MDMA alters the response of the circadian clock to a photic and non-photic stimulus

3,4-Methylenedioxymethamphetamine (MDMA or 'Ecstasy') is a widely used recreational drug that damages serotonin 5-HT neurons in animals and possibly humans. Published literature has shown that the serotonergic system is involved in photic and non-photic phase shifting of the circadian clock, which is located in the suprachiasmatic nuclei. Despite the dense innervation of the circadian system by 5-HT and the known selective neurotoxicity of MDMA, little is known about the effects of MDMA on the circadian oscillator. This study investigated whether repeated exposure to the serotonin neurotoxin MDMA alters the behavioural response of the Syrian hamster to phase shift to the serotonin 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT). This agonist was administered under an Aschoff Type I (CT8) and Aschoff Type II (ZT8) paradigm (5 mg/kg) and was given before and after treatment with MDMA (10, 15 and 20 mg/kg administered on successive days). Pre-treatment with MDMA significantly attenuated phase shifts to 8-OH-DPAT. We also tested the ability of the clock to phase shift to a photic stimulus after treatment with MDMA. A 15-min light pulse (mean lux 125 at CT14 or ZT14) was administered before and after treatment with MDMA. Phase shifts to a photic stimulus were significantly attenuated by pre-treatment with MDMA. Our study demonstrates that repeated exposure to MDMA may alter the ability of the circadian clock to phase shift to a photic and non-photic stimulus in the hamster. Disruption of circadian function has been linked with a variety of clinical conditions such as sleep disorders, mood, concentration difficulties and depression, consequently outlining the potential dangers of long-term ecstasy use.     

Phase-resetting effect of 8-OH-DPAT, a serotonin1A receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro.

The 5-HTergic neurons in the mesencephalic raphe nuclei provide a robust projection to the hypothalamic suprachiasmatic nucleus (SCN), the site of a putative neuronal circadian pacemaker. Although it has been suggested that 5-HT neurons may play a role in the circadian timing system, this role has not yet been specified. Prosser et al. (Brain Res., 534 (1990) 336-339) reported that 1 h treatments with quipazine induce robust phase shifts in vitro, and that this effect depends upon the circadian time of treatment. However, quipazine is a non-specific 5-HT agonist. Besides, it is reported that the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetraline hydrobromide (8-OH-DPAT) affected a circadian rhythm of hamster wheel-running activity. In the present study we investigated whether the 5-HT1A agonist 8-OH-DPAT can reset the phase of the SCN clock when it is isolated in vitro. The present results show that 1 h treatments with 8-OH-DPAT induce robust phase advances in vitro when it was administered during the subjective day. This result suggests that 5HT1A receptor functioning may play a role in modulating the phase of SCN clock, especially during the subjective day.     

Serotonin phase-shifts the mouse suprachiasmatic circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) receives multiple afferent signals that could potentially modulate its phase. One input, the serotonin (5-HT) projection from the raphe nuclei, has been extensively investigated in rats and hamsters, yet its role(s) in modulating circadian clock phase remains controversial. To expand our investigation of 5-HT modulation of the SCN clock, we investigated the phase-shifting effects of 5-HT and its agonist, (+)8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), when applied to mouse SCN brain slices. 5-HT induced 2-3 h phase advances when applied during subjective day, while non-significant phase shifts were seen after 5-HT application at other times. These phase shifts were completely blocked by the 5-HT antagonist, metergoline. DPAT also induced phase shifts when applied during mid-subjective day, and this effect appeared dose-dependent. Together, these results demonstrate that the mouse SCN, like that of the rat, is directly sensitive to in vitro phase-resetting by 5-HT.     

Short-term constant light potentiation of large-magnitude circadian phase shifts induced by 8-OH-DPAT: effects on serotonin receptors and gene expression in the hamster suprachiasmatic nucleus

Nonphotic phase-shifting of mammalian circadian rhythms is thought to be mediated in part by serotonin (5-HT) acting in the suprachiasmatic nucleus (SCN) circadian clock. Previously we showed that brief (1-3 days) exposure to constant light (LL) greatly potentiates nonphotic phase-shifting induced by the 5-HT agonist, (+/-)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT). Here we investigated potential mechanisms for this action of LL, including 5-HT receptor upregulation and SCN clock gene and neuropeptide gene expression. Autoradiographic analysis of ritanserin inhibition of [3H]8-OH-DPAT binding indicated that LL (approximately 2 days) did not affect 5-HT7 receptor binding in the SCN or dorsal raphe. Measurement of 5-HT1A autoreceptors in the median raphe and 5-HT1B receptors in the SCN also showed no effect of LL. In experiment 2, hamsters held under a 14-h light : 10-h dark photocycle (LD) or exposed to LL for approximately 2 days received an intraperitoneal injection of 8-OH-DPAT or vehicle at zeitgeber time (ZT) 6 or 0 and were killed after 2 h of dark exposure. 8-OH-DPAT suppressed SCN Per1 and Per2 mRNAs at both ZTs, as assessed by in situ hybridization. Per1 mRNA was also suppressed by LL alone. In addition, in situ hybridization of arginine vasopressin (AVP) mRNA and vasoactive intestinal polypeptide mRNA showed that LL significantly suppressed the former but not the latter. The LL-induced suppression of SCN Per1 mRNA and AVP mRNA may be involved in LL-induced potentiation of pacemaker resetting, especially as these data provide additional evidence that LL suppresses circadian pacemaker amplitude, thus rendering the clock more susceptible to phase-shifting stimuli.     

Serotonergic activation potentiates light resetting of the main circadian clock and alters clock gene expression in a diurnal rodent

The main circadian clock, localized in the suprachiasmatic nuclei (SCN) in mammals, can be synchronized by light and non-photic factors such as serotonergic cues. In nocturnal rodents, injections during the subjective day of the 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or its positive enantiomer, induce behavioral phase-advances in correlation with decreased expression of two clock genes, Per1/2. In addition, 8-OH-DPAT and the selective serotonin reuptake inhibitor fluoxetine reduce light-induced phase-shifts during the subjective night. Beside the chronobiotic effects of serotonin, changes of serotonergic activity in humans have been involved in mood disorders, that are often associated with alterations in circadian rhythmicity. To get insights into the circadian role of serotonin in diurnal species, we investigated its modulation of the SCN in Arvicanthis ansorgei housed in constant darkness. In striking contrast to nocturnal rodents, daily serotonin content in Arvicanthis SCN peaked during daytime while the sensitivity window of its SCN to (+)8-OH-DPAT occurred essentially during the subjective night. Moreover, fluoxetine produced behavioral phase-advances at circadian time (CT) 0 and CT12. Expression of Per1/2, Rev-erbalpha/beta and Roralpha/beta in the SCN was not modified after fluoxetine or (+)8-OH-DPAT injection. Furthermore, both treatments enhanced light-induced phase-advances and delays. Light responses of Per1 and Rorbeta expression at CT0 and those of Per2 and Rev-erbalpha at CT12 were markedly altered by serotonergic activation. The present findings demonstrate that the serotonergic modulation of the SCN clock appears to differ between nocturnal species and the diurnal Arvicanthis. The potentiating effects of fluoxetine on light resetting in a diurnal rodent may be clinically relevant.     

A serotonin agonist phase-shifts the circadian clock in the suprachiasmatic nuclei in vitro

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) receives a large serotonergic (5-HTergic) projection from the raphe nuclei. Whether the SCN pacemaker can be modulated by this afferent projection is a question of considerable theoretical and practical interest. In this study we investigated whether the 5-HT agonist, quipazine, can reset the phase of the SCN clock when it is isolated in vitro. Our results show that 1 h treatments with quipazine induce robust phase shifts in vitro, and that this effect depends upon the circadian time of treatment. We further show that the ability of quipazine to induce phase shifts is dose-dependent. These results suggest that the SCN circadian pacemaker is sensitive to 5-HTergic stimulation, and therefore that the 5-HTergic projection to the SCN may play a role in modulating the phase of the SCN clock in the intact animal.     

Light and melatonin inhibit in vivo serotonergic phase advances without altering serotonergic-induced decrease of Per expression in the hamster suprachiasmatic nucleus

In the Syrian hamster a serotonergic (5-HTergic) stimulation during daytime acts on the circadian timing system by inducing behavioral phase advances and by decreasing Per1 and Per2 (Period) mRNA levels in the suprachiasmatic nuclei, containing the main circadian clock in mammals. The present study was conducted in Syrian hamsters, housed in constant darkness, to investigate the interactions between light or melatonin with serotonergic stimulation in terms of phase resetting and clock gene expression. Both light exposure and systemic administration of melatonin prior to the injection of a 5-HT(1A/7) receptor agonist, 8-OH-DPAT, in the middle of the day blocked behavioral phase advances. In contrast, neither light nor melatonin treatment during daytime prevented serotonergic-induced down-regulation of Per1 and/or Per2 mRNA levels in the suprachiasmatic nuclei. Taken together, the results show that interactions between afferent cues to the suprachiasmatic nuclei differentially modulate phase adjustment and clock gene expression during daytime.     

Short-term exposure to constant light promotes strong circadian phase-resetting responses to nonphotic stimuli in Syrian hamsters

Behavioral (nonphotic) stimuli can shift circadian rhythms by serotonin (5-HT) and/or neuropeptide Y (NPY) inputs to the suprachiasmatic nucleus (SCN) circadian clock. Based on the idea that behavioral phase resetting is modulated by endogenous changes in postsynaptic sensitivity to such transmitters, hamsters were exposed to constant light (LL; approximately 250 lx) for 1-3 days, which suppresses locomotor activity and eliminates the daily rhythm of SCN 5-HT release measured by microdialysis. Groups subjected to brief LL or maintained under a light/dark cycle (LD) received phase-resetting treatments with the 5-HT(1A,7) agonist (+/-)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) or sleep deprivation (SD). Animals were released to constant darkness at the start of the treatments. Phase advances to 8-OH-DPAT and SD during the day were 11 and 3 h for LL vs. 2 and 1 h for LD, respectively. Phase delays during the night were -12 and -5 h for LL vs. no responses for LD, respectively. Phase-transition curves for both LL treatments had slopes approximating 0, indicative of Type 0 phase resetting. For all treatments, the degree of locomotor suppression by LL was not correlated with the phase shift magnitude. Re-establishing locomotor activity by overnight food deprivation did not prevent potentiated shifting to SD. However, re-establishing peak extracellular 5-HT levels by intra-SCN 5-HT reverse microdialysis perfusion in LL did significantly reduce potentiated 8-OH-DPAT phase advances. Constant light also enhanced intra-SCN NPY-induced phase advances during the day (6 vs. 2 h for LD). These results suggest that LL promotes Type 0 phase resetting by supersensitizing 5-HT and/or NPY postsynaptic responses and possibly by attenuating the amplitude of the circadian pacemaker, thus enhancing circadian clock resetting nonspecifically.     

MDMA alters the response of the mammalian circadian clock in hamsters: effects on re-entrainment and triazolam-induced phase shifts

Serotonin (5-hydroxytryptamine or 5-HT) is a neurotransmitter that is involved in a wide range of behavioural and physiological processes. Previous work has indicated that serotonin is important in the regulation of the circadian clock, which is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. 3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy'), which is widely used as a recreational drug of abuse, is a serotonin neurotoxin in animals and non-human primates. Previous work has shown that MDMA exposure can alter circadian clock function both in vitro and in vivo. Evidence shows that 5-HT may have a modulatory role in the regulation of the circadian clock by non-photic stimuli, such as the benzodiazepine triazolam (TRZ). Triazolam is a short-acting benzodiazepine that results in phase advances of the wheel running activity in hamsters when administered during the mid-subjective day. In the present study, male Syrian hamsters treated with TRZ (5 mg/kg) at ZT6 significantly phase advanced their clock. Treatment with MDMA significantly diminished the TRZ induced phase shift in hamsters. Previous evidence shows the involvement of 5-HT in the re-synchronisation of the endogenous clock to a new shifted light-dark cycle. Untreated animals were successfully entrained to a new, 6 h advanced light-dark cycle within an average of 4.5 +/- 0.1 days. Following treatment with MDMA, these animals took an average of 8.3 +/- 0.1 days to re-entrain to a shifted environmental cycle. Immunohistochemical analysis revealed that animals treated with MDMA showed reduced serotonin staining, as evidenced by a decrease in innervation density in the SCN. No significant differences were found in cell counts within the raphe nuclei. These results demonstrate the importance of the serotonergic system in the modulation of photic and non-photic responses of the circadian pacemaker.     

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.     

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.     

Serotonin‐induced phase advances of SCN neuronal firing in vitro: A possible role for 5‐HT5A receptors?

Spontaneous firing rates of neurons in the suprachiasmatic nuclei (SCN) follow a consistent pattern, peaking near the midpoint of the light phase in a 12:12 light/dark schedule, and repeating this brief period of increased activity in subsequent circadian cycles. These carefully timed fluctuations reflect the output signal of the SCN, long recognized as the site of the endogenous biological clock in mammals. In rat hypothalamic slices, bath incubations of 8-OH-DPAT had previously been shown to elicit phase advances when applied at ZT6 (or 6 h following the onset of light), an action that could readily be attributed to 5-HT7 receptor activation. The present studies set out with the simple goal of establishing that the same receptor mechanism was responsible for the phase-shifting actions of 5-HT itself. Surprisingly, the phase advances elicited by 5-HT (0.5 microM, 1 h) at ZT6 were reduced by one 5-HT7 antagonist, ritanserin (10 microM), but not by another, mesulergine (10 microM). Receptor binding studies demonstrated a 25-fold greater affinity of ritanserin for h5-HT5A sites compared to mesulergine (Ki = 71 nM vs. 1,800 nM), an observation suggestive of a 5-HT5A mechanism for 5-HT and consistent with earlier observations of robust labeling of 5-HT5A sites in the SCN. 5-HT generated by the addition of L-tryptophan (10 microM, 1 h) to the slices displayed the same pattern of sensitivity, that is, blockade by ritanserin but not by mesulergine. Rp-cAMPS, a cAMP antagonist, failed to block the phase shifts elicited by 5-HT at a concentration (1 microM) previously shown to be effective against 8-OH-DPAT-induced phase shifts, in keeping with the proposed negative coupling of 5-HT5A receptors to cAMP production. Taken together, these results suggest that activation of both 5-HT5A and 5-HT7 receptors can produce phase advances of the circadian clock in vitro when they occur during mid-subjective day.     

Effects of 21 days treatment with fluoxetine on stimulated endogenous 5-hydroxytryptamine overflow in the rat dorsal raphe and suprachiasmatic nucleus studies using fast cyclic voltammetry in vitro

Changes in the extracellular concentration of 5-HT evoked by electrical stimulation of brain slices containing either dorsal raphe nucleus (DRN) or suprachiasmatic nucleus (SCN) from rats treated for 21 days with fluoxetine (5 mg/kg; i.p.) or water were monitored using fast cyclic voltammetry (FCV). Stimulated 5-HT overflow was enhanced significantly in both brain regions after 21 days treatment with fluoxetine but there was no change in the half time for re-uptake (t1/2). Concentration response curves for inhibition of electrically stimulated 5-HT overflow by 8-OH-DPAT (5-HT_1a receptor agonist) or RU24969 (5-HT_1b receptor agonist) in the DRN or SCN respectively were obtained in slices prepared from both groups of animals. There was a significant shift to the right in the dose-response curve for RU24969 in the SCN in fluoxetine treated animals but a shift to the left for the dose-response curve for 8-OH-DPAT in the DRN. These data suggest that down regulation of the 5-HT_1b autoreceptors occurs in an axon terminal region (SCN) but that there is a sensitisation of 5-HT_1a autoreceptor mechanisms controlling 5-HT overflow in the DRN.     

Effects of serotonergic agonists on firing rates of photically responsive cells in the hamster suprachiasmatic nucleus

Serotonergic neurons from the midbrain raphe nuclei innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, which functions as the dominant pacemaker for mammalian circadian rhythms. We investigated the effects of serotonin (5-HT) on firing rates of light-activated SCN cells in urethane-anesthetized hamsters. Micro-iontophoretic application of 5-HT or 5-HT_1A agonists (8-OH-DPAT and 5-CT) causeda dose-dependent inhibition of spontaneous activity and photic responses in the majority of SCN cells tested. Application of metergoline alone, a non-selective 5-HT antagonist, slightly increased firing rates during darkness and light exposure, suggesting a tonic serotonergic suppression of SCN activity. Metergoline also effectively attenuated suppression induced by the three 5-HT agonists. In addition, the effects of 8-OH-DPAT were blocked by a 5-HT_1A antagonist, SDZ 216-525. However, other putative 5-HT antagonists were weak (propranolol and NAN-190) or ineffective (ketanserin) in blocking the action of 8-OH-DPAT. These results indicate that serotonin has a potent role in reducing photic effects on retinally activated SCN cells in hamsters, and that these effects are mediated by a receptor with properties similar to those of the 5-HT_1A subtype.     

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.     

Neuropeptide Y blocks serotonergic phase shifts of the suprachiasmatic circadian clock in vitro

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) can be reset in vitro by various neurochemical stimuli. This study investigated the phase-shifting properties of neuropeptide Y (NPY) and serotonin (5-HT) agonists when applied alone, as well as their combined effects on clock resetting. These neurotransmitters have both been shown to advance the SCN clock in vitro when applied during the daytime. By monitoring the SCN neuronal activity rhythm in vitro, I first confirm that the 5HT_1A/5HT₇ agonist (+)DPAT maximally advances the SCN clock when applied at zeitgeber time 6 (ZT6). Conversely, NPY only phase advances the neuronal activity rhythm when applied at ZT 10. This effect occurs through stimulation of Y₂ receptors. NPY, again acting through Y₂ receptors, blocks (+)DPAT-induced phase shifts at ZT 6, while neither (+)DPAT nor 5-HT affect NPY-induced phase shifts at ZT 10. NPY appears to block (+)DPAT-induced phase shifts by preventing increases in cyclic AMP. These data are the first to demonstrate in vitro interactions between daytime resetting stimuli in the rat, and provide critical insights into mechanisms controlling circadian clock phase.     

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.     

Altered prolactin response to M-chlorophenylpiperazine in monkeys previously treated with 3,4-methylenedioxymethamphetamine (MDMA) or fenfluramine

3,4-Methylenedioxymethamphetamine ("Ecstasy," MDMA) and fenfluramine, widely used by humans, are potent brain serotonin (5-HT) neurotoxins in animals. Thus, there is concern that humans previously exposed to these amphetamine derivatives may have incurred brain 5-HT neurotoxicity. However, assessing the status of brain 5-HT neurons in the living organism is challenging. To determine whether MDMA- and/or fenfluramine-induced 5-HT neurotoxicity can be detected during life using neuroendocrine methods, groups of monkeys previously treated with neurotoxic regimens of MDMA or fenfluramine, along with saline-treated controls, underwent neuroendocrine challenge with the direct 5-HT agonist and 5-HT-releasing drug, m-chlorophenylpiperazine (m-CPP). Animals treated 2 weeks previously with MDMA exhibited a nonsignificant reduction in the prolactin response to m-CPP. In contrast, monkeys treated 3 1/2 years previously with MDMA or 2 years previously with fenfluramine exhibited significantly increased prolactin responses to m-CPP. No significant differences in cortisol concentrations were noted between groups at any time point. These data indicate that neuroendocrine challenge with m-CPP is capable of detecting substituted amphetamine-induced 5-HT neurotoxicity in living primates, but that the recency of drug exposure is an important consideration. Changes in the neuroendocrine response to m-CPP over time in animals with substituted amphetamine-induced neurotoxicity may be related to aberrant 5-HT reinnervation of the basal forebrain that occurs over time in monkeys previously treated with neurotoxic doses of MDMA or fenfluramine.     

Nonphotic entrainment by 5-HT1A/7 receptor agonists accompanied by reduced Per1 and Per2 mRNA levels in the suprachiasmatic nuclei.

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock gene, Period (Per), are involved in photic entrainment. Therefore, we examined whether phase advances elicited by 8-OH DPAT were associated with a change of Period mRNA levels in the SCN. In this experiment, we cloned partial cDNAs encoding hamster Per1, Per2, and Per3 and observed both circadian oscillation and the light responsiveness of Period. Furthermore, we found that the inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurred only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT.