Melanin-concentrating hormone (MCH) in the median raphe nucleus: Fibers, receptors and cellular effects.

Serotonergic neurons of the median raphe nucleus (MnR) and hypothalamic melanin-concentrating hormone (MCH)-containing neurons, have been involved in the control of REM sleep and mood. In the present study, we examined in rats and cats the anatomical relationship between MCH-containing fibers and MnR neurons, as well as the presence of MCHergic receptors in these neurons. In addition, by means of in vivo unit recording in urethane anesthetized rats, we determined the effects of MCH in MnR neuronal firing. Our results showed that MCH-containing fibers were present in the central and paracentral regions of the MnR. MCHergic fibers were in close apposition to serotonergic and non-serotonergic neurons. By means of an indirect approach, we also analyzed the presence of MCHergic receptors within the MnR. Accordingly, we microinjected MCH conjugated with the fluorophore rhodamine (R-MCH) into the lateral ventricle. R-MCH was internalized into serotonergic and non-serotonergic MnR neurons; some of these neurons were GABAergic. Furthermore, we determined that intracerebroventricular administration of MCH induced a significant decrease in the firing rate of 53 % of MnR neurons, while the juxtacellular administration of MCH reduced the frequency of discharge in 67 % of these neurons. Finally, the juxtacellular administration of the MCH-receptor antagonist ATC-0175 produced an increase in the firing rate in 78 % of MnR neurons. Hence, MCH produces a strong regulation of MnR neuronal activity. We hypothesize that MCHergic modulation of the MnR neuronal activity may be involved in the promotion of REM sleep and in the pathophysiology of depressive disorders.

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons.

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ±â€¯0.7% of serotonergic and 48.9 ±â€¯5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.

Injections of the of the α1-adrenoceptor antagonist prazosin into the median raphe nucleus increase food intake and Fos expression in orexin neurons of free-feeding rats.

Previously, we showed that the blockade of α1-adrenoreceptors in the median raphe nucleus (MnR) increased food intake in free-feeding rats, indicating that adrenergic mechanisms in the MnR participate in the regulation of food intake. However, the impact of such a pharmacological manipulation on other neural circuits related to food intake remains unknown. In the current study, we sought to identify forebrain regions which are responsive to α1-adrenergic receptor blockade and presumably involved in the modulation of the feeding response. For this purpose, we examined the induction of c-Fos immunoreactivity in forebrain structures following injections of the α1-adrenoceptor antagonist prazosin into the MnR of free-feeding rats. To determine the chemical identity of hypothalamic c-Fos-positive cells, we then conducted double-label immunohistochemistry for Fos/orexin (OX) or Fos/melanin-concentrating hormone (MCH). Finally, we combined anterograde tracing from the MnR with immunohistochemical detection of orexin. Prazosin injections into the MnR significantly increased food intake. The ingestive response was accompanied by an increase in Fos expression in the basolateral amygdala (BLA) and lateral hypothalamic area (LHA). In the LHA, Fos expression occurred in neurons expressing OX, but not MCH. Combined anterograde tracing experiments revealed that LHA OX neurons are prominently targeted by MnR axons. These findings suggest that intra-MnR injection of prazosin, via activation of orexinergic neurons in the LHA and non-orexinergic neurons in the BLA, evoked a motivational response toward food intake.

Effects of perinatal protein malnutrition and fenfluramine action on food intake and neuronal activation in the hypothalamus and raphe nuclei of neonate rats.

In neonatal rats, hunger and satiety responses occur particularly via dehydration and gastric distention, respectively. The control of food intake in newborns is yet to be fully consolidated, particularly with respect to the participation of the hypothalamic nuclei and their relationship with the serotonergic pathway. Moreover, it is unclear how the environmental stressors in early life, like undernutrition, interfere in these events. Therefore, this study examined the serotonin-system's impact on food intake in rat neonates at postnatal day (P) 10 and P18 and the manner in which protein undernutrition during pregnancy and lactation interferes in this behavior. To accomplish this, Wistar rats were used, nutritionally manipulated by a diet having two protein levels, (8% and 17%) during pregnancy and lactation, to form the Control (n=10) and Low protein groups (n=10). At 10 and 18 postnatal days pups received an acute dose of fenfluramine (3mg/kg) or saline (0.9% NaCl) and subjected to milk consumption testing and then perfused to obtain the brains for the analysis of cell activation of the immunoreactive c-Fos in the hypothalamic and raphe nuclei. At 10days a reduction in weight gain was observed in both groups. On comparison of the neuronal activation for the paraventricular nucleus, an increased activation in response to fenfluramine was observed. At 18days, the weight gain percentage differed between the groups according to the nutritional manipulation, in which the control animals had no significant change while the undernourished presented increased weight gain with the use of fenfluramine. The marking of c-Fos in response to fenfluramine in the hypothalamic and raphe nuclei revealed, an especially lower activation of the PVN, MnR and DR compared intra-group. However when evaluating the effect of undernutrition, marking activation was observed to increase in all the nuclei analyzed, in the hypothalamus and raphe. Data from this study indicate that the action of serotonin via food intake in the neonates may have been delayed by early protein undernutrition.

Antidepressant Potential of Chlorogenic Acid-Enriched Extract from Eucommia ulmoides Oliver Bark with Neuron Protection and Promotion of Serotonin Release through Enhancing Synapsin I Expression.

Eucommia ulmoides Oliver (E. ulmoides) is a traditional Chinese medicine with many beneficial effects, used as a tonic medicine in China and other countries. Chlorogenic acid (CGA) is an important compound in E. ulmoides with neuroprotective, cognition improvement and other pharmacological effects. However, it is unknown whether chlorogenic acid-enriched Eucommia ulmoides Oliver bark has antidepressant potential through neuron protection, serotonin release promotion and penetration of blood-cerebrospinal fluid barrier. In the present study, we demonstrated that CGA could stimulate axon and dendrite growth and promote serotonin release through enhancing synapsin I expression in the cells of fetal rat raphe neurons in vitro. More importantly, CGA-enriched extract of E. ulmoides (EUWE) at 200 and 400 mg/kg/day orally administered for 7 days showed antidepressant-like effects in the tail suspension test of KM mice. Furthermore, we also found CGA could be detected in the the cerebrospinal fluid of the rats orally treated with EUWE and reach the level of pharmacological effect for neuroprotection by UHPLC-ESI-MS/MS. The findings indicate CGA is able to cross the blood-cerebrospinal fluid barrier to exhibit its neuron protection and promotion of serotonin release through enhancing synapsin I expression. This is the first report of the effect of CGA on promoting 5-HT release through enhancing synapsin I expression and CGA-enriched EUWE has antidepressant-like effect in vivo. EUWE may be developed as the natural drugs for the treatment of depression.

Dorsal and medial raphe nuclei participate differentially in reproductive functions of the male rat.

BACKGROUND: Innervation of the hypothalamus and median eminence arise from the dorsal and medial raphe nuclei (DRN and MRN, respectively). The hypothalamus regulates the secretion of gonadotropins, which in turn regulate the reproductive function of males and females. However, it is not known the role of raphe nuclei in male reproductive function. Our goal was to investigate the role of the DRN and MRN in the regulation of the testicular function and secretion of gonadotropins in prepubertal rats. METHODS: Dihydroxytryptamine (5,6-DHT) in ascorbic acid was used to chemically lesion the DRN or MRN. Rats were treated at 30 days-of-age and sacrificed at 45 or 65 days-of-age. Sham-treated controls were injected with ascorbic acid only. Negative controls were untreated rats. The damage induced by the 5,6-DHT was monitored in coronal serial sections of DRN and MRN; only the animals in which lesion of the DRN or MRN was detected were included in this study. As output parameters, we measured the concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in the anterior (AH) and medial (MH) hypothalamus by high performance liquid chromatography (HPLC); whereas, circulating concentrations of gonadotropins and sexual steroids were measured by radioimmunoassay. Seminiferous epithelium and sperm quality were also evaluated. RESULTS: Lesion of DRN or MRN does not induced changes in concentrations of LH, progesterone, and testosterone. Compared with the control group, the sham or lesion of the DRN or MRN did not modify noradrenaline or dopamine concentrations in the AH and MH at 45 or 65 days of age. Meanwhile, serotonin concentrations decreased significantly in lesioned rats. Lesion of DRN induced significantly lower concentrations of FSH regardless of age; similar lesion in the MRN had no impact on FSH levels. Sperm concentration and motility were significantly decreased in the same animals. The lesion of the MRN does not induced changes in the seminiferous epithelium or gonadotropin levels. Our results suggest that raphe nuclei regulate differentially the male reproductive functions. CONCLUSIONS: The DRN but not the MRN regulates the secretion of gonadotropins and testicular function.

Altered expression of 5-HT1A receptors in adult rats induced by neonatal treatment with clomipramine.

Chronic administration of clomipramine (CMI) to neonatal rats produces behaviors that resemble a depressive state in adulthood. Dysfunctions in the activity of the central nervous system's serotonergic function are important in understanding the pathophysiology of depression. The serotonin system is implicated in major depression and suicide and is negatively regulated by somatodendritic 5-HT1A autoreceptors. Desensitization of 5-HT1A autoreceptors is implicated in the long latency of some antidepressant treatments. Alterations in 5-HT1A receptor levels are reported in depression and suicide. In this study, we analyzed the effect of neonatal administration of CMI on the activity of 5-HT1A receptors, both pre- and post-synaptically, by administering an agonist of 5-HT1A receptors, 8-OH-DPAT, and then subjecting the rats to the forced swimming test (FST) a common procedure used to detect signs of depression in rats. Also measured were levels of the mRNA expression of 5-HT1A receptors in the dorsal raphe (DR), the hypothalamus and the hippocampus. Wistar rats were injected twice daily with CMI at doses of 15mgkg(-1) or saline as vehicle (CON) via s.c. from postnatal day 8 for 14days. At 3-4months of age, one set of rats from each group (CON, CMI) was evaluated for the effect of a selective agonist to the 5-HT1A receptor subtype, 8-OH-DPAT, by testing in the FST. Also determined was the participation of the pre- or post-synaptic 5-HT1A receptor in the antidepressant-like action of 8-OH-DPAT. This involved administering an inhibitor of tryptophan hydroxylase, parachlorophenylalanine (PCPA), and pretreatment with 8-OH-DPAT before the FST test and to evaluate the rectal temperature and locomotor activity. The expression of the mRNA of the 5-HT1A receptors was examined in the dorsal raphe nucleus, the hypothalamus and the hippocampus using the semi-quantitative RT-PCR method. The results from this study corroborate that neonatal treatment with clomipramine induces a pronounced immobility in the FST when animals reach adulthood, manifested by a significant decrease in swimming behavior, though counts of climbing behavior were not modified. This effect was similar in magnitude when 8-OH-DPAT was administered to CON group. Furthermore, the administration of 8-OH-DPAT induces a significant and similar increase in rectal temperature and locomotor activity in both the CON as in the CMI group. Neonatal treatment with CMI resulted in a significant decrease in the expression of the mRNA of the 5-HT1A receptors in the DR (% more than vehicle) in adulthood. In the case of the postsynaptic receptors located in the hypothalamus and hippocampus, neonatal treatment with CMI induced a significant increase in the mRNA expression of the 5-HT1A receptors. These data suggest that neonatal treatment with CMI induces a downregulation of the mRNA of the 5-HT1A autoreceptors in the DR, and an increment in the expression of the postsynaptic 5-HT1A receptors. The results after the administration of PCPA and 8-OH-DPAT on FST, rectal temperature and locomotor activity for both groups suggest that the function of postsynaptic receptors remains unchanged. All together these data show that the depressive behavior observed in adulthood in this animal model may be associated with long-term alterations in the expression of the mRNA of the 5-HT1A receptors.

Effects of early serotonin programming on behavior and central monoamine concentrations in an avian model.

Serotonin (5-HT) acts as a neurogenic compound in the developing brain; however serotonin altering drugs such as SSRIs are often prescribed to pregnant and lactating mothers. Early agonism of 5-HT receptors could alter the development of serotonergic circuitry, altering neurotransmission and behaviors mediated by 5-HT signaling, including memory, fear and aggression. This study was designed to investigate the effects of early serotonin agonism on later behaviors. An extremely aggressive White leghorn strain (15I5) was used in the study. The chicks were injected with 5-MT (a serotonin agonist) at 2.5mg/kg (low dose), 10mg/kg (high dose) or saline (control) on the day of hatch and a second dose 24h later (n=9/sex/trt). Chicks' fear response and memory were tested at 2 weeks of age. In the fear test, chicks were subjected to a social isolation test for 20min, time to first vocalization and numbers of vocalizations were recorded. In the memory test, chicks were placed in a running wheel and presented with an imprinted object (white box with a red light) and a similar shaped novel object (blue box with a white light), respectively. The distance traveled in the wheel toward each object was measured. At 10 weeks of age birds were tested for aggression and concentrations of catecholamines were determined from the raphe nucleus and hypothalamus by HPLC (n=12). Expression of 5-HT1A and 5-HT1B receptor genes were measured by RT-PCR. Both high and low dose chicks tended to have shorter latency to first vocalization and a greater number of vocalizations compared with control chicks. Memory test showed that chicks from all groups traveled a similar distance toward a familiar object. However, control chicks walked the least toward a novel object, low dose chicks tended to walk further, and high dose chicks walked significantly further for a novel object. In aggression tests, both high and low dose males exhibited greater frequency of aggressive behaviors compared to controls, while no difference in aggression was evident in the females. Norepinephrine concentrations were also reduced in the low dose birds in the hypothalamus and in the raphe nucleus. Serotonin concentrations tended to be lower only in the both hypothalamus and raphe nucleus of the low dose birds. 5-HT1A expression was greatest in the hypothalamus and raphe nucleus of low dose birds. The agonism of the serotonin system during neural development of birds genetically predisposed to aggression alters both the dopaminergic and serotonergic systems further increasing their aggressiveness.

Brain-derived neurotrophic factor-mediated downregulation of brainstem K+-Cl- cotransporter and cell-type-specific GABA impairment for activation of descending pain facilitation.

Chronic pain is thought to be partly caused by a loss of GABAergic inhibition and resultant neuronal hyperactivation in the central pain-modulating system, but the underlying mechanisms for pain-modulating neurons in the brain are unclear. In this study, we investigated the cellular mechanisms for activation of brainstem descending pain facilitation in rats under persistent pain conditions. In the nucleus raphe magnus (NRM), a critical relay in the brain's descending pain-modulating system, persistent inflammatory pain induced by complete Freund's adjuvant decreased the protein level of K(+)-Cl(-) cotransporter (KCC2) in both total and synaptosomal preparations. Persistent pain also shifted the equilibrium potential of GABAergic inhibitory postsynaptic current (EIPSC) to a more positive level and increased the firing of evoked action potentials selectively in µ-opioid receptor (MOR)-expressing NRM neurons, but not in MOR-lacking NRM neurons. Microinjection of brain-derived neurotrophic factor (BDNF) into the NRM inhibited the KCC2 protein level in the NRM, and both BDNF administration and KCC2 inhibition by furosemide mimicked the pain-induced effects on EIPSC and excitability in MOR-expressing neurons. Furthermore, inhibiting BDNF signaling by NRM infusion of tyrosine receptor kinase B-IgG or blocking KCC2 with furosemide prevented these pain effects in MOR-expressing neurons. These findings demonstrate a cellular mechanism by which the hyperactivity of NRM MOR-expressing neurons, presumably responsible for descending pain facilitation, contributes to pain sensitization through the signaling cascade of BDNF-KCC2-GABA impairment in the development of chronic pain.

Chronic effects of corticosterone on GIRK1-3 subunits and 5-HT1A receptor expression in rat brain and their reversal by concurrent fluoxetine treatment.

Dysregulation of the serotonergic system and abnormalities of the hypothalamic-pituitary-adrenal axis have been demonstrated in major depression. Animal studies indicate that 5-HT1A receptor expression may be reduced by long-term administration of corticosterone. However, similar studies on the regulation of GIRK channels, one of the most important effectors of the neuronal 5-HT1A receptor, are limited. In order to address these issues, slow-release corticosterone pellets were implanted subcutaneously to adrenal intact male rats (200mg pellets, 35 days release). Starting on day 15, animals were treated for 21 days with fluoxetine (5mg/kg/day, i.p.), or vehicle. Using in situ hybridization histochemistry and receptor autoradiography, we found that chronic corticosterone treatment was accompanied by a significant decrease on the mRNAs coding for mineralocorticoid receptors in hippocampal areas. Under these conditions, 5-HT1A receptor mRNA expression decreased in dorsal raphe nucleus and dentate gyrus. However, 5-HT1A receptor levels, as measured by [(3)H]-8-OH-DPAT binding, diminished significantly only in dentate gyrus. It is noteworthy that chronic treatment with fluoxetine reversed the alterations on 5-HT1A receptor mRNA levels only in dorsal raphe. Finally, chronic corticosterone treatment produced an increase on the mRNA coding for the GIRK2 subunit in several hypothalamic and thalamic areas, which was reversed by fluoxetine. Measurements of cell density and volume of the granular layer of the dentate gyrus did not reveal significant changes after corticosterone or corticosterone plus fluoxetine treatments. These data are relevant for a better understanding of the differential regulation of pre- and postsynaptic 5-HT1A receptors by corticosterone flattened rhythm.

Adolescence fluoxetine increases serotonergic activity in the raphe-hippocampus axis and improves depression-like behaviors in female rats that experienced neonatal maternal separation.

This study was conducted to examine if fluoxetine, a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor, would reverse adverse behavioral effects of neonatal maternal separation in female rats. Sprague-Dawley pups were separated from dam daily for 3h during postnatal day (PND) 1-14 (maternal separation; MS) or left undisturbed (non-handled; NH). Female NH and MS pups received intraperitoneal injection of fluoxetine (10mg/kg) or vehicle daily from PND 35 until the end of the whole experimental period. Rats were either subjected to behavioral tests during PND 44-54, or sacrificed for neurochemical analyses during PND 43-45. Daily food intake and weight gain of both NH and MS pups were suppressed by fluoxetine, with greater effects in MS pups. MS experience increased immobility and decrease swimming in forced swim test. Swimming was increased, although immobility was not significantly decreased, in MS females by adolescence fluoxetine. However, adolescence fluoxetine increased immobility during forced swim test and decreased time spent in open arms during elevated plus maze test in NH females. Fluoxetine normalized MS-induced decrease of the raphe 5-HT levels and increased 5-HT metabolism in the hippocampus in MS females, and increased the hypothalamic 5-HT both in NH and MS. Fluoxetine decreased the raphe 5-HT and increased the plasma corticosterone in NH females. Results suggest that decreased 5-HTergic activity in the raphe nucleus is implicated in the pathophysiology of depression-like behaviors, and increased 5-HTergic activities in the raphe-hippocampus axis may be a part of anti-depressant efficacy of fluoxetine, in MS females. Also, an extra-hypothalamic 5-HTergic activity may contribute to the increased anorectic efficacy of fluoxetine in MS females. Additionally, decreased 5-HT in the raphe and elevated plasma corticosterone may be related with fluoxetine-induced depression- and/or anxiety-like behaviors in NH females.

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse.

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

Cannabidiol, a non-psychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT(1A) somatodendritic autoreceptors in the dorsal raphe nucleus.

BACKGROUND AND PURPOSE: To evaluate the hypothesis that activation of somatodendritic 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis. EXPERIMENTAL APPROACH: The potential of systemic and intra-DRN administration of 5-HT(1A) receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT(1A) receptors and to modify the ability of the 5-HT(1A) agonist, 8-OH-DPAT, to stimulate [(35) S]GTPγS binding in rat brainstem membranes. KEY RESULTS: CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg(-1) , but not 40 mg·kg(-1) )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose-response curve) at enhancing the ability of 8-OH-DPAT to stimulate [(35) S]GTPγS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping. CONCLUSIONS AND IMPLICATIONS: These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT(1A) autoreceptors in the DRN. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

An orexinergic projection from perifornical hypothalamus to raphe pallidus increases rat brown adipose tissue thermogenesis.

Orexin (hypocretin) neurons, located exclusively in the PeF-LH, which includes the perifornical area (PeF), the lateral hypothalamus (LH), and lateral portions of the medial hypothalamus, have widespread projections and influence many physiological functions, including the autonomic regulation of body temperature and energy metabolism. Narcolepsy is characterized by the loss of orexin neurons and by disrupted sleep, but also by dysregulation of body temperature and by a strong tendency for obesity. Heat production (thermogenesis) in brown adipose tissue (BAT) contributes to the maintenance of body temperature and, through energy consumption, to body weight regulation. We identified a neural substrate for the influence of orexin neurons on BAT thermogenesis in rat. Nanoinjection of orexin-A (12 pmol) into the rostral raphe pallidus (rRPa), the site of BAT sympathetic premotor neurons, produced large, sustained increases in BAT sympathetic outflow and in BAT thermogenesis. Activation of neurons in the PeF-LH also enhanced BAT thermogenesis over a long time course. Combining viral retrograde tracing from BAT, or cholera toxin subunit b tracing from rRPa, with orexin immunohistochemistry revealed synaptic connections to BAT from orexin neurons in PeF-LH and from rRPa neurons with closely apposed, varicose orexin fibers, as well as a direct, orexinergic projection from PeF-LH to rRPa. These results indicate a potent modulation of BAT thermogenesis by orexin released from the terminals of orexin neurons in PeF-LH directly into the rRPa and provide a potential mechanism contributing to the disrupted regulation of body temperature and energy metabolism in the absence of orexin.

Sexually dimorphic serotonergic dysfunction in a mouse model of Huntington's disease and depression.

Depression is the most common psychiatric disorder in Huntington's disease (HD) patients. In the general population, women are more prone to develop depression and such susceptibility might be related to serotonergic dysregulation. There is yet to be a study of sexual dimorphism in the development and presentation of depression in HD patients. We investigated whether 8-week-old male and female R6/1 transgenic HD mice display depressive-like endophenotypes associated with serotonergic impairments. We also studied the behavioral effects of acute treatment with sertraline. We found that only female HD mice exhibited a decreased preference for saccharin as well as impaired emotionality-related behaviors when assessed on the novelty-suppressed feeding test (NSFT) and the forced-swimming test (FST). The exaggerated immobility time displayed by female HD in the FST was reduced by acute administration of sertraline. We also report an increased response to the 5-HT(1A) receptor agonist 8-OH-DPAT in inducing hypothermia and a decreased 5-HT(2A) receptor function in HD animals. While tissue levels of serotonin were reduced in both male and female HD mice, we found that serotonin concentration and hydroxylase-2 (TPH2) mRNA levels were higher in the hippocampus of males compared to female animals. Finally, the antidepressant-like effects of sertraline in the FST were blunted in male HD animals. This study reveals sex-specific depressive-related behaviors during an early stage of HD prior to any cognitive and motor deficits. Our data suggest a crucial role for disrupted serotonin signaling in mediating the sexually dimorphic depression-like phenotype in HD mice.

Diltiazem potentiates pentobarbital-induced hypnosis via 5-HT1A and 5-HT2A/2C receptors: role for dorsal raphe nucleus.

It has been reported that the sedative component of pentobarbital is mediated by GABA receptors in an endogenous sleep pathway and the ventrolateral preoptic area (VLPO)-tuberomammillary nucleus (TMN) or VLPO-dorsal raphe nucleus (DRN) neural circuit is important in the sedative response to pentobarbital. Our previous findings indicated that the VLPO-TMN neuronal circuit may play crucial part in the augmentative effect of diltiazem on pentobarbital sleep and the serotonergic system may be involved. This study was designed to investigate the role of DRN and the serotonergic receptors 5-HT(1A) and 5-HT(2A/2C) in the augmentative effect of diltiazem on pentobarbital-induced hypnosis in rats. The results showed that diltiazem (5mg/kg, i.g.) significantly reversed pentobarbital-induced (35 mg/kg, i.p.) reduction of c-Fos expression in 5-HT neurons of DRNV (at -7.5mm Bregma), DRND, DRNVL and MRN (at -8.0mm Bregma). However it did not influence this reducing effect of pentobarbital on non-5-HT neurons either in DRN or in MRN. Moreover, the effect of diltiazem (1 or 2mg/kg, i.g.) on pentobarbital-induced (35 mg/kg, i.p.) hypnosis was significantly inhibited by 5-HT(1A) agonist 8-OH-DPAT (0.5mg/kg, i.p.) and 5-HT(2A/2C) agonist DOI (0.5mg/kg, i.p.), and potentiated by 5-HT(1A) antagonist p-MPPI (2mg/kg, i.p.) and 5-HT(2A/2C) antagonist ritanserin (2mg/kg, i.p.), respectively. From these results, it should be presumed that the augmentative effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT(1A) and 5-HT(2A/2C) receptors, and DRN may be involved. In addition, it also suggested that the DRN may play a multi-modulating role in sleep-wake regulation rather than being recognized simply as arousal nuclei.

Ca²+ modulation in dorsal raphe plays an important role in NREM and REM sleep regulation during pentobarbital hypnosis.

Our previous studies indicated that L-type calcium channel blocker diltiazem could potentiate pentobarbital-induced hypnosis through serotonergic system. In view of the important role of dorsal raphe nucleus (DRN) on the sleep regulation and the pharmacological actions of calcium channel blocker, we presumed that Ca(2+) in the DRN may play an important role in sleep regulation in pentobarbital treated rats. Therefore, we investigated whether the Ca(2+) modulation in DRN by the microinjection of L-type Ca(2+) channel antagonist diltiazem, agonist BAY-K-8644, Ca(2+) chelator EGTA and CaCl(2) would alter the sleep parameters in pentobarbital treated rats. Results showed that perfusion of the agents attenuating Ca(2+) function, such as diltiazem (5 or 20 nmol) or EGTA (3 or 6 pmol) into DRN significantly increased pentobarbital (35 mg/kg, i.p.)-induced total sleep (TS), non-rapid eye movement (NREM) sleep and the slow wave sleep (SWS) ratio in NREM sleep. On the contrary, the DRN injection of the agents improving Ca(2+) function, such as BAY-K-8644 (10 nmol) or CaCl(2) (50 or 100 nmol) significantly reduced pentobarbital (35 mg/kg, i.p.)-induced TS, NREM sleep, rapid eye movement (REM) sleep and REM sleep ratio in TS without influence on SWS. These results suggested that the suppression of Ca(2+) function in DRN could increase NREM sleep including SWS, and the elevation of Ca(2+) function could reduce both NREM and REM sleep in pentobarbital treated rats.

Organization of two independent kisspeptin systems derived from evolutionary-ancient kiss genes in the brain of zebrafish.

Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.

Characterization of the electrophysiological properties of triple reuptake inhibitors on monoaminergic neurons.

Triple reuptake inhibitors represent a potential new class of antidepressant drugs that block norepinephrine (NE), dopamine (DA) and serotonin [5-hydroxytryptamine (5-HT)] transporters. The present in-vivo electrophysiological study was undertaken to determine the effects of the triple reuptake inhibitors SEP-225289 and DOV216303 on the neuronal activities of locus coeruleus (LC) NE, ventral tegmental area (VTA) DA and dorsal raphe (DR) 5-HT neurons. Administered acutely, SEP-225289 and DOV216303 dose-dependently decreased the spontaneous firing rate of LC NE, VTA DA and DR 5-HT neurons through the activation of α2, D2 and 5-HT(1A) autoreceptors, respectively. Both compounds predominantly inhibited the firing rate of LC NE neurons while producing only a partial decrease in VTA DA and DR 5-HT neuronal discharge. SEP-225289 was equipotent at inhibiting 5-HT and NE transporters since it prolonged to the same extent the time required for a 50% recovery (RT50) of the firing activity of dorsal hippocampus CA3 pyramidal neurons from the inhibition induced by microiontophoretic application of 5-HT and NE. Finally, in the presence of WAY100635, a 5-HT(1A) receptor antagonist, SEP-225289 activated 5-HT neurons at doses that normally did not inhibit them. Taken together, the present results indicate that reciprocal interactions among NE, DA and 5-HT inputs need to be considered to anticipate the net effect of triple reuptake inhibitors on the enhancement of brain monoamine transmission. The results also suggest that the therapeutic action of triple reuptake inhibitors may be potentiated by antagonizing the cell body 5-HT(1A) autoreceptors.

Nucleus raphe pallidus participates in midbrain-medullary cardiovascular sympathoinhibition during electroacupuncture.

We have shown that electroacupuncture (EA) inhibits sympathoexcitatory rostral ventrolateral medulla (rVLM) neurons and reflex responses following activation of a long-loop pathway in the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG). Additionally, EA at P 5-6 acupoints (overlying the median nerve) activates serotonin-containing neurons in the nucleus raphé pallidus (NRP), which, in turn, inhibit rVLM neurons. Although direct projections from the vlPAG to the rVLM exist, it is uncertain whether an indirect pathway through the NRP serves an important role in vlPAG-rVLM cardiovascular modulation. Therefore, the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes, and EA was applied at P 5-6 acupoints in α-chloralose-anesthetized cats. A single-barreled recording electrode was inserted into the NRP or rVLM. Microinjection of DL-homocysteic acid (DLH) into the vlPAG increased the NRP neuronal response to SN stimulation (5 ± 1 to 12 ± 2 spikes/30 stim). Likewise, EA at P 5-6 for 30 min increased the NRP response to SN stimulation (3 ± 1 to 10 ± 2 spikes/30 stim), an effect that could be blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in blood pressure induced by application of bradykinin to the gallbladder and the rVLM cardiovascular presympathetic neuronal response to SN stimulation was inhibited by injection of DLH into the vlPAG, a response that was reversed by injection of KYN into the NRP. These results indicate that EA activates the vlPAG, which excites the NRP to, in turn, inhibit rVLM presympathetic neurons and reflex cardiovascular sympathoexcitatory responses.