Daily rhythms of serotonin metabolism in the medial hypothalamus of the chicken: effects of pinealectomy and exogenous melatonin

Indoleamine levels in punches of the medial hypothalamus containing the suprachiasmatic nuclei (SCN) of 4-week-old cockerels were determined by HPLC-EC. Melatonin levels in punches were determined by radioimmunoassay (RIA). Daily rhythms of serotonin (5-HT) and of its metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were observed; levels were higher at midnight than at midday. A daily rhythm with the same phase in punch melatonin content was also observed. Pinealectomy at 1 week after hatching abolished the 5-HIAA and melatonin rhythm in 4-week-old birds but did not abolish the 5-HT rhythm. Injections of melatonin (0.5 mg/kg) increased 5-HT, 5-HIAA and melatonin levels in the hypothalamic punches. These results indicate that circulating melatonin of pineal origin may act to increase 5-HT turnover and/or release in the SCN. They suggest a link between the circadian secretion of pineal melatonin and the regulation of 5-HT projections to the hypothalamus from the raphe nuclei in the brainstem of the chicken. We have previously shown that the rhythmic secretion of melatonin by the pineal is influenced by oscillators in the brain via the superior cervical ganglia. The results reported here indicate that melatonin in turn may regulate brain oscillators, suggesting a neuroendocrine loop within the avian circadian system.     

Persistence of nonphotic phase shifts in hamsters after serotonin depletion in the suprachiasmatic nucleus

Serotonin-containing fibres (5-HT) project from the raphe complex to the suprachiasmatic nucleus (SCN). Previous studies have suggested that this pathway may be involved in nonphotic resetting of the circadian clock. For example, 5-HT agonists are capable of phase shifting the biological clock both in vivo and in vitro, producing phase response curves (PRCs) similar in shape to those of other nonphotic stimuli. Therefore we studied the role of the serotonergic projection to the SCN in nonphotic phase shifts by bilateral injection of the selective 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) onto the SCN of hamsters. About 50 days after the administration of the neurotoxin, the 5-HT and 5-HIAA (5-hydroxyindole acetic acid) levels were severely depleted in the SCN, as revealed by high performance liquid chromatography (HPLC), and immunocytochemistry (ICC). The average level of 5-HT depletion was 88% in Experiment 1 and 95% in Experiment 2. This treatment had no effect on the magnitude of phase shifts produced by 3 h of novelty-induced wheel-running starting at circadian time (CT) 4, the peak of the advance region of the PRC to this stimulus. The effect of 5-HT depletion on shifts produced by running at CT 22 were inconclusive because of changes in the behavior of control animals. No changes in the phase angle of entrainment of animals in a 14:10 light:dark (LD) cycle were detected in depleted animals. The results suggest that the 5-HT projection from the raphe to the SCN is not essential for activity-induced phase shifts in hamsters.     

Voltammetric measurements of 5-hydroxyindole compounds in the suprachiasmatic nuclei: Circadian fluctuations

This study concerns the voltammetric signal appearing at a +300 mV potential (peak 3) recorded from the suprachiasmatic nuclei (SCN) of rats under acute and chronic conditions. In acute conditions, and in order to accurately localize the working electrode, a topographical study of the peak 3 height was first realized in the frontal plane containing the SCN by use of differential pulse voltammetry and monocarbon fiber electrodes. In the same conditions, the effects of clorgyline and reserpine were studied. Clorgyline decreased peak 3 while reserpine increased it. Contrary to 5-HT, 5-HIAA contents of the SCN, measured with high performance liquid chromatography, demonstrate analogous variations. All these data suggest that it is essentially 5-HIAA which is responsible for peak 3 from the SCN. In unanesthetized, freely moving rats, under a 12/12 h light-dark cycle, spontaneous and circadian variations of the SCN's peak 3 were studied, during the rest-activity cycle. Multifiber working electrodes were used for this purpose. During spontaneous and successive periods of rest and activity peak 3 height was always found to be higher during activity. Further, these spontaneous variations were superimposed with a circadian variation exhibiting its acrophasis during the dark period. Our data suggest that the release and catabolism of serotonin is greater in waking than in sleeping animals. They also suggest, that the rostral raphe system, phase locked with the SCN, plays an important role in the circadian variations measured.     

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.     

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.     

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.     

Suprachiasmatic nuclei monoamine metabolism of glucose tolerant versus intolerant hamsters.

A critical role for temporal organization of dopaminergic and serotonergic activities within the suprachiasmatic nuclei (SCN) in the regulation of peripheral glucose metabolism has been postulated. This study employed in vivo microdialysis to investigate the temporal extracellular profiles of dopamine and serotonin metabolites in the SCN of freely behaving naturally glucose tolerant and intolerant Syrian hamsters. Microdialysis samples from the right SCN of awake, glucose tolerant or intolerant hamsters held on 14 h daily photoperiods were collected every 2 h over a 24 h period and assayed via HPLC for the metabolites of dopamine: homovanillic acid (HVA) and serotonin (5-hydroxyindolacetic acid, 5-HIAA). Among glucose tolerant hamsters, daily rhythms of SCN HVA and 5-HIAA were observed with coincident plateaus throughout the nocturnal phase of the day (both p<0.01). Relative to glucose tolerant hamsters, glucose intolerant animals exhibited a loss in the daily rhythm of SCN HVA (p<0.0001) and 5-HIAA (p<0.02) due to marked reductions (70%) throughout the 24 h period in HVA levels and comparative decreases (35%) in nocturnal peak levels of 5-HIAA. These findings demonstrate that daily profiles of extracellular dopamine and serotonin activities in the SCN, known to influence glucose metabolism, differ between glucose tolerant and intolerant hamsters.     

Effects of 5-hydroxytryptophan on extracellular serotonin in the spinal cord of rats with experimental allergic encephalomyelitis.

Serotonin (5-HT) and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were collected by in vivo dialysis in the lumbar spinal cord of control rats and rats with hindlimb paralysis induced by experimental allergic encephalomyelitis (EAE). Both 5-HT and 5-HIAA were significantly decreased in baseline samples from EAE rats compared to controls. This decrease in extracellular 5-HT and 5-HIAA in the EAE rats was accompanied by marked morphological changes in spinal cord axons and axon terminal plexuses that were stained for 5-HT-like immunoreactivity. The 5-HT precursor, 5-hydroxytryptophan (5-HTP)-increased 5-HT and 5-HIAA levels in dialysate samples from both control and EAE animals. However, the 5-HTP-induced increase in extracellular 5-HT was significantly greater in the EAE rats than in the controls, despite a lower baseline 5-HT level in the EAE animals. In contrast to 5-HT, both baseline and post-5-HTP levels of 5-HIAA were significantly higher in control animals than in EAE animals. The decreased extracellular 5-HT and 5-HIAA in baseline samples from the EAE rats compared to controls is probably a consequence of the damage to descending 5-HT axons and axon terminals that occurs during the disease. The larger increase in extracellular 5-HT in EAE animals after precursor injection may reflect both decreased 5-HT reuptake from the extracellular space by damaged 5-HT terminals and disruption of the blood-brain barrier that allows entry into the central nervous system of 5-HT that was synthesized from 5-HTP in the periphery.     

Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5-HT) afferents from the median raphe. 5-HT(1B) and 5-HT(7) receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5-HT(1B) and 5-HT(7) receptors in the SCN. 5-HT(1B) receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5-HT(1B) receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5-HT(1B) immunoreaction product was noted in dendrites and somata of SCN neurons. 5-HT(7) receptor immunoreactivity in gamma-aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double-label procedures. 5-HT(7) receptor immunoreaction product was often observed in GABA-, VIP-, and VP-immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5-HT(7) receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA-, VIP-, and VP-immunoreactive perikarya, 5-HT(7) receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5-HT(1B) receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5-HT(7) receptors in both pre- and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity.     

Effects of tryptophan and/or acute running on extracellular 5-HT and 5-HIAA levels in the hippocampus of food-deprived rats

The present microdialysis study has examined whether exercise-elicited increases in brain tryptophan availability (and in turn 5-HT synthesis alter 5-HT release in the hippocampus of food-deprived rats. To this end, we compared the respective effects of acute exercise, administration of tryptophan, and the combination of both treatments, upon extracellular 5-HT and 5-hydroxyindoleacetic acid (5-HLAA) levels. All rats were trained to run on a treadmill before implantation of the microdialysis probe and 24 h of food deprivation. Acute exercise (12 m/min for 1 h) increased in a time-dependent manner extracellular 5-HT levels (maximal increase: 47%). these levels returning to their baseline levels within the first hour of the recovery period. Besides, exercise-induced increases in extracellular 5-HIAA levels did not reach significance. Acute administration of a tryptophan dose (50 mg/kg i.p.) that increased extracellular 5-HIAA (but not 5-HT) levels in fed rats, increased within 60 min extracellular 5-HT levels (maximal increase: 55%) in food-deprived rats. Whereas 5-HT levels returned toward their baseline levels within the 160 min that followed tryptophan administration, extracellular 5-HIAA levels rose throughout the experiment (maximal increase: 75%). Lastly, treatment with tryptophan (60 min beforehand) before acute exercise led to marked increases in extracellular 5-HT and 5-HIAA levels (maximal increases: 100% and 83%, respectively) throughout the 240 min that followed tryptophan administration. This study indicates that exercise stimulates 5-HT release in the hippocampus of fasted rats, and that a pretreatment with tryptophan (at a dose increasing extracellular 5-HT levels) amplifies exercise-induced 5-HT release.     

An improved liquid chromatographic method with electrochemical detection for direct determination of serotonin in microdialysates from Caudate-putamen and pineal gland regions of rat brain

A liquid chromatography method coupled with electrochemical detection has been developed for the direct measurement 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in brain microdialysates. The separation conditions have been optimized to detect only the 5-HT and 5-HIAA in dialysates and elute the other monoamines and their metabolites in the void. Linear regression analysis of chromatographic peak area as a function of concentration in the range 5-1000 pg/mL gave correlation coefficients over 0.995. Sample stability and an assay validation for precision and accuracy were also performed. The limit of detection (S/N = 3) for 5-HT was 12 femtomol/mL. The method has been applied to simultaneously measure extracellular 5-HT and 5-HIAA in brain microdialysates from the pineal gland and caudate-putamen of awake and freely-moving rats under basal conditions.     

Melatonin inhibits in vitro serotonergic phase shifts of the suprachiasmatic circadian clock

The suprachiasmatic (SCN) circadian pacemaker generates 24 h rhythms of spontaneous neuronal activity when isolated in an acute brain slice preparation. The isolated pacemaker also retains its capacity to be reset, or phase-shifted by exogenous stimuli. For example, serotonin (5-HT) agonists advance the SCN pacemaker when applied during mid subjective day, while neuropeptide Y (NPY) agonists and melatonin advance the pacemaker when applied during late subjective day. Previous work has demonstrated interactions between NPY and 5-HT agonists, such that NPY can block 5-HTergic phase advances, while 5-HT agonists do not prevent NPY-induced advances. Due to a number of similarities in the actions of melatonin and NPY in the SCN, it seemed possible that melatonin and 5-HT might interact in the SCN as well. Therefore, in this study potential interactions between melatonin and 5-HT agonists were explored. Melatonin inhibited phase advances by the 5-HT agonist, (+)DPAT, and this inhibition was decreased by co-application of tetrodotoxin. Conversely, melatonin was unable to block phase advances by the cyclic AMP analog, 8BA-cAMP. Finally, neither 5-HT agonists nor 8BA-AMP were able to block melatonin-induced phase advances. These results demonstrate a clear interaction between melatonin and 5-HT in the SCN, and suggest that melatonin and NPY may play similar roles with respect to modulating the phase of the SCN circadian pacemaker in rats.     

Aging regulates 5-HT(1B) receptors and serotonin reuptake sites in the SCN

Middle age is associated with changes in circadian rhythms (e.g., alterations in the timing of the circadian wheel running rhythm) which resemble changes induced by selective destruction of the serotonergic input to the suprachiasmatic nucleus (SCN), the principal mammalian circadian pacemaker. We hypothesized that serotonergic neurotransmission in the SCN is decreased in middle-aged hamsters, as compared to young adults. This hypothesis was tested indirectly by investigating the effect of aging on two markers of serotonin neurotransmission, 5-HT(1B) receptors and serotonin reuptake sites, which are regulated by serotonin. Previous studies have shown that experimentally induced decreases in serotonergic neurotransmission increase 5-HT(1B) receptors but decrease serotonin reuptake sites. Quantitative autoradiography was conducted using [125I]iodocyanopindolol ([125I]ICYP) and [3H]paroxetine, selective radioligands for the 5-HT(1B) receptors and the serotonin reuptake sites, respectively. Consistent with the hypothesis, specific ([125I]ICYP binding was significantly elevated in the SCN of middle-aged hamsters, as compared to young hamsters. The results also showed that serotonin reuptake sites in the SCN were significantly increased in both middle-aged and old hamsters, as compared to young controls. This result could not have been caused by decreased serotonin release. Alternatively, increased serotonin reuptake, which would reduce serotonin levels in the synaptic cleft, may cause or contribute to the increase in 5-HT(1B) receptor binding in the SCN in middle aged animals. These results show that the SCN exhibits changes in serotonergic function during middle age, which has been characterized by changes in the expression of circadian rhythms. Because these changes occur during middle age, they probably reflect the aging process, rather than senescence or disease.     

Localization of serotonin(5A) receptors in discrete regions of the circadian timing system in the Syrian hamster

Endogenous serotonin and serotonergic drugs influence many aspects of circadian rhythms, including phase shifts, onset of locomotor activity, and period length and integrity of rhythms during exposure to constant light. The receptor subtype(s) mediating all of these circadian effects of serotonin has (have) not been identified. Immunoreactivity for the serotonin(5A) (5-HT(5A)) receptor has recently been identified in the rat suprachiasmatic nucleus (SCN). In this study, we investigated the distribution of the 5-HT(5A) receptors in four neural components of the circadian timing system (the SCN, the intergeniculate leaflet, and the median and dorsal raphe nuclei), in the Syrian hamster. Single and dual immunohistochemistry were conducted using an affinity-purified rabbit antibody generated against a peptide sequence unique to the 5-HT(5A) receptor, guinea pig anti-5-HT antisera and guinea pig anti-GABA antisera. For single labeling, immunoreactivity was visualized using DAB-nickel as the chromagen. All four regions showed strong, yet distinct, immunoreactivity for the 5-HT(5A) receptor. No specific labeling was present in the absorption or omission controls. For double labeling, immunoreactivity was visualized using immunofluorescence with Cy5- and FITC-labeled second antibodies followed by confocal microscopy. In the raphe nuclei, 5-HT-immunoreactivity and 5-HT(5A)-immunoreactivity were co-localized in cell bodies and axons. GABA-immunoreactive fibers surrounded some of the 5-HT(5A) receptor-immunoreactive cell bodies in the raphe nuclei. In conclusion, the 5-HT(5A) receptors are localized within several important neuroanatomical substrates of the circadian timekeeping system, and within the raphe nuclei, appear to be present on serotonin neurons. These findings suggest that some of the circadian effects of 5-HT may be mediated by the 5-HT(5A) receptor, which may function as a presynaptic autoreceptor.     

Extracellular serotonin levels change with behavioral state but not with pyrogen-induced hyperthermia.

Extracellular 5-HT in the anterior hypothalamus/preoptic area (AH/POA) and caudate nucleus of the freely moving cat was measured using in vivo brain microdialysis. Administration of 8-OH-DPAT, a 5-HT1A receptor agonist that decreases 5-HT neuronal activity, decreased extracellular 5-HT in both brain areas. Extracellular 5-HT levels were also examined in relationship to the sleep-wake cycle, because previous data from our laboratory have indicated that behavioral state is the primary determinant of 5-HT neuronal discharge. As with 5-HT neuronal discharge, extracellular 5-HT was increased during active behavioral states and decreased during somnolent periods. These first two sets of findings confirm the ability of the microdialysis technique to measure physiological fluctuations in extracellular 5-HT levels and support the hypothesis that neuronal discharge is a major determinant of extracellular 5-HT levels. Levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) in the AH/POA were also responsive to changes in behavioral state and administration of 8-OH-DPAT, though fluctuations in extracellular 5-HIAA were less robust and temporally delayed. Finally, extracellular 5-HT and 5-HIAA were examined in the AH/POA during fever induced by systemic injection of the synthetic pyrogen muramyl dipeptide. Previous data from our laboratory have indicated that 5-HT neuronal activity is unaffected by this manipulation, though 5-HT has been implicated specifically in thermoregulation. Pyrogen-induced hypothermia produced no specific change in 5-HT efflux, because any changes noted could be accounted for by behavioral state changes. These data are consistent with the hypothesis that the brain serotonergic system is closely linked to the sleep-wake-arousal cycle. However, extracellular 5-HT may be involved in thermoregulatory processes as part of a global role in modulating neuronal activity in coordination with the behavioral state of the animal.     

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.     

Functional meaning of tryptophan-induced increase of 5-HT metabolism as clarified by in vivo voltammetry

Differential pulse voltammetry with carbon fiber electrodes was used to study serotonin (5-HT) metabolism in freely moving rats. The electrodes implanted in the striatum recorded the extracellular 5-hydroxyindoleacetic acid (5-HIAA) oxidation peak after oral tryptophan (150 mg/kg). This 5-HT precursor did not modify the 5-HIAA peak in any rat tested, but it raised 5-HIAA levels determined in total tissue by a classical biochemical method (HPLC). The administration of 5-hydroxytryptophan (5-HTP) (25 mg/kg i.p.) induced an increase of 5-HIAA detectable both in the extracellular medium by voltammetry and in tissue samples. As previously shown, dorsal raphe electrical stimulation raises extracellular 5-HIAA in the striatum and this effect is enhanced by pretreatment with tryptophan. The results suggest that tryptophan in 'normal' conditions enhances 5-HT metabolism without affecting 5-HT release unless such release is stimulated. 5-HTP increases 5-HT metabolism and release.     

Measurement of 5-hydroxytryptamine synthesis and metabolism in selected discrete regions of the rat brain using high performance liquid chromatography and electrochemical detection: Pharmacological manipulations

High performance liquid chromatography coupled with electrochemical detection (LCEC) was employed to measure 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophan (5-HTP) in the suprachiasmatic (SCN), medial preoptic (MPO) and arcuate (AN) nuclei as well as the median eminence (ME) and striatum (ST) of individual rat brains. Biochemical estimations of changes in 5-HT neuronal activity were made by measuring: (1) concentrations of 5-HT and 5-HIAA and (2) the rate of 5-HT synthesis (5-HTP accumulation following the administration of NSD 1015, an inhibitor of aromatic L-amino acid decarboxylase) after the administration of pharmacological agents known to influence these neurons. Pargyline increased the concentration of 5-HT and decreased the concentration of 5-HIAA while probenecid increased the concentration of 5-HIAA in all 5 brain regions. At both 2 and 24 hours after reserpine the concentration of 5-HT decreased, 5-HIAA increased or did not change, and the rate of 5-HT synthesis increased. In most of the brain regions blockers of 5-HT neuronal uptake (fluoxetine, chlorimipramine) did not influence 5-HT or 5-HIAA concentrations dramatically, but increased the rate of 5-HT synthesis. L-tryptophan generally increased the concentrations of 5-HT and 5-HIAA as well as the rate of accumulation of 5-HTP in all regions except the ME where 5-HIAA and 5-HTP concentrations both were unaffected. These results reveal that the method using LCEC is sensitive enough to detect pharmacologically-induced changes in 5-HT metabolism and synthesis in discrete regions of rat brain. The drugs examined in the present study generally caused similar changes in 5-HT dynamics in all 5 brain regions examined.     

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.     

Characterization of monoamine release in the lateral hypothalamus of awake, freely moving rats using in vivo microdialysis

Extracellular levels of serotonin (5-HT), dopamine (DA) and their major metabolites 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), were measured in the lateral hypothalamus of awake, freely moving rats using microdialysis combined with HPLC and electrochemical detection. To characterize the factors which control 5-HT release, the effects of various drugs were assessed. TTX had a reversible inhibitory effect on the basal levels of 5-HT, 5-HIAA, DOPAC and HVA. Infusion of K+ concomitantly increased 5-HT and DA and decreased 5-HIAA and HVA. Imipramine increased extracellular levels of 5-HT and DA and decreased 5-HIAA levels; this effect was TTX-sensitive. Systemic pargyline increased extracellular 5-HT and markedly decreased the metabolic levels. Pargyline pretreatment in the presence of imipramine, infused through the dialysis probe, slowly increased 5-HT levels above that produced by the reuptake blocker alone. Infusion with AMPH produced a dramatic, TTX-insensitive, increase in 5-HT and DA and a decrease in the metabolic levels. These results provide evidence that (1) basal release of 5-HT in the lateral hypothalamus results from neuronal activity, (2) the metabolites in the extracellular fluid derive primarily from intracellular monoamine oxidase (MAO) activity, (3) 5-HT is mainly removed from the extracellular space by a reuptake mechanism, with minimal contribution of an extracellular MAO, and (4) the AMPH-evoked release of 5-HT and DA is a Na+ channel-independent process.