Sleep/waking effects of a selective 5-HT1A receptor agonist given systemically as well as perfused in the dorsal raphe nucleus in rats

Sleep/waking stages and behavior were studied following the selective 5-HT_1A agonist 8-OH-DPAT given subcutaneously (s.c.) (0.010–0.375 mg/kg) as well as perfused continuously (10 μM) for 6 h into the dorsal raphe nucleus (DRN) using microdialysis. Given systemically, 8-OH-DPAT at 0.375 mg/kg s.c. induced 5-HT behavioral syndrome, increased waking to 149% and reduced slow wave sleep (SWS) to 86%, transition to 76% and rapid eye movement (REM) sleep to 73%. The effect on deep SWS (SWS-2) was biphasic, with an increase after 2 h. 8-OH-DPAT at 0.010 mg/kg did not have any vigilance effects. 8-OH-DPAT perfusion in DRN produced a fourfold increase in REM sleep compared to perfusion of artificial cerebrospinal fluid. This is consistent with the hypothesis that reduced 5-HT neurotransmission following 5-HT_1A autoreceptor stimulation will disinhibit cholinergic REM-promoting mesopontine neurons and thereby lead to a REM sleep increase. The other sleep/waking stages were not significantly affected by 8-OH-DPAT perfusion in DRN.     

Long-term fluoxetine, but not desipramine, inhibits the ACTH and oxytocin responses to the 5-HT1A agonist, 8-OH-DPAT, in male rats

The present studies determined whether serotonin 5-HT_1A receptor-mediated function is modified by chronic exposure to antidepressants. Hormone responses to the 5-HT_1A agonist, 8-OH-DPAT, were evaluated after long-term exposure to two antidepressants, the 5-HT uptake blocker, fluoxetine, and the norepinephrine uptake blocker, desipramine (DMI). In addition, the density and affinity of 5-HT_1A receptors in the hypothalamus and cerebral cortex were measured. Male rats received fluoxetine (10 mg/kg i.p.), DMI (5 mg/kg i.p) or saline injections once daily for 21 days. 8-OH-DPAT (0–500 μg/kg s.c.) was administered 18 h after the final antidepressant injection and 15 min before sacrifice. 8-OH-DPAT significantly increased plasma ACTH, corticosterone, oxytocin and prolactin, but not renin or vasopressin concentrations. Chronic injections of fluoxetine inhibited the ACTH, corticosterone and oxytocin responses to 8-OH-DPAT. suggesting reduced 5-HT_1A receptor function. In contrast, chronic DMI did not alter the hormone responses to 8-OH-DPAT. The density and affinity of 5-HT_1A receptors in the frontal cortex or hypothalamus were not altered by either fluoxetine or DMI. To verify that the observed effects require prolonged exposure to fluoxetine, rats received a single injection of fluoxetin (10 mg/kg, i.p.), 3 h before 8-OH-DPAT (0–500 μg/kg s.c.). Acute fluoxetine did not reduce any of the hormone responses to 8-OH-DPAT. In conclusion, the results suggest that chronic, but not acute, exposure to fluoxetine decreases 5-HT_1A receptor function. This effects is not seen in rats chronically exposed to DMI. The mechanism of the effects of chronic fluoxetine on 5-HT_1A receptor-mediated hormone responses is not likely due to reduced density of 5-HT_1A receptors, because no changes were observed in [³H]8-OH-DPAT binding in hypothalamus and cortex, but could be due to changes in signal transduction.     

Involvement of 5-HT1A receptor mechanisms in the inhibitory effects of methamphetamine on photic responses in the rodent suprachiasmatic nucleus

We examined the role of serotonin 1A (5-HT_1A) receptors in the inhibitory effects of methamphetamine (MA) on photic entrainment to the circadian pacemaker in the suprachiasmatic nucleus (SCN) of rodents. MA inhibited optic nerve stimulation-evoked field potential in the SCN, light-induced Fos expression in the SCN and light-induced phase shift of hamster wheel-running rhythm. NAN-190, a 5-HT_1A receptor antagonist, eliminated the inhibitory effects of MA. NAN-190 has also been reported to antagonize ₁ adrenergic receptors. However, prazosin, which selectively antagonizes ₁ adrenergic receptors, did not affect the inhibitory action of MA on light-induced Fos expression. In addition, parachloroamphetamine, which is known to be a 5-HT releaser, dose-dependently inhibited light-induced phase shift of wheel-running rhythm. These findings suggest that elevation of endogenous 5-HT levels by MA inhibits the photic entraining responses of the circadian pacemaker in the SCN via 5-HT_1A receptor stimulation of the 5-HT released by MA.     

Serotonergic regulation of inhibitory avoidance and one-way escape in the rat elevated T-maze

It has been proposed that distinct 5-HT pathways modulate different types of anxiety. Activation of the ascending dorsal raphe (DR)-5-HT pathway, innervating the amygdala and frontal cortex, would facilitate learned defensive behaviors. On the other hand, activation of the DR-periventricular 5-HT pathway, which innervates the dorsal periaqueductal gray matter (DPAG), would inhibit innate flight or fight reactions. Dysfunction of these pathways has been suggested to relate to generalized anxiety disorder (GAD) and panic disorder (PD) in humans, respectively. The elevated T-maze has been developed to separate conditioned (inhibitory avoidance) from unconditioned (escape) defensive responses in the same rat. Pharmacological validation of this model has shown that the GAD-effective serotonergic anxiolytic buspirone or the putative anxiolytic ritanserin selectively impaired inhibitory avoidance while leaving one-way escape unchanged. Chronic injection of the 5-HT/noradrenaline reuptake inhibitor imipramine impaired inhibitory avoidance and prolonged escape, an effect that may be related to the therapeutic action of this drug on both GAD and PD. Like imipramine, intra-DPAG injection of the 5-HT_1A agonist 8-OH-DPAT impaired both inhibitory avoidance and one-way escape. Intra-DPAG administration of the 5-HT_2A/2C agonist DOI prolonged escape, without affecting inhibitory avoidance. The reversible inactivation of the DRN by muscimol impaired inhibitory avoidance, while facilitating escape from the open arm. Taken together, these results suggest that 5-HT exerts differential control on inhibitory avoidance and escape response in the elevated T-maze, mobilizing different types of 5-HT receptors in key structures implicated in fear/anxiety.     

Destruction of the nucleus raphe obscurus and potentiation of serotonin-mediated behaviors following administration of the neurotoxin 3-acetylpyridine

Systemic administration of the neurotoxin 3-acetylpyridine (3-AP) to rats produced spontaneous episodes of spasmodic movement involving the trunk and limbs including torticollis, contortions of the trunk and rigid extension of the limbs. Because the neurotransmitter serotonin (5-HT) has been implicated in various human involuntary movement disorders, the functional and anatomical integrity of the 5-HT system in rats treated with 3-AP were examined. 5-HT-containing neurons in the brain stem were studied using immunohistochemical labeling with antiserum to 5-HT. Cells in the nucleus raphe obscurus were found to be altered following 3-AP treatment as shown by a decrease in 5-HT immunoreactivity as compared to control rats. No changes in 5-HT immunoreactivity were observed in any other region containing 5-HT cell bodies. Behaviorally, rats treated with 3-AP were 2.5-fold more sensitive to the ability of the 5-HT_1A agonist 8-OH-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.33–3.3 mg/kg) to produce the 5-HT syndrome. Similarly, 3-AP-treated rats were 2-fold more sensitive to the selective 5-HT₂ agonist 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB; 0–1.0 mg/kg) at producing the head shake response. Although these behaviors associated with brain stem 5-HT receptors were potentiated by 3-AP, the hypothermic effect of 8-OH-DPAT which involves ascending mesencephalic 5-HT neurons was unchanged following 3-AP treatment. Treatment with 3-AP did not produce significant alterations of 5-HT or 5-hydroxyindoleacetic acid (5-HIAA) content in any brain region studied. Quantitative autoradiographic analysis of the density of 5-HT_1A receptors labeled with [³H]8-OH-DPAT revealed that these sites were largely unchanged in regions of the brain (frontal cortex, hippocampus and brain stem) and in the spinal cord. Similarly, few changes in the density of 5-HT₂ receptors measured with [³H]ketanserin were observed in various brain regions. These results suggest that neurons from the nucleus raphe obscurus are involved in the elicitation of 5-HT-mediated behavioral resonses by 5-HT_1A and 5-HT₂ receptor agonists that are thought to be mediated through brain stem and spinal cord mechanisms. In addition, because of the close neuroanatomical relationship of the nucleus raphe obscurus with various brain regions known to be involved in motor control, the destruction of this region by 3-AP may contribute to the spasmodic motor behaviors observed following 3-AP treatment.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

5-HT-Dependent myoclonus in guinea pigs: Mediation through 5-HT1A-5-HT2 receptor interaction

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-HT_1A/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-HT_1A 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-HT,A and 5-HT2 receptors may be required for the induction of myoclonus in the guinea pig.     

A comparison of the effects of the 5-HT1 agonists TFMPP and RU 24969 on feeding following peripheral or medial hypothalamic injection.

Experiments were conducted to compare the food intake suppressant effects of the 5-hydroxytryptamine (5-HT)₁ agonists 1-3-trifluoro-methylphenylpiperazine hydrochloride (TFMPP) and 5-methoxy-3-(1,2,3,6-tetrahydropyridinyl)1H indole (RU 24969) following either peripheral or medial hypothalamic injections. The effects of these manipulations were examined in 3 different paradigms involving the stimulation of feeding by: (1) infusion of 25 nmol noradrenaline (NA) into the medial hypothalamus, (2) adaptation to a 20 h food deprivation schedule, and (3) the presentation of a palatable wet mash diet for 1 h each day to ad libitum-fed rats. In all 3 paradigms TFMPP and RU 24969 (0.31–5 mg/kg, i.p.) induced dose-dependent reductions of food intake. Both drugs were somewhat less potent at inhibiting feeding that resulted from food deprivation. In contrast to these results medial hypothalamic infusion of TFMPP or RU 24969 (12.5–50 nmol) failed to affect food intake in any of the 3 tests. This occured in spite of the fact that both 5-HT (12.5–50 nmol) and fluoxetine (12.5–50 nmol) mildly attenuated the feeding that resulted from NA infusion into the same site. The results provide clear evidence that the food intake suppressant effects of peripherally injected TFMPP and RU 24969 are not mediated in the medial hypothalamus. They also suggest that even though manipulations of serotonergic function within the medial hypothalamus can alter food intake, this probably does not involve selective activation of 5-HT_1C and/or 5-HT_1B receptors.     

Chronic paroxetine desensitises 5-HT1D but not 5-HT1B autoreceptors in rat lateral geniculate nucleus

The present study examined the effect of chronic paroxetine (10 mg/kg p.o., 21 days) on the 5-HT_1B and 5-HT_1D autoreceptors controlling 5-HT efflux in slices of rat ventrolateral geniculate nucleus. Electrically stimulated 5-HT efflux (10 pulses, 200 Hz, 0.1 ms, 10 mA) was measured using fast cyclic voltammetry. Peak 5-HT efflux was greater (P<0.01) after chronic paroxetine (22.2±1.4 nM, mean±S.E.M.) than water (15.8±1.4 nM). 5-HT efflux was inhibited by CP 93129 (1 nM–10 μM) and sumatriptan (1 nM–1 μM), agonists at 5-HT_1B and 5-HT_1D receptors, respectively. Chronic paroxetine did not affect the sensitivity of the 5-HT_1B autoreceptor but shifted the sumatriptan concentration-response curve to the right (P<0.05). These data suggest that chronic paroxetine increases evoked 5-HT efflux. This may be the result of desensitisation of 5-HT_1D but not 5-HT_1B autoreceptors.     

A reliable method for the preferential activation of C- or A-fibre heat nociceptors

The thyroid hormone triiodothyronine (T3) augments and accelerates the effects of antidepressant drugs. Although the majority of studies showing this have used tricyclics, a few studies have shown similar effects with the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. In this study we investigated the effects of fluoxetine (5 mg/kg), T3 (20 μg/kg) and the combination of these drugs, each administered daily for 7 days, on serotonergic function in the rat brain, using in vivo microdialysis. Fluoxetine alone induced a trend towards desensitization of 5-HT_1A autoreceptors as shown by a reduction in the effect of 8-OH-DPAT to lower 5-HT levels in frontal cortex, and desensitized 5-HT_1B autoreceptors in frontal cortex. The combination of fluoxetine and T3 induced desensitization of 5-HT_1B autoreceptors in hypothalamus. Since there is evidence linking hypothalamic function and depression, we suggest that this effect may partly account for the therapeutic efficacy of the combination of an SSRI and T3.     

Excitatory and indirect inhibitory actions of 5-hydroxytryptamine on sympathetic preganglionic neurones in the neonate rat spinal cord in vitro

The action of 5-hydroxytryptamine (5-HT) on sympathetic preganglionic neurones (SPN) was studied by intracellular recordings in thin slices of neonatal rat spinal cord in vitro. Superfusion of 5-HT (1–270 μM) to SPN caused a concentration dependent slow depolarization or inward current and an increase in synaptic activity consisting of both EPSPs and IPSPs. The slow depolarization was still present after superfusion with TTX. Similar effects were seen during superfusion with 5-carboxamidotryptamine (5-CT) or -methyl-5-hydroxytryptamine (-me-5-HT). A comparison with the potency of 5-HT was made for 5-CT or -me-5-HT on the same neurone by determining the magnitude of the slow depolarization to different concentrations of agonist. This showed that the apparent potency of the agonists was 5-CT> 5-HT> -me-5-HT even in the presence of fluoxetine, a 5-HT uptake inhibitor. The 5-HT-induced slow depolarization was partially blocked by ketanserin but full recovery was not observed. The results suggest that the excitatory action of 5-HT on SPN is mediated via an atypical 5-HT₂ receptor or a 5-HT_1C-like receptor. The 5-HT-induced IPSPs were reversibly blocked by superfusion with strychnine, suggesting they were mediated by glycine.     

Role of the thalamic intergeniculate leaflet and its 5-HT afferences in the chronobiological properties of 8-OH-DPAT and triazolam in syrian hamster

The 5-HT(1A/7) receptor agonist 8-hydroxy-2-[di-n-propylamino]-tetralin (8-OH-DPAT) has chronobiological effects on the circadian system and, in the Syrian hamster, it is known that serotonergic (5-HT) projections connecting the median raphe nucleus to the suprachiasmatic nuclei (SCN) of the hypothalamus are a prerequisite for the expression of 8-OH-DPAT-induced phase advance of locomotor activity rhythm. We examined the possible involvement of the thalamic intergeniculate leaflet (IGL) in the phase-shifting properties of 8-OH-DPAT injections at CT7. Bilateral electrolytic lesions of the IGL blocked phase-shift responses to 8-OH-DPAT of the activity rhythm. Phase changes induced by injections of 8-OH-DPAT at CT7 and triazolam (Tz), a short-acting benzodiazepine, at CT6 were also studied after bilateral chemical lesion of the 5-HT fibres connecting the dorsal raphe nucleus (DR) to IGL. Destruction of 5-HT fibres within the IGL blocked the phase-shift response to Tz, but not the phase-shift response to 8-OH-DPAT. In conclusion, (a) IGL is essential for the phase-shifting effect of peripheral 8-OH-DPAT injections; (b) 5-HT fibres connecting DR to IGL are necessary for the expression of the phase-shifting effect of Tz but not of 8-OH-DPAT.     

MDMA and fenfluramine alter the response of the circadian clock to a serotonin agonist in vitro

The substituted amphetamine drugs, 3,4-methylenedioxymethamphetamine (MDMA or ‘Ecstasy’) and fenfluramine, are known to damage 5-HT neurons in the brain of animals. However, little is known about the drugs’ effects on circadian rhythmicity which is known to be influenced by serotonergic input to the suprachiasmatic nuclei. In the present study, we tested the ability of MDMA and fenfluramine treatment to alter the ability of the circadian clock to reset in response to an agonist of the 5-HT1A and 5-HT7 receptor subtypes soon after treatment with the drugs, and then again at 20 weeks. Coronal hypothalamic slices containing the suprachiasmatic nuclei (SCN) were prepared from rats and 3-min recordings of the firing rate of individual cells were performed throughout a 12-h period. The ability of the 5-HT agonist, 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), to cause a phase advance in the firing pattern of SCN neurons was assessed in slices from control animals and those pretreated with MDMA or fenfluramine (10, 15 and 20 mg/kg administered on successive days) 6–10 days or 20 weeks previously. Phase advances to 8-OH-DPAT in the slice were attenuated by pretreatment with MDMA or fenfluramine at both drug-test intervals. Our study demonstrates that repeated exposure to MDMA or fenfluramine may interfere with the ability of serotonin to phase shift the circadian clock in the rat. It is possible that such an effect may be responsible for some of the clinical changes, such as sleep disorders and mood changes, sometimes reported by human users of the substituted amphetamines.     

5-HT system and cognition

The study of 5-hydroxytryptamine (5-HT) system has benefited from the identification, classification and cloning of multiple 5-HT receptors (5-HT₁ to 5-HT₇). Growing evidence suggests that 5-HT is important in learning and memory and all its receptors might be implicated in this. Actually, 5-HT pathways, 5-HT reuptake site/transporter complex and 5-HT receptors show regional distribution in brain areas implicated in learning and memory. Likewise, the stimulation or blockade of presynaptic 5-HT_1A, 5-HT_1B, 5-HT_2A/2C and 5-HT₃ receptors, postsynaptic 5-HT_2B/2C and 5-HT₄ receptors and 5-HT uptake/transporter sites modulate these processes. Available evidence strongly suggests that the 5-HT system may be important in normal function, the treatment and/or pathogenesis of cognitive disorders. Further investigation will help to specify the 5-HT system nature involvement in cognitive processes, pharmacotherapies, their mechanisms and action sites and to determine under which conditions they could operate. In this regard, it is probable that selective drugs with agonists, neutral antagonist, agonists or inverse agonist properties for 5-HT_1A, 5-HT_1B/1D, 5-HT_2A/2B/2C, 5-HT₄ and 5-HT₇ receptors could constitute a new therapeutic opportunity for learning and memory alterations.     

Activation of the serotonin 5-HT3 receptor in the dorsal raphe nucleus suppresses REM sleep in the rat

The effects of the selective 5-HT₃ receptor agonist and antagonist m-chlorophenylbiguanide (m-CPBG) and ondansetron, respectively, were studied in adult male Wistar rats implanted for chronic sleep recordings. Microinjection of m-CPBG (2.0 and 4.0 mM) into the dorsal raphe nucleus (DRN) decreased rapid-eye-movement sleep (REMS) and the number of REM periods during the first, second, and third 2-h recording period. On the other hand, direct infusion of ondansetron (0.5–1.0 mM) into the DRN induced no significant changes in sleep variables over the 6 h of recording. Pretreatment with ondansetron (0.5 mM) antagonized the m-CPBG (2.0 mM)-induced reduction of REMS and of the number of REM periods. The data are consistent with the hypothesis that the 5-HT₃ receptor is involved in the effect of DRN serotonergic neurons on brainstem structures that act to promote and induce REMS.

It is suggested that the suppression of REMS after the microinjection of m-CPBG into the DRN is related, at least in part, to the stimulation of glutamatergic interneurons that express 5-HT₃ receptors. Activation of these receptors facilitates the release of glutamate, which, in turn, acts on postsynaptic N-methyl-d-aspartate and non-N-methyl-d-aspartate receptors expressed by serotonergic neurons of the DRN and increases the release of 5-HT at postsynaptic sites.     

Muscarinic regulation of Ca2+ oscillation frequency in GH3 cells

The circadian timekeeping system exhibits many functional changes with aging, including a loss of sensitivity to time cues such as systemic injections of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In order to elucidate the neurochemical mechanisms responsible for this age-related loss of sensitivity of the circadian pacemaker to serotonin agonists, the present study used quantitative autoradiography to determine whether aging decreases serotonin receptor populations in male Syrian hamsters. Four neuroanatomical regions that regulate circadian timekeeping were studied (the suprachiasmatic nuclei [SCN], the lateral geniculate nuclei [LGN], and the median raphe nucleus [MRN] and dorsal raphe nucleus [DRN]). The specific binding of [3H]8-OH-DPAT to serotonin7 (5-HT7) and serotonin1A (5-HT1A) receptors was investigated by competitive inhibition with ritanserin and pindolol, respectively. The results showed that the SCN, IGL, MRN, and DRN of the male Syrian hamster exhibited specific binding of [3H]8-OH-DPAT to both the 5-HT7 and 5-HT1A receptors, and that the latter receptor subtype is more abundant in all of these regions. At 17-19 months of age, a 50% decrease in 5-HT7 receptors was found in the DRN but not in any other regions. No significant age-related changes in 5-HT1A receptors were observed in any regions examined. The finding that a marked decrease in 5-HT7 receptors occurs in the DRN at the age previously characterized by loss of sensitivity to 8-OH-DPAT suggests that this region and this receptor subtype play important roles in 8-OH-DPAT induction of circadian phase shifts in vivo and that they constitute an important locus of aging in the circadian timing system.     

Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting

Serotonin (5-HT) is strongly implicated in the regulation of mammalian circadian rhythms. However, little is known of the functional relationship between the circadian clock located in the suprachiasmatic nucleus (SCN) and its source of serotonergic innervation, the midbrain raphe nuclei. In previous studies, we reported that electrical stimulation of the dorsal or median raphe nuclei (DRN and MRN, respectively) induced 5-HT release in the SCN. Notably, DRN- but not MRN-stimulated 5-HT release was blocked by the 5-HT(1,2,7) antagonist, metergoline, suggesting that the DRN signals to the SCN indirectly via the activation of a 5-HT-responsive multisynaptic pathway. In the present study, pretreatment with the 5-HT(2,7) antagonist, ritanserin, also significantly inhibited DRN-electrically stimulated SCN 5-HT release. However, pretreatment with the 5-HT(1A) antagonist, NAN-190, or the 5-HT(2) antagonists ketanserin and cinanserin had little suppressive effect on this DRN-stimulated 5-HT release. In complementary behavioral trials, electrical stimulation of the DRN during subjective midday caused a 1.3-h advance in the free-running circadian activity rhythm under constant darkness, which was inhibited by metergoline. Collectively, these results are evidence that: (1) DRN-stimulated 5-HT release in the SCN requires the activation of an intermediate target with receptors having 5-HT(7) pharmacological characteristics; (2) electrical stimulation of the DRN induces phase-resetting of the circadian activity rhythm; and (3) activation of 5-HT receptors is necessary for this DRN-stimulated circadian phase-resetting. In view of the dynamic changes in DRN neuronal activity incumbent with the daily sleep-activity cycle, and its functional linkages to the SCN and intergeniculate leaflet, the DRN could serve to provide behavioral/arousal state information to various sites comprising the brain circadian system.     

Fast cyclic voltammetry can be used to measure stimulated endogenous 5-hydroxytryptamine release in untreated rat brain slices

Fast cyclic voltammetry at a carbon fibre microelectrode was used to monitor the time course of 5-hydroxytryptamine (5-HT) overflow in slices of rat dorsal raphe (DRN) and suprachiasmatic nuclei (SCN), incubated in a brain slice chamber for over 6 h. 5-HT overflow was detected in response to electrical brain stimulation in both regions. Voltammetric evidence showed that the released substance was identical to exogenously applied 5-HT. Overflow was reversibly abolished when Ca2+ was removed from the incubating medium or when TTX was added. Ro4-1284, a reserpine like agent, irreversibly abolished 5-HT overflow from both nuclei. The 5-HT uptake blockers, citalopram, clomipramine, fenfluramine and fluvoxamine dose dependently increased overflow and slowed the rate of removal of 5-HT from the extracellular space in both regions. Benztropine had no effect on overflow in the DRN and SCN whereas it caused a significant increase in dopamine overflow in slices of caudate putamen (CPu). Xylamine had no effect on 5-HT overflow in the DRN and SCN. This evidence indicates that the release of endogenous 5-HT can be measured reliably for long periods and that FCV can be used in brain slices for quantitative studies of 5-HT release and uptake.     

Phorbol-12,13-dibutyrate antagonizes while forskolin potentiates the presynaptic autoreceptor-mediated inhibition of [3H]-5-hydroxytryptamine release in rat hypothalamic slices

In superfused rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), the 5-HT autoreceptor agonists 5-methoxytryptamine and RU 24969 inhibited in a concentration-dependent manner the electrically evoked release of [3H]-5-HT. Exposure to phorbol-12,13-dibutyrate increased in a concentration-dependent manner the stimulation-evoked overflow of [3H]-5-HT and shifted to the right the 5-methoxytryptamine inhibition curve. Exposure to forskolin, a potent activator of adenylate cyclase, increased the stimulation-evoked [3H]-5-HT overflow and shifted to the left the 5-methoxytryptamine or RU 24969 inhibitory curves on transmitter release. A similar interaction was observed in the presence of 1-isobutyl,3-methylxanthine (IBMX), a phosphodiesterase inhibitor, or 8-bromo-cyclic AMP and the serotonin autoreceptor agonist 5-methoxytryptamine. In the presence of phorbol-12,13-dibutyrate or forskolin, the enhancing effect of the 5-HT autoreceptor antagonist methiothepin on the stimulation-evoked [3H]-5-HT overflow was significantly less pronounced than in the absence of these compounds. These results indicate that the presynaptic 5-HT autoreceptors that modulate the release of [3H]-5-HT in rat hypothalamic slices may be coupled to the phosphoinositide cycle and protein kinase C-dependent mechanisms. In addition, the increase in intracellular level of cyclic AMP by forskolin, IBMX, and 8-bromo-cyclic AMP potentiates the inhibitory effects of the autoreceptor agonist 5-methoxytryptamine on [3H]-5-HT release, although the mechanism of the interaction remains to be elucidated.