A combination therapy of neural and glial restricted precursor cells and chronic quipazine treatment paired with passive cycling promotes quipazine-induced stepping in adult spinalized rats.

INTRODUCTION: In order to develop optimal treatments to promote recovery from complete spinal cord injury (SCI), we examined the combination of: (1) a cellular graft of neural and glial restricted precursor (NRP/GRP) cells, (2) passive exercise, and (3) chronic quipazine treatment on behavioral outcomes and compared them with the individual treatment elements. NRP/GRP cells were transplanted at the time of spinalization. METHODS: Daily passive exercise began 1 week after injury to give sufficient time for the animals to recover. Chronic quipazine administration began 2 weeks after spinalization to allow for sufficient receptor upregulation permitting the expression of its behavioral effects. Behavioral measures consisted of the Basso, Beattie, and Bresnahan (BBB) locomotor score and percent of weight-supported steps and hops on a treadmill. RESULTS: Rats displayed an increased response to quipazine (BBB ≥ 9) beginning at 8 weeks post-injury in all the animals that received the combination therapy. This increase in BBB score was persistent through the end of the study (12 weeks post-injury). CONCLUSION: Unlike the individual treatment groups which never achieved weight support, the combination therapy animals were able to perform uncoordinated weight-supported stepping without a body weight support system while on a moving treadmill (6.5 m per minute) and were capable of supporting their own weight in stance during open field locomotion testing. No regeneration of descending serotonergic projections into and through the lesion cavity was observed. Furthermore, these results are a testament to the capacity of the lumbar spinal cord, when properly stimulated, to sustain functioning locomotor circuitry following complete SCI.

Presynaptic modulation of 5-HT release in the rat septal region.

5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [(3)H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S(1)), S(2), S(3); 360 pulses, 3 Hz, 2 ms, 26-28 mA). In all septal regions the Ca(2+)-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [(3)H] was inhibited by the 5-HT(1B) agonist CP-93,129 and the alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the mu- and kappa-opioid receptor agonists (d-Ala(2), N-Me-Phe(4), glycinol(5))-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the delta-opioid receptor agonist (d-Pen(2), d-Pen(5))-enkephalin (DPDPE), the dopamine D(2) receptor agonist quinpirole and the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine were all ineffective; the GABA(B) receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S(1). It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT(1B) autoreceptors, as well as by alpha(2)-, mu- and kappa-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.

Behavioral and neuropharmacological evidence that serotonin crosses the blood-brain barrier in Coturnix japonica (Galliformes; Aves)

This study was carried out aiming to reach behavioral and neuropharmacological evidence of the permeability of the blood-brain barrier (BBB) to serotonin systemically administered in quails. Serotonin injected by a parenteral route (250-1000 æg.kg-1, sc) elicited a sequence of behavioral events concerned with a sleeping-like state. Sleeping-like behaviors began with feather bristling, rapid oral movements, blinking and finally crouching and closure of the eyes. Previous administration of 5-HT2C antagonist, LY53857 (3 mg.kg-1, sc) reduced the episodes of feather bristling and rapid oral movements significantly but without altering the frequency of blinking and closure of the eyes. Treatment with the 5-HT2A/2C antagonist, ketanserin (3 mg.kg-1, sc) did not affect any of the responses evoked by the serotonin. Quipazine (5 mg.kg-1, sc) a 5-HT2A/2C/3 agonist induced intense hypomotility, long periods of yawning-like and sleeping-like states. Previous ketanserin suppressed gaping responses and reduced hypomotility, rapid oral movements and bristling but was ineffective for remaining responses induced by quipazine. Results showed that unlike mammals, serotonin permeates the BBB and activates hypnogenic mechanisms in quails. Studies using serotoninergic agonist and antagonists have disclosed that among the actions of the serotonin, feather bristling, rapid oral movements and yawning-like state originated from activation of 5-HT2 receptors while blinking and closure of the eyes possibly require other subtypes of receptors.

Este estudo foi desenvolvido objetivando ampliar as evidências comportamentais e neurofarmacológicas da permeabilidade da barreira hematoencefálica (BHE) à serotonina administrada sistemicamente em codornas. A serotonina injetada por via parenteral (250-1000 æg.kg-1, sc) produziu uma seqüência de eventos relacionados com um estado semelhante ao sono. Comportamentos semelhantes ao sono começaram com o eriçamento das penas, movimentos orais rápidos, piscadelas e finalmente agachamento e fechamento dos olhos. A administração prévia do antagonista do receptor 5-HT2C, LY53857 (3 mg.kg-1, sc) reduziu significativamente os episódios de eriçamento das penas e movimentos orais rápidos, mas não alterou a freqüência de piscadelas e fechamento dos olhos. Tratamento com o antagonista do receptor 5-HT2A/2C, quetanserina (3 mg.kg-1, sc) não afetou nenhuma das respostas evocadas pela serotonina. A quipazina (5 mg.kg-1, sc), um agonista dos receptores 5-HT2A/2C/3, induziu intensa hipomotilidade e longos períodos de comportamentos semelhantes ao bocejo e ao sono. O tratamento prévio com quetanserina suprimiu as reações de bocejo e reduziu a hipomotilidade, os movimentos orais rápidos e as piscadelas, mas foi sem efeito para as demais respostas induzidas pela quipazina. Os resultados mostraram que, diferentemente dos mamíferos, a serotonina atravessa a BHE e ativa mecanismos hipnogênicos em codornas. Estudos com agonistas serotoninérgicos e antagonistas revelaram que, entre as ações da serotonina, o eriçamento das penas, os movimentos orais rápidos e o comportamento semelhante ao bocejo foram originados pela ativação de receptores 5-HT2, enquanto o piscar e o fechamento dos olhos possivelmente requereu outros subtipos de receptores.

Presynaptic serotonergic modulation of 5-HT and acetylcholine release in the hippocampus and the cortex of 5-HT1B-receptor knockout mice.

Lesioning of serotonergic afferents increases hippocampal ACh release and attenuates memory deficits produced by cholinergic lesions. Improved memory performance described in 5-HT1B-knockout (KO) mice might thus be due to a weaker 5-HT1B-mediated inhibitory influence of 5-HT on hippocampal ACh release. The selective delay-dependent impairment of working memory observed in these KO mice suggests, however, that cortical regions also participate in task performance, possibly via indirect influences of 5-HT on ACh release. To provide neuropharmacological support for these hypotheses we measured evoked ACh and 5-HT release in hippocampal and cortical slices of wild-type (WT) and 5-HT1B KO mice. Superfused slices (preincubated with [3H]choline or [3H]5-HT) were electrically stimulated in the absence or presence of 5-HT1B receptor ligands. In hippocampus and cortex, 5-HT1B agonists decreased and antagonists increased 5-HT release in WT, but not in 5-HT1B KO mice. In 5-HT1B KO mice, 5-HT release was enhanced in both structures, while ACh release (in nCi) was reduced. ACh release was inhibited by 5-HT1B agonists in hippocampal (not cortical) slices of WT but not of 5-HT1B KO mice. Our data (i) confirm the absence of autoinhibition of 5-HT release in 5-HT1B-KO mice, (ii) demonstrate a reduced release of ACh, and the absence of 5-HT1B-receptor-mediated inhibition of ACh release, in the hippocampus and cortex of 5-HT1B-KO mice, and (iii) are compatible with an indirect role of cortical ACh in the working memory impairment observed in these KO mice.

Involvement of the retinohypothalamic tract in the photic-like effects of the serotonin agonist quipazine in the rat.

Light is the major synchronizer of the mammalian circadian pacemaker located in the suprachiasmatic nucleus. Photic information is perceived by the retina and conveyed to the suprachiasmatic nucleus either directly by the retinohypothalamic tract or indirectly by the intergeniculate leaflet and the geniculohypothalamic tract. In addition, serotonin has been shown to affect the suprachiasmatic nucleus by both direct and indirect serotonin projections from the raphe nuclei. Indeed, systemic as well as local administrations of the serotonin agonist quipazine in the region of the suprachiasmatic nucleus mimic the effects of light on the circadian system of rats, i.e. they induce phase-advances of the locomotor activity rhythm as well as c-FOS expression in the suprachiasmatic nucleus during late subjective night. The aim of this study was to localize the site(s) of action mediating those effects. Phase shifts of the locomotor activity rhythm as well as c-FOS expression in the suprachiasmatic nucleus after s.c. injection of quipazine (10 mg/kg) were assessed in Lewis rats, which had received either radio-frequency lesions of the intergeniculate leaflet or infusions of the serotonin neurotoxin 5,7-dihydroxytryptamine into the suprachiasmatic nucleus (25 microg) or bilateral enucleation. Lesions of intergeniculate leaflet and serotonin afferents to the suprachiasmatic nucleus did not reduce the photic-like effects of quipazine, whereas bilateral enucleation and the subsequent degeneration of the retinohypothalamic tract abolished both the phase-shifting and the FOS-inducing effects of quipazine. The results indicate that photic-like effects of quipazine are mediated via the retinohypothalamic tract.

Effect of valproic acid on serotonin-2A receptor signaling in C6 glioma cells.

Valproic acid (VPA), which has demonstrated efficacy in the treatment of bipolar disorder, has been shown to alter components of the phosphoinositide (PI) signaling cascade and to increase gene expression mediated by the transcription factor activator protein 1 (AP-1). Central serotonin-2A (5-HT2A) receptors, which have been implicated in the pathophysiology of manic-depressive illness, are coupled to PI hydrolysis. The promoter region of the 5-HT2A receptor gene contains AP-1 binding sites. We examined in C6 glioma cells the effect of VPA on 5-HT2A receptor signaling. Treatment of cells with VPA (100 microg/mL) for 20 h, but not 1.5 h, resulted in an enhancement of 5-HT2A receptor-stimulated PI hydrolysis. This effect of 20-h VPA exposure appeared not to be at the level of G protein or effector (i.e. phospholipase C: PLC) as inositol phosphate accumulation stimulated by aluminum fluoride or the PLC activator 2,4,6-trimethyl-N-(m-3-trifluromethylphenyl) benzenesulfonamide was not increased. The number of 5-HT2A receptors, as determined in saturation binding experiments using [3H]ketanserin, was increased by 20-h VPA treatment, with no change in affinity (KD). Taken together, our data suggest that the increase in 5-HT2A receptor-mediated PI hydrolysis following 20-h VPA exposure is not due to a general effect of VPA on this signaling cascade, but due to the up-regulation of 5-HT2A receptor number.

Response of the mouse circadian system to serotonin 1A/2/7 agonists in vivo: surprisingly little.

Serotonin (5-HT) is thought to play a role in regulating nonphotic phase shifts and modulating photic phase shifts of the mammalian circadian system, but results with different species (rats vs. hamsters) and techniques (in vivo vs. in vitro; systemic vs. intracerebral drug delivery) have been discordant. Here we examined the effects of the 5-HT1A/7 agonist 8-OH-DPAT and the 5-HT1/2 agonist quipazine on the circadian system in mice, with some parallel experiments conducted with hamsters for comparative purposes. In mice, neither drug, delivered systemically at a range of circadian phases and doses, induced phase shifts significantly different from vehicle injections. In hamsters, quipazine intraperitoneally (i.p.) did not induce phase shifts, whereas 8-OH-DPAT induced phase shifts after i.p. but not intra-SCN injections. In mice, quipazine modestly increased c-Fos expression in the SCN (site of the circadian pacemaker) during the subjective day, whereas 8-OH-DPAT did not affect SCN c-Fos. In hamsters, both drugs suppressed SCN c-Fos in the subjective day. In both species, both drugs strongly induced c-Fos in the paraventricular nucleus (within-subject positive control). 8-OH-DPAT did not significantly attenuate light-induced phase shifts in mice but did in hamsters (between-species positive control). These results indicate that in the intact mouse in vivo, acute activation of 5-HT1A/2/7 receptors in the circadian system is not sufficient to reset the SCN pacemaker or to oppose phase-shifting effects of light. There appear to be significant species differences in the susceptibility of the circadian system to modulation by systemically delivered serotonergics.

Evidence for serotonergic modulation of progesterone-induced hyperphagia, depression and algesia in female mice.

The acute administration of the neurosteroid precursor, progesterone (10 mg/kg, s.c.) produced significant hyperphagia in female mice as observed at 0.5-, 1-, 2- and 3-h time intervals. At this dose progesterone also produced significant increase in immobility period duration in Porsolt's forced swim test and nociceptive response in hot-plate and tail-flick tests. Treatment with direct (quipazine, 5 mg/kg, i.p.) and indirect (fluoxetine, 10 mg/kg, i.p.) acting serotonergic agents per se produced significant hypophagia, decrease in immobility period and induced analgesic effect in hot-plate and tail-flick test. Further, treatment with both fluoxetine (10 mg/kg, i.p.) and quipazine (5 mg/kg, i.p.) significantly reversed progesterone-induced hyperphagia, depression and algesia in the female mice. Pretreatment with seganserin, a 5-HT(2) receptor antagonist (2 mg/kg, i.p.) significantly reversed fluoxetine and quipazine-induced antidepressant and analgesic effects. Seganserin reversed quipazine-induced hypophagia but in a replicate study it failed to reverse fluoxetine-induced hypophagia. Further, seganserin, 2 mg/kg, i.p., significantly reversed the suppressive effect of fluoxetine and quipazine on progesterone-induced hyperphagia, depression and algesia in hot-plate test. Seganserin also reversed the suppressive effect of fluoxetine and quipazine on progesterone-induced algesia in hot-plate test. These data suggest that the modulation of progesterone-induced effects by these serotonergic agents possibly involve 5-HT(2) receptor mechanisms. Further, the study underscores the use of serotonergic agents for the treatment of eating and affective disorders caused by the regular changes or disturbances of ovarian steroid levels in females.

Neural circuits regulating pulsatile luteinizing hormone release in the female guinea-pig: opioid, adrenergic and serotonergic interactions.

We studied three neurotransmitters involved in the regulation of pulsatile luteinizing hormone (LH) release: opioid peptides, serotonin and norepinephrine, using the ovariectomized guinea-pig. This is an attractive animal model due to the regularity of its LH pulses, enabling any disruptions to be clearly ascertained. In all experiments, a specific agonist or antagonist was administered, either alone or serially to enable detection of interactions, and effects on mean LH concentrations, pulse amplitude and interpulse interval were determined by PULSAR analysis. In the ovariectomized guinea-pig, catecholamines are stimulatory (acting through the alpha1 and alpha2 but not beta receptors, unlike other species), opioids inhibitory and serotonin permissively stimulatory to pulsatile LH release. Stimulatory effects of the opiate antagonist were not blocked by pretreatment with an alpha1- or alpha2-adrenergic antagonist. Similarly, pretreatment with the opiate antagonist did not prevent the suppression of LH release by alpha1 and alpha2 antagonists. This suggests that, in the guinea-pig, effects of opiates and catecholamines on LH release are exerted by independent pathways to luteinizing hormone releasing hormone (LHRH) neurones. For the opiate-serotonin interactions, pretreatment with the serotonergic antagonist did not block the stimulatory effect of the opiate antagonist on LH release. However, pretreatment with the opiate agonist could not be overcome by the serotonergic agonist. This suggests that the effects of the serotonin system on LHRH release may be indirectly mediated by opioid neurones. Taken together, these studies demonstrate that the three neurotransmitter systems studied are critically involved in normal pulsatile LH release in the female guinea-pig, and demonstrate novel functional relationships between the opioid and the adrenergic and serotonergic systems.

Local effects of the serotonin agonist quipazine on the suprachiasmatic nucleus of rats.

In mammals, circadian rhythms of locomotor activity and many other behavioral and physiological functions are controlled by an endogenous pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). One of the SCN's afferents is a dense serotonergic input from the mesencephalic raphe complex. Previous work from this laboratory demonstrated that systemic administrations of the serotonin agonist quipazine mimic the effects of light on the circadian system of rats, i.e. they induce photic-like phase shifts of the circadian activity rhythm as well as c-Fos expression in the SCN. In contrast, no such effect has been demonstrated so far in the isolated rat SCN slice preparation. In this study we demonstrate that local injections of quipazine (0.5 microg/kg) into the region of the SCN induce photic-like effects similar to those induced by systemic injections. These findings suggest a role for 5-HT in the transmission of photic information to the rat circadian system through a direct action at the level of the SCN.

Serotonin agonist quipazine induces photic-like phase shifts of the circadian activity rhythm and c-Fos expression in the rat suprachiasmatic nucleus.

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice, and serotonin is thought to be involved in the phase-resetting effects of these stimuli. In the present study, the authors examined the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Furthermore, they investigated the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduced the amount of running wheel activity for 3 h after treatment, however, no long-term changes in tau and in the activity level were observed. More important, quipazine induced significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction were observed during the subjective day. Quipazine injections also resulted in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats.

Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin.

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5-HT) release and 5-HT reuptake activity, hippocampal extracellular 5-HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5-HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5-HT levels. Adenosine and A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), decreased extracellular 5-HT levels, whereas non-selective antagonist, caffeine, and A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased them. When 5-HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5-HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and A3 receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not affect 5-HT levels; however, when A1 receptor was blocked by CPT, 5-HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5-HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5-HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5-HT reuptake activity might be modulated by hippocampal A3 receptor.

Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus.

There are many examples of a single receptor coupling directly to more than one cellular signal transduction pathway. Although traditional receptor theory allows for activation of multiple cellular effectors by agonists, it predicts that the relative degree of activation of each effector pathway by an agonist (relative efficacy) must be the same. In the current experiments, we demonstrate that agonists at the human serotonin2A (5-HT2A) and 5-HT2C receptors activate differentially two signal transduction pathways independently coupled to the receptors [phospholipase C (PLC)-mediated inositol phosphate (IP) accumulation and phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release]. The relative efficacies of agonists differed depending on which signal transduction pathway was measured. Moreover, relative to 5-HT, some 5-HT2C agonists (e.g., 3-trifluoromethylphenyl-piperazine) preferentially activated the PLC-IP pathway, whereas others (e.g., lysergic acid diethylamide) favored the PLA2-AA pathway. In contrast, when two dependent responses were measured (IP accumulation and calcium mobilization), agonist relative efficacies were not different. These data strongly support the hypothesis termed "agonist-directed trafficking of receptor stimulus" recently proposed by Kenakin [Trends Pharmacol Sci 16:232-238 (1995)]. Concentration-response curves to 5-HT2C agonists were fit well by a three-state model of receptor activation, suggesting that two active receptor states may be sufficient to explain pathway-dependent agonist efficacy. Rational drug design that optimizes preferential effector activity within a group of receptor-selective drugs holds the promise of increased selectivity in clinically useful agents.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

Quipazine and light have similar effects on c-fos induction in the rat suprachiasmatic nucleus.

The effects of the serotonin agonist, quipazine, on the induction of c-fos in the suprachiasmatic nucleus of the rat was examined at different times of the 24 h cycle. Quipazine administered at night induced Fos production in a dose dependent manner (1, 3, 10, 30 mumol/kg) in the ventrolateral portion of the suprachiasmatic nucleus at ZT18. Administration of the highest dose at other times resulted in c-fos induction at ZT15 but not at other times of the day or subjective day examined (CT6 and ZT12). When compared to the effects of light pulses (2 lux/1 min), quipazine only caused c-fos induction at times when light caused induction. Our results support a role of serotonergic pathways in the transmission or modulation of photic information from the retina to the suprachiasmatic nucleus of the rat.

Characterisation of human 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptors expressed in the human neuroblastoma cell line SH-SY5Y: comparative stimulation by hallucinogenic drugs.

Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine2A (5-HT2A) or 5-HT2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT2C receptor (pEC50 = 7.80 +/- 0.06) compared with the 5-HT2A receptor (pEC50 = 7.30 +/- 0.08). Activation of the 5-HT2A receptor caused a transient fourfold increase in intracellular Ca2+ concentration. Whole-cell recordings of cells clamped at -50 mV demonstrated a small inward current (2 pA) in response to 10 microM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT2C receptor, whereas activity at the 5-HT2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.

5-hydroxytryptaminergic receptor-mediated regulation of growth hormone secretion in Holstein steers occurs via alpha 2-adrenergic-dependent and -independent mechanisms.

In vitro and in vivo experiments were used to determine the relationship between 5-hydroxytryptaminergic and alpha 2-adrenergic receptors in regulation of growth hormone secretion in cattle. Activation of 5-hydroxytryptaminergic receptors (10(-8), 10(-6), 10(-4) M quipazine) or alpha 2-adrenergic receptors (10(-8), 10(-6), 10(-4) M clonidine) had no effect on secretion of growth hormone from perifused anterior pituitary cells. In vivo, quipazine (0.2 mg/kg body wt, i.v.) and clonidine (8 micrograms/kg body wt, i.v.), when injected separately, each maximized secretion of growth hormone in Holstein steers. However, concurrent administration of quipazine and clonidine at these doses additively increased secretion of growth hormone (mean areas under curves = 439, 914, 1425, and 2359 +/- a pooled SEM of 246 ng.ml-1.min for vehicle, clonidine, quipazine, and quipazine plus clonidine treatments, respectively). Blockade of 5-hydroxytryptaminergic receptors with cyproheptadine (0.2 or 1.0 mg/kg body wt, s.c., 0740 hr) decreased basal concentrations of growth hormone but had no effect on the ability of clonidine (8 micrograms/kg body wt, i.v., 0840 hr) to increase secretion of growth hormone (mean areas under curves = 591, 1218, 363, 1087, and 1002 +/- a pooled SEM of 177 ng.ml-1.min for vehicle-vehicle, vehicle-clonidine, 0.2 mg cyproheptadine-vehicle, 0.2 mg cyproheptadine-clonidine and 1.0 mg cyproheptadine-clonidine treatments, respectively). Blockade of alpha 2-adrenergic receptors with either yohimbine (5 mg/kg body wt, s.c., 0740 hr) or idazoxan (20 mg/kg body wt, s.c., 0740 hr) suppressed both basal and 5-hydroxytryptaminergic receptor-stimulated (0.2 mg quipazine/kg body wt, i.v., 0840 hr) secretion of growth hormone (mean areas under curves = 568, 1252, 410, and 558 +/- a pooled SEM of 108 ng.ml-1.min for vehicle-vehicle, vehicle-quipazine, yohimbine-vehicle, and yohimbine-quipazine treatments, respectively, and means of 553, 1468, 194, and 686 +/- a pooled SEM of 221 ng.ml-1.min for vehicle-vehicle, vehicle-quipazine, idazoxan-vehicle, and idazoxan-quipazine treatments, respectively). We conclude that two mechanisms in the central nervous system mediate 5-hydroxytryptaminergic receptor-stimulated secretion of growth hormone in cattle; one independent and another dependent on alpha 2-adrenergic receptors, possibly via regulation of basal growth hormone secretion. In contrast, alpha 2-adrenergic receptor-induced secretion of growth hormone occurs independently of 5-hydroxytryptaminergic receptors.

Growth-inhibitory effects of serotonin uptake inhibitors on human prostate carcinoma cell lines.

Growth stimulation of a variety of cell types by the neurotransmitter serotonin has been reported. We have examined the effects of three serotonin-uptake inhibitors, 6-nitroquipazine, zimelidine and fluoxetine (Prozac, Eli Lilly Co., Indianapolis, Indiana) on human prostate carcinoma cell lines. In vitro, all 3 of these compounds inhibited the proliferation of PC-3, DU-145 and LNCaP cells in a dose-dependent manner. Also, all 3 compounds blocked the uptake of a radiolabeled analog of serotonin by the prostate carcinoma cell lines. The order of potency for inhibition of growth as well as for serotonin uptake was fluoxetine > zimelidine > 6-nitroquipazine. The growth of subcutaneous, PC-3 xenografts in athymic nude mice was significantly inhibited by fluoxetine. These results implicate biogenic amines such as serotonin in the growth of prostate carcinoma cells and indicate the potential use of serotonin-uptake inhibitors for the treatment of prostate cancer.

Serotonergic stimulation of prolactin secretion is inhibited by vasoactive intestinal peptide immunoneutralization in the turkey.

The neuronal mechanisms that govern prolactin (PRL) secretion in the turkey appear to involve monoaminergic systems. Considerable evidence indicates that serotonin (5-HT), acting centrally, is a potent stimulator of PRL secretion. This study, using birds actively immunized against VIP, tests the hypothesis that 5-HT stimulates PRL secretion by releasing vasoactive intestinal peptide (VIP). Nonimmunized turkeys were injected ip with saline, quipazine (5-HT agonist; 5 mg/kg), methysergide (5-HT antagonist; 8 mg/kg), or methysergide plus quipazine, and VIP-immunized birds were injected with saline or quipazine. Quipazine increased plasma PRL levels from 26.8 +/- 7.1 ng/ml at Time 0 to a peak value of 148.1 +/- 31.4 ng/ml 2 hr after infection. Pretreatment with methysergide or VIP-immunoneutralization abolished the PRL response to quipazine. Intraventricular infusion of 5-HT (1 nmol/min) caused plasma PRL to rise from a baseline of 16.3 +/- 2.6 ng/ml to 85.2 +/- 14.3 ng/ml after 30 min in nonimmunized control birds. Serotonin infusion did not induce PRL secretion in the VIP-immunized birds. These findings suggest that serotonergic stimulation of PRL secretion in the female turkey requires a functional VIPergic system.

A radioimmunoassay for recombinant-derived chicken prolactin suitable for the measurement of prolactin in other avian species.

A homologous radioimmunoassay for chicken prolactin is described. The assay is based on recombinant-derived chicken prolactin that has been used to raise an antibody and produce 125I-labeled tracer and assay standards. The radioimmunoassay measures a minimum of 0.03 +/- 0.01 ng of the recombinant prolactin with 50% displacement of binding by 0.6 +/- 0.1 ng. Chicken plasma and pituitary immunoreactivity dilute in parallel with the standard curve. The mean (+/- SE) concentrations of plasma prolactin were 12.1 +/- 1.5, 433.2 +/- 5.5, and 1609.8 +/- 18.5 ng/ml in out-of-lay, laying, and incubating bantam hens, respectively. The binding of recombinant prolactin was not displaced by extracts of neural lobe or plasma from a hypophysectomized hen. Plasma prolactin concentrations were increased after intravenous administration of chicken vasoactive intestinal polypeptide or quipazine, a 5-HT agonist. The assay measures immunoreactive prolactin in other birds. Plasma from quail, turkey, great tit, and wryneck displaced the 125I-labeled prolactin tracer parallel to the standard curve. Starling, ring dove, and penguin plasma displaced the binding in a nonparallel manner.