administration of Fos-inducing agents produces a neuroadaptive downregulation of the 5-HT 7 receptor in the hypothalamus. The current studies extend the previous observations to include several pharmacologically distinct antidepressants. In addition, these studies provide further evidence to support the role of the 5-HT 7 receptor in the mechanism of antidepressant action and in the regulation of circadian rhythms controlled by the SCN.

Melatonin, the principal hormone of the vertebrate pineal gland, has been implicated in a variety of neurobiological processes such as circadian rhythmicity and reproductive function. One of the earliest described actions of melatonin was its ability to cause pigment translocation in the dermal melanophores of amphibians. Melatonin binding sites have been identified in the brain of many species and in pigmented tumour cell lines; however, the dermal melanophores of the frog Xenopus Laevis possess the highest known density of melatonin binding sites. These cells are the source from which a melatonin receptor has been cloned and provide an excellent model to study melatonin-mediated signal transduction in an isolated cell system. In Xenopus melanophores, melatonin induces a rapid perinuclear aggregation of intracellular pigment which is associated with a pertussis toxin-sensitive inhibition of cAMP. We have previously demonstrated that a subtype of melatonin binding sites found in selected regions of the pigeon brain and in Syrian Hamster RPMI 1846 melatonin cells are functionally coupled to phosphoinositide hydrolysis as a second messenger. Here we now present evidence to suggest that Xenopus Laevis melanophores also possess melatonin binding sites which are functionally linked to phosphoinositide hydrolysis. Melatonin agonists induced phosphoinositide hydrolysis in melanophores in a concentration-dependent manner with a rank order of potency of 2-iodomelatonin > 6-chloromelatonin > N-acetylserotonin > melatonin. Stimulatory response of 2-iodomelatonin was blocked by the melatonin antagonist N-acetyltryptamine and the alpha-adrenergic antagonist prazosin, which has been shown to have high affinity for melatonin binding sites. Phosphoinositide hydrolysis induced by melatonin agonists was not blocked by the serotonin antagonist ketanserin or by phentolamine, an alpha-adrenergic antagonist, indicating that the response observed was not due to stimulation of 5-HT2a/2c receptors or alpha-adrenergic receptors. Furthermore, incubation of melanophores with the non-hydrolyzable G-protein source GTP-gamma-S attenuated the phosphoinositide dose response induced by 2-iodomelatonin, and pre-incubation of the cells with pertussis toxin had no effect on 2-iodomelatonin-induced phosphoinositide hydrolysis. The present data suggest that Xenopus Laevis Melanophores possess G-protein linked pertussis toxin-insensitive melatonin binding sites which are functionally coupled to phosphoinositide hydrolysis as a signal transduction mechanism.

Numerous investigations have studied in vivo regulation of central 5-HT2A receptors. The majority of pharmacological studies point to non-classical regulation of this site. Serotonergic denervation does not modify 5-HT2A receptor density or second messenger responses (phosphoinositide hydrolysis). 5-HT2A receptor downregulation is produced by the chronic administration of 5-HT2A receptor agonists and uniquely among monoamine receptors by antagonists. Several classes of psychotherapeutic agents also downregulate 5-HT2A receptors with chronic administration including classical antidepressants and antipsychotics. 5-HT2A receptor downregulation produced by 5-HT2A antagonists and antidepressants occurs after presynaptic 5-HT denervation, suggesting that 5-HT2A receptors are postsynaptically localized and emphasizing that they are regulated differently than traditional monoaminergic receptors. Interestingly, the behavioral and biochemical effects of 5-HT2A receptor activation are modulated by activity at other 5-HT receptor subtypes (5-HT1A), as well as by stimulation of receptors for other neurotransmitters and hormones such as norepinephrine (beta-adrenergic) and melatonin. It is suggested that these diverse modulatory influences on 5-HT2A receptor regulation and function may meaningfully impact the therapeutic actions of drugs, including pharmacologically distinct antidepressants.

Nefazodone is a new antidepressant drug with a pharmacologic profile distinct from that of the tricyclic, monoamine oxidase inhibitor, and serotonin selective reuptake inhibitor antidepressants. Nefazodone was initially discovered for its ability to block 5-HT2A receptors and its reduced potency as an alpha 1-adrenergic blocker. It was later shown to inhibit both serotonin and norepinephrine uptake in vitro, attributes which most likely impart its clinical efficacy and which differentiate nefazodone from its chemical predecessor trazodone. The combination of these two mechanisms may ultimately result in a facilitation of 5-HT1A-mediated neurotransmission, which may be beneficial for treating symptoms of depression as evidenced by recent clinical findings. In addition, the preclinical profile of nefazodone demonstrates that it has decreased anticholinergic and antihistaminic activity relative to traditional agents. Clinical findings to date are consistent with these observations.

Interactions between melatonin and serotonin type 2A (5-HT2A) receptors in the regulation of the sleep-wakefulness cycle in the rat have been reported. We studied the acute effects of melatonin and related agonists on 5-HT2A neurotransmission as reflected in behavioral (head shake) and biochemical [phosphoinositide (PI) hydrolysis] responses to 5-HT2A receptor stimulation. Like 5-HT1A agonists and antidepressants, acute administration of melatonin and related agonists inhibited the 5-HT2A-mediated (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane-induced head shake in a dose-dependent manner. Consistent with these behavioral findings, in vitro incubation of cortical slices with melatonin agonists robustly inhibited 5-HT2A receptor-mediated PI hydrolysis in a noncompetitive manner. 2-Iodomelatonin-induced reductions in 5-HT2A-stimulated PI hydrolysis were blocked by preincubation with the melatonin antagonist N-acetyltryptamine. Further, pretreatment of rats in vivo with melatonin and related agonists reduced the cortical PI hydrolysis response to the 5-HT2A agonist alpha methyl-5-HT but did not alter cortical 5-HT2A receptor density. The present data support an interaction between melatonin and 5-HT2A receptors in the central nervous system.

1. Anatomical, behavioral, neurochemical and electrophysiological evidence collectively support a role for central 5-HT in the modulation of anxiety and the anti-anxiety action of the benzodiazepines. 2. The advent of selective agonists and antagonists for 5-HT receptor subtypes (5-HT1, 5-HT2, 5-HT3) has rekindled investigation of the role of 5-HT in anxiety mechanisms. 3. The azapirones represent a new class of agent which possesses affinity for 5-HT1A receptors (partial agonists) and is active in anxiolytic animal models as well as in the clinic (buspirone) 4. While preclinical data supporting the anxiolytic potential of 5-HT2 antagonists remains controversial, a recent clinical study supports ritanserin's anxiolytic effects. 5. Several animal models support the anxiolytic potential of the 5-HT3 antagonist odansetron (GR38032F). Confirmation of it's therapeutic utility awaits clinical results.

High densities of [125I]-iodomelatonin binding sites have been demonstrated in pigeon brain. Melatonin binding sites have been shown to be linked to signal transduction mechanisms in other species. The present study investigated the melatonin-mediated second messenger response of phosphoinositide hydrolysis in slices of telencephalon, optic tectum, cerebellum, hypothalamus, and pons medulla of pigeon brain. The highest rates of melatonin-mediated phosphoinositide hydrolysis were observed in telencephalon and pons/medulla. Relative potencies of melatonin agonists to induce phosphoinositide hydrolysis were as follows: 2-iodomelatonin > 6-chloromelatonin > N-acetylserotonin > melatonin > > serotonin (5-HT). Agonist-induced phosphoinositide hydrolysis was blocked by N-acetyltryptamine (NAT), a melatonin antagonist, but not by ketanserin, a 5HT2A/2C receptor antagonist, demonstrating that phosphoinositide hydrolysis did not result from 5HT2A or 5HT2C receptor stimulation. In addition, the effects of melatonin agonists were sensitive to prazosin, an alpha-adrenergic antagonist reported to exhibit nanomolar affinity for melatonin binding sites in hamster brain, but not to phentolamine, an alpha-adrenergic antagonist that shows no affinity for melatonin binding sites. These data provide evidence that signal transduction associated with melatonin in pigeon brain involves the induction of phosphoinositide hydrolysis as a second messenger.

Investigations utilizing agonists for 5-HT receptor subtypes have been conducted to determine which 5-HT receptor subtype(s) subserve myoclonus in the guinea pig. Administration of a nonselective 5-HT agonist such as 5-MeODMT (5-HT1A/5-HT2 agonist) induces a dose-dependent behavior characterized by head jerking at low doses (1-2 mg/kg, SC) and full-blown myoclonus (continuous rhythmic whole-body jerking) at higher doses (2.5-5 mg/kg, SC). In contrast, the selective 5-HT1A receptor agonist 8-OH-DPAT and the selective 5-HT2 receptor agonist DOI do not induce myoclonus, and elicit only limited head jerking across an otherwise behaviorally active range of doses (1-5 mg/kg, SC). Importantly, the coadministration of both 8-OH-DPAT and DOI results in the emergence of dose-dependent myoclonic behavior. These data suggest that coactivation of 5-HT1A and 5-HT2 receptors may be required for the induction of myoclonus in the guinea pig.

The compound BMY 33462 (4-amino-N-(1-azabicyclo-[2,2,2]oct-3-yl)-2-(butan-2-one-3-yl) oxy-5-chlorobenzamine 1.25 fumerate hydrate) has been shown to be a selective and potent serotonin type-3 (5-HT3) receptor antagonist. Its receptor binding profile includes subnanomolar affinity for the 5-HT3 receptor (K(i), 0.22 +/- 0.06 nM) and no affinity for other serotonergic, dopaminergic, and adrenergic receptors (K(i) > 1,000 nM). BMY 33462 was also shown to be a potent inhibitor of the Bezold-Jarisch reflex in the anesthetized rat (ED50, 0.09 mug/kg, iv), a functional correlate of 5-HT3 receptor antagonism in vivo. It has been reported that ondansetron and other 5-HT3 antagonists have the ability to block hyperactivity in rats induced by DiMe-C7 ([pGlu5, Me-Phe8, Sar9]SP5-11), a metabolically stable analogue of substance P [Hagan et al. (1990): Br J Pharmacol 99:227-232]. DiMe-C7-induced hyperactivity is thought to occur through activation of mesolimbic dopamine (DA) neurons which project to the nucleus accumbens [West and Michael (1991): Brain Res Bull 26:229-233]. BMY 33462 was examined for its ability to block DiMe-C7-induced hyperactivity as was haloperidol and ondansetron. The dopamine-2 (D2) antagonist haloperidol produced the greatest inhibition of DiMe-C7-induced hyperactivity at a dose of 0.025 mg/kg (53.7%). Ondansetron significantly inhibited the DiMe-C7-induced increase in locomotor activity at doses of 0.10 mg/kg and 0.50 mg/kg (42% and 30%, respectively). BMY 33462, at doses of 0.025 mg/kg and 0.05 mg/kg, significantly decreased DiMe-C7-induced hyperactivity by 41% and 37%, respectively. Haloperidol proved to be highly efficacious and potent in blocking increases in DiMe-C7-induced locomotor activity, a phenomenon which may correlate with its clinical effectiveness as a neuroleptic agent. BMY 33462 and ondansetron were equally efficacious, however, BMY 33462 was more potent. The ability of 5-HT3 antagonists to alter a mesolimbic DA-mediated behavior indicates a complex interaction between these two neurotransmitter systems and perhaps a role for central 5-HT3 receptors and 5-HT in the regulation of DA transmission. In addition, BMY 33462 and ondansetron were unable to inhibit stereotypy induced by apomorphine (inactive at 100 mg/kg, orally) suggesting a lack of interaction with the nigrostriatal DA pathway. These results support the notion that 5-HT3 antagonists may be potential therapeutic agents for the treatment of hyperdopaminergic disease states, such as schizophrenia, without the side effect liability generally associated with classical neuroleptics.

Recent pharmacological studies demonstrate that Syrian hamster melanoma (RPMI 1846) cells possess a nanomolar-affinity binding site for melatonin. Inhibition of 2-[125|]-iodomelatonin binding to RPMI membranes by melatonergic ligands exhibit a rank order relationship similar to that observed in hamster hypothalamus. Biochemical studies indicate that the melatonin binding site in RPMI 1846 cells is not coupled in either a stimulatory or inhibitory fashion to adenylate cyclase as a second messenger. We now report that stimulation of RPMI 1846 melanoma cells with melatonergic agonists induces phosphoinositide hydrolysis in a concentration-dependent manner EC50 : N-acetylserotonin = 0.29 μM; 2-l-Melatonin = 0.39 μM; 6-Cl-Melatonin = 0.38 μM; Melatonin = 0.45 μM. Further, phosphoinositide hydrolysis induced by 2-l-melatonin and N-acetylserotonin was blocked by pre-incubation with the melatonin antagonist N-acetyltryptamine and prazosin, an antagonist which exhibits potency at the nanomolar affinity melatonin binding site. 2-l-melatonin and N-acetylserotonin-induced phosphoinositide hydrolysis were not blocked by the serotonin type 2 antagonist ketanserin or alpha-adrenergic antagonist, phentolamine. These data suggest that melatonin binding sites on RPMI 1846 cells are linked to phosphoinositide hydrolysis as a second messenger.

Chronic treatment with the full 5-hydroxytryptamine1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), either by twice daily subcutaneous injection (0.2 or 2.0 mg/kg) or continuous subcutaneous administration via osmotic minipumps (0.4 or 4.0 mg/kg/day), for 14 days, had no effect on the dose-response curves for inhibition of 5-hydroxytryptophan (5-HTP) accumulation in rat cortex or hippocampus by 8-OH-DPAT or the partial 5-HT1A agonist BMY 7378. In contrast, chronic treatment with the nonbenzodiazepine putative anxiolytic gepirone via osmotic minipumps (20 mg/kg/day) resulted in a small but significant rightward shift (1.8-fold) in the dose-response curve to 8-OH-DPAT challenge in the cortex and a slightly larger shift (2.4-fold) in the hippocampus. Similarly, chronic treatment with another putative anxiolytic, ipsapirone, administered via twice daily subcutaneous injections (20 mg/kg), also resulted in a rightward shift (2.6-fold) in the dose-response curve to 8-OH-DPAT in the cortex and a slightly smaller shift (2.3-fold) in the hippocampus. Neither drug, however, decreased the maximal response. The present results are consistent with the suggestion that the clinical anxiolytic effects of gepirone and ipsapirone, and not of 8-OH-DPAT, may be related to their ability to desensitize somatodendritic 5-HT1A autoreceptors; other potential mechanisms are discussed.

Although the ability of agonists at specific serotonin (5-HT) receptor subtypes to induce distinct behaviors has been well documented in the rat, similar studies have not been reported in the Mongolian gerbil. We have found that the 5-HT1A/5-HT2 agonist 5-methoxy, N-N dimethyltryptamine (5-MeODMT) (0.5–8 mg/kg, SC), the specific 5-HT1A agonist 8-hydroxy (di-n-propylamino)tetralin (8-OH-DPAT) (0.125–16 mg/kg, SC), and the 5-HT precursor L-5-hydroxytryptophan (L-5-HTP) (100–250 mg/kg, SC) all elicit a 5-HT syndrome in the gerbil. This syndrome, analogous to the 5-HT syndrome in the rat, consists of reciprocal forepaw treading (RFT), hindleg abduction (HA), body tremors (BT), and Straub tail (ST). The putative 5-HT1A antagonist NAN-190 (0.25–8 mg/kg, SC) when doses 15 min prior to either 5-MeODMT (4 mg/kg, SC) or 8-OH-DPAT (16 mg/kg, SC) blocked both RFT and HA in a dose-dependent manner, suggesting these 5-HT syndrome behaviors are mediated via 5-HT1A receptor activation. We also identified a unique, dose-responsive behavior in the gerbil, induced selectively by 5-HT2 agonists such as quipazine (2–16 mg/kg, SC) and (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.125–8 mg/kg, SC). This reciprocal hindleg body scratch (RHBS) behavior is dose dependently inhibited by pretreatment with the selective 5-HT2 antagonist ritanserin (0.0125–0.2 mg/kg, SC). RHBS behavior is also potently inhibited by pretreament with the selective 5-HT1A agonist 8-OH-DPAT (0.005–0.04 mg/kg, SC), demonstrating a 5-HT1A/5-HT2 receptor subtype interaction. Simultaneous administration of the 5-HT2 agonist DOI (1 mg/kg, SC) with 8-OH-DPAT (0.5 mg/kg, SC) significantly potentiated both RFT and BT behaviors of the 5-HT syndrome in gerbils, as has been reported in rats. Furthermore, pretreatment with the selective 5-HT2 antagonist ritanserin (0.5 mg/kg, SC) resulted in nearly complete inhibition of the potentiation of RFT and BT by DOI and 8-OH-DPAT. Ritanserin pretreatment also shifted the dose-response curve for the 5-MeODMT-induced 5-HT syndrome to the right. These data suggest that 5-HT2 receptor activation facilitates the expression of some 5-HT1A receptor-mediated behaviors of the 5-HT syndrome and may explain why 5-MeODMT has greater potency than 8-OH-DPAT in the gerbil.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.

The effect of continuous treatment with the selective 5-HT1A agonist gepirone upon 5-HT2-mediated behavior and cortical 5-HT2 receptor binding sites was examined in naive rats or rats receiving noradrenergic (DSP4) or serotonergic (5,7-DHT) lesions. Continuous administration of gepirone in non-lesioned rats for 3, 7, or 14 days enhanced the head shake response to the 5-HT agonist quipazine. This enhancement of 5-HT2-mediated behavior occurred despite concomitant down-regulation of cortical 5-HT2 binding sites. However, 28 days of gepirone administration significantly reduced behavioral responsiveness to quipazine. The gepirone-induced facilitation of 5-HT2-mediated behavior observed after 7 days of continuous treatment was blocked in both DSP4 and 5,7-DHT-lesioned rats. However, both noradrenergic and serotonergic denervation failed to modify the down-regulation of 5-HT2 receptor binding sites produced by continuous gepirone administration. These results suggest that the curious dissociation of behavioral and biochemical indices of 5-HT2 receptor function produced by continuous gepirone treatment may be the result of a dual yet separate action of the drug on central presynaptic noradrenergic and serotonergic mechanisms and postsynaptic 5-HT receptors. Furthermore, the postsynaptic action of gepirone which reduces the maximal number of cortical 5-HT2 receptor binding sites may be the result of gepirone's agonist action at postsynaptic 5-HT1A receptors.

Chronic (14 day) administration of several pharmacologically-distinct antidepressant drugs resulted in marked reductions in the serotonin2 (5-HT2)-mediated quipazine-induced head shake response which were accompanied by significant reductions in the density of cortical beta-adrenergic and 5-HT2 binding sites. Noradrenergic (DSP4[N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine]-induced) and serotonergic (5,7-DHT[5,7-dihydroxytryptamine]-induced) lesions either attenuated or blocked antidepressant-induced reductions in 5-HT2-mediated behavior. DSP4- and 5,7-DHT lesions did not alter the down-regulation of 5-HT2 binding sites produced by imipramine, desipramine, phenelzine or iprindole. To a large extent, the antagonism of antidepressant-induced reductions in 5-HT2-mediated behavior was coincident with the blockade of down-regulation of beta-adrenergic binding sites by both noradrenergic and serotonergic denervation. The functional interrelationship of 5-HT2 and beta-adrenergic receptors suggested by the present findings may provide insight into a common mechanism underlying the action of pharmacologically-distinct antidepressant drugs.

The azapirones, a unique pharmacologic class that includes buspirone and gepirone appear to offer the promise of both antidepressant and anxiolytic activity. Their singular affinity for the serotonin (5-hydroxytryptamine [5-HT]) type 1A (5-HT1A) receptor subtype may be the factor responsible for the therapeutic activity of these agents. It is hoped that the distinctive characteristics of this new class of drugs will lead to effective therapy for the treatment of mood disorders without the adverse or ancillary effects associated with currently available agents such as the benzodiazepines. The development of drugs for the treatment of anxiety has gradually evolved from less selective agents, such as alcohol, opiates, and the bromides, to progressively more specific drugs, leading ultimately to the development of the benzodiazepine anxiolytics in 1959. The approval of the azapirone buspirone in 1986 marked a new era of psychotherapeutic drug therapy. There is now evidence that this new generation of anxiolytic drugs may offer antidepressant potential in addition to its anxiolytic properties.

Recent pharmacologic studies suggest that nefazodone may possess antidepressant activity. Nefazodone is active in behavioral models predictive of antidepressant potential. It is active in reversing learned helplessness, prevents reserpine-induced ptosis, and enhances response efficiency in the differential reinforcement for low rates of response paradigm. In in vitro studies, nefazodone inhibits the binding of [3H]ketanserin to cortical serotonin2 (5-HT2) binding sites, whereas in vivo, it antagonizes the 5-HT2-mediated quipazine-induced head shake in rats. In ex vivo studies, acute oral administration of nefazodone inhibits cortical serotonin uptake and occupies frontal cortical 5-HT2 receptor binding sites. Chronic administration of nefazodone produces a reduction in 5-HT2-mediated behavior and decreases cortical 5-HT2 receptor binding site density. Further, a chronic high-dose nefazodone regimen significantly potentiates 5-HT1A-mediated behavioral responses in rats. Nefazodone exhibits decreased anticholinergic, alpha-adrenolytic, and sedative activity relative to other antidepressants.