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Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 ( Tph2 ) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 ( Vglut3 ) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2 + MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1 -neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2- Pet1 Tph2 -high versus r2- Pet1 Vglut3 -high Using male and female mice, we found r2- Pet1 axonal boutons segregated anatomically largely by serotonin + versus VGLUT3 + identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2- Pet1 Tph2- high and r2- Pet1 Vglut3- high neurons likely regulate distinct brain regions and behaviors. Some r2- Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3 + or VGLUT3 + /5-HT + identity; this suggests that some r2- Pet1 Vglut3- high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations. SIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1 + precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2- Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2- Pet1 boutons-those that are VGLUT3 + or VGLUT3 + /5-HT + co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration., (Copyright © 2021 the authors.)

Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anxiety disorders may be mediated in part by disruptions in serotonin (5-hydroxytryptamine, 5-HT) system function. Behavioral measures of approach-avoidance conflict suggest that serotonin neurons within the median raphe nucleus (MRN) promote an anxiogenic state, and some evidence indicates this may be mediated by serotonergic signaling within the dorsal hippocampus. Here, we test this hypothesis using an optogenetic approach to examine the contribution of MRN 5-HT neurons and 5-HT innervation of the dorsal hippocampus (dHC) to anxiety-like behaviours in female mice. Mice expressing the excitatory opsin ChR2 were generated by crossing the ePet-cre serotonergic cre-driver line with the conditional Ai32 ChR2 reporter line, resulting in selective expression of ChR2 in 5-HT neurons. Electrophysiological recordings confirmed that this approach enabled reliable optogenetic stimulation of MRN 5-HT neurons, and this stimulation produced downstream 5-HT release in the dHC as measured by in vivo microdialysis. Optogenetic stimulation of the MRN elicited behavioral responses indicative of an anxiogenic effect in three behavioural tests: novelty-suppressed feeding, marble burying and exploration on the elevated-plus maze. These effects were shown to be behaviourally-specific. Stimulation of 5-HT terminals in the dHC recapitulated the anxiety-like behaviour in the novelty-suppressed feeding and marble burying tests. These results show that activation of 5-HT efferents from the MRN rapidly induces expression of anxiety-like behaviour, in part via projections to the dHC. These findings reveal an important neural circuit implicated in the expression of anxiety in female mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb-IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state-setting modulatory projection systems., (© 2019 Wiley Periodicals, Inc.)

The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine., (© 2017 Wiley Periodicals, Inc.)

Background: To study serotonergic volume neurotransmission at cellular level it needs to investigate neurotransmitter release and re-uptake sites in serotonergic neurons. However, due to the low number of cell bodies in the raphe nuclei and their widely branching neurites, serotonergic neuronal cultures are not accessible ex vivo., New Method: We have combined differentiation protocols for the generation of stem cell-derived serotonergic neurons together with confocal microscopy to study the uptake and release of fluorescent substrates known to be selectively taken up by monoaminergic neurons. These substances include: (i) 4-(4-(dimethylamino)styryl)-N-methylpyridiunium (ASP+), an analog of the neurotoxin MPP+; (ii) the fluorescent false neurotransmitter (FFN511); and (iii) serotonin (5-hydroxytryptamine; 5-HT) itself, which is known to emit fluorescence upon excitation at 320-460nm., Result: ASP+ is taken up into living serotonergic neurons through the serotonin transporter, but not accumulated into synaptic vesicles; FFN511 diffuses in a SERT-independent way into serotonergic neurons and accumulated into synaptic vesicles. KCl-induced release of FFN511 and 5-HT can be visualized and quantified in living serotonergic neurons., Comparison With Existing Methods: Application of ASP+ so far has been used to investigate substrate/transporter interactions; studies on FFN511 uptake and release have only been performed in dopaminergic neurons; quantitative studies on uptake and release of 5-HT in living serotonergic neurons have not been reported yet., Conclusion: The differentiation protocols for the generation of stem cell-derived serotonergic neurons combined with the application of different fluorescent dyes allow to quantify neurotransmitter uptake and release in living serotonergic neurons in vitro., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The medullary raphé (MR) of the medulla oblongata contains chemosensitive neurons that respond to increases in arterial [CO₂], by altering firing rate, with increases being associated with serotonergic (5-hydroxytryptamine [5HT]) neurons and decreases, with GABAergic neurons. Both types of neurons contribute to increased alveolar ventilation. Decreases in intracellular pH are thought to link the rise in [CO₂] to increased ventilation. Because electroneutral Na(+)-coupled HCO₃(-) transporters (nNCBTs), which help protect cells from intracellular acidosis, are expressed robustly in the neurons of the central nervous system, a key question is whether these transporters are present in chemosensitive neurons. Therefore, we used an immunocytochemistry approach to identify neurons (using a microtubule associated protein-2 monoclonal antibody) and specifically 5HT neurons (TPH monoclonal antibody) or GABAergic neurons (GAD2 monoclonal antibody) in freshly dissociated cells from the mouse MR. We also co-labeled with polyclonal antibodies against the three nNCBTs: NBCn1, NDCBE, and NBCn2. We exploited ePet-EYFP (enhanced yellow fluorescent protein) mice (with EYFP-labeled 5HT neurons) as well as mice genetically deficient in each of the three nNCBTs. Quantitative image analysis distinguished positively stained cells from background signals. We found that >80% of GAD2(+) cells also were positive for NDCBE, and >90% of the TPH(+) and GAD2(+) cells were positive for the other nNCBTs. Assuming that the transporters are independently distributed among neurons, we can conclude that virtually all chemosensitive MR neurons contain at least one nNCBT., (Published by Elsevier Ltd.)

The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.

Altered brain serotonin activity is implicated in schizophrenia. We have previously shown differential involvement of serotonergic projections from the dorsal or median raphe nucleus in phencyclidine-induced hyperlocomotion in rats, a behavioral model of aspects of schizophrenia. Here we further investigated the effects of serotonergic lesions of the raphe nuclei on phencyclidine-induced hyperlocomotion by parallel assessment of Fos-like immunoreactivity (FLI), a marker of neuronal activation in the brain. Male Sprague-Dawley rats were anesthetized with pentobarbitone and stereotaxically microinjected with 5 μg of the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into either the dorsal raphe (DRN) or median raphe nucleus (MRN). Two weeks after the surgery, rats with lesions of the MRN, but not those with lesions of the DRN, showed significant enhancement of the hyperlocomotion induced by injection of 2.5 mg/kg of phencyclidine. Rats with MRN lesions also showed significantly higher levels of FLI in the polymorphic layer of the dentate gyrus in the dorsal hippocampus (PoDG) when compared with sham-operated controls. Rats with lesions of the DRN showed significantly higher levels of FLI in the nucleus accumbens (NAcc). These results indicate that FLI in the PoDG, but not the NAcc, correlates with enhanced phencyclidine-induced locomotor hyperactivity in MRN-lesioned rats. These results support our previous studies suggesting a role of serotonergic projections from the MRN to the dorsal hippocampus in some of the symptoms of schizophrenia., (Copyright © 2012 Wiley Periodicals, Inc.)

Dehydration is a powerful stimulus causing disequilibrium in homeostasis of water and electrolytes resulting from depletion in total body water. Most studies have focused on domestic and laboratory animals; however, the study of desert animals allows improved understanding about water balance and resistance to dehydration and associated behavioral changes, including those related to mood disorders. Meriones shawi (Shaw's Jird) is a desert rodent characterized by its resistance to long periods of thirst that can extend for several months. In the present study, M. shawi were subjected to water deprivation for 1 and 3 months. We used 5-HT immunohistochemistry to evaluate the effects of prolonged dehydration on the serotoninergic system in both dorsal and median raphe nuclei (DRN, MRN), which are the main sources of 5-HT input to several brain areas. In addition, a dark/light box was used to evaluate the anxiolytic-like or anxiogenic-like effects of dehydration on M. shawi. The results showed a reduction in the 5-HT immunolabelling in both DRN and MRN following 1 and 3 months of dehydration. This diminution of serotonin immunoreactivity was accompanied by noticeable changes in anxiety behavior of Meriones, with animals spending more time in the light box, suggesting anxiogenic-like effects caused by dehydration. Overall, the results indicate that dehydration is able to reduce serotoninergic neurotransmission, which might be involved in generating anxiety behavior in this desert animal., (Copyright © 2011. Published by Elsevier GmbH.)

We have demonstrated marked differences in the neurobiology of the serotonin system between stress-sensitive (SS) and stress-resilient (SR) cynomolgus macaques characterized in a model of stress-induced amenorrhea, also called functional hypothalamic amenorrhea (FHA). Dysfunction of the serotonin system in SS monkeys suggested that administration of a selective serotonin reuptake inhibitor (SSRI) might correct FHA. This study examines the effect of escitalopram (CIT) administration to SS and SR monkeys on corticotrophin-releasing factor (CRF) receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2) gene expression in the serotonin cell body region of the midbrain dorsal raphe. CRF-R1 was not significantly different between groups. There was a significant effect of treatment and a significant interaction between treatment and stress sensitivity on the average CRF-R2-positive pixel area (P < .004 and P < .006, respectively) and on the average number of CRF-R2-positive cells (P < .023 and P < .025, respectively). CIT significantly increased CRF-R2-positive pixel area and cell number in the SS group (pixel area P < .001; cell number P < .01; Bonferoni) but not in the SR group. In summary, CIT administration tended to decrease CRF-R1, but the small animal number precluded significance. CIT administration significantly increased CRF-R2 only in SS animals. These data suggest that the administration of CIT reduces anxiogenic components and increases anxiolytic components of the CRF system in the midbrain serotonin network, which in turn leads to improved ovarian function. Moreover, these data raise the possibility that SSRIs may be effective in the treatment of stress-induced infertility.

Serotonin, or 5-hydroxytryptamine (5-HT), is a substance found in many tissues of the body, including as a neurotransmitter in the nervous system, where it can exert different post-synaptic actions. Inside the neuro-axis, 5-HT neurons are almost entirely restricted to the raphe nuclei of the brainstem. As such, 5-HT-immunoreactivity has been considered a marker of the raphe nuclei, which are located in the brainstem, at or near the midline. The present study investigated distribution of serotonergic neurons in the brain of the rock cavy (Kerodon rupestris), a rodent species inhabiting the Brazilian Northeast. The cytoarchitectonic location of serotonergic neurons was established through a series of 5-HT immunostained sections, compared with diagrams obtained from adjacent coronal and sagittal sections stained by the Nissl method. The following nuclei were defined: the rostral group, consisting of rostral linear raphe, caudal linear raphe, median and paramedian raphe, dorsal raphe, and pontine raphe nuclei, and the caudal group composed of raphe magnus, raphe pallidus and raphe obscurus nuclei. Other serotonergic neuronal clusters, such as the supralemniscal group and the rostral and caudal ventrolateral medulla oblongata clusters, were found outside the midline. Rare 5-HT-producing neurons were identified in the lateral parabrachial nucleus and in the pontine reticular formation, mostly along fibers of the lateral lemniscus. Despite exhibiting some specializations, the picture outlined for serotonergic groups in the rock cavy brain is comparable to that described for other mammalian species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The ability to sense and respond appropriately to increases in ambient and body temperatures is critical for the survival of all animals. Although evidence suggests that brain serotonergic systems play a role in thermoregulation, including thermoregulatory cooling, evidence for activation of brainstem serotonergic neurons in vivo, in unanesthetized animals, during heat exposure is lacking. In this experiment we tested the hypothesis that populations of serotonergic neurons in the midbrain and medullary raphe complex are activated following exposure to warm ambient temperature. Rats were exposed to an incubation chamber at either warm ambient temperature (37°C) or room temperature (RT; 23°C) for 105 min. Brains then were removed and processed for immunohistochemical detection of the protein product of the immediate-early gene c-fos (as a marker of neuronal activation) and tryptophan hydroxylase (as a marker of serotonergic neurons). Exposure to warm ambient temperature increased body temperature and c-Fos expression in topographically organized populations of serotonergic neurons in the dorsal raphe nucleus. Activation of the dorsal raphe nucleus serotonergic system was positively correlated with body temperature following exposure to the incubation chamber. In the medulla, exposure to warm ambient temperature, compared with exposure to RT, decreased c-Fos expression in serotonergic neurons in the raphe pallidus nucleus and in non-serotonergic cells in the rostral ventrolateral medulla. Together, these results provide evidence for multiple but anatomically discrete thermosensitive serotonergic systems that may have implications for the regulation of body temperature, as well as, via projections to forebrain targets, cognitive and affective functions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Serotonin neurons play a major role in many normal and pathological brain functions. In the rat these neurons have a varying number of cotransmitters, including neuropeptides. Here we studied, with histochemical techniques, the relation between serotonin, some other small-molecule transmitters, and a number of neuropeptides in the dorsal raphe nucleus (DRN) and the adjacent ventral periaqueductal gray (vPAG) of mouse, an important question being to establish possible differences from rat. Even if similarly distributed, the serotonin neurons in mouse lacked the extensive coexpression of nitric oxide synthase and galanin seen in rat. Although partly overlapping in the vPAG, no evidence was obtained for the coexistence of serotonin with dopamine, substance P, cholecystokinin, enkephalin, somatostatin, neurotensin, dynorphin, thyrotropin-releasing hormone, or corticotropin-releasing hormone. However, some serotonin neurons expressed the gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD). Work in other laboratories suggests that, as in rat, serotonin neurons in the mouse midline DRN express the vesicular glutamate transporter 3, presumably releasing glutamate. Our study also shows that many of the neuropeptides studied (substance P, galanin, neurotensin, dynorphin, and corticotropin-releasing factor) are present in nerve terminal networks of varying densities close to the serotonin neurons, and therefore may directly or indirectly influence these cells. The apparently low numbers of coexisting messengers in mouse serotonin neurons, compared to rat, indicate considerable species differences with regard to the chemical neuronatomy of the DRN. Thus, extrapolation of DRN physiology, and possibly pathology, from rat to mouse, and even human, should be made with caution.

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.

Deficient levels of serotonin are associated with suicide and depression. Paradoxically, in the dorsal raphe nucleus (DRN) there are more serotonin neurons and more neuronal tryptophan hydroxylase-2 (TPH2) expression postmortem in depressed suicides. In this study, we sought to determine whether greater TPH2 expression in depressed suicides was the result of more TPH2 expression per neuron. In situ hybridization and computer-assisted image analysis were performed on tissue sections throughout the extent of the raphe nuclei at the level of silver grains per neuron to systematically quantify TPH2 neuronal expression. Depressed suicides have 26.5% more TPH2 grain density per neuron in the DRN compared with matched controls (P=0.04). The difference in grain density is greater at mid- and caudal anatomical levels across the rostrocaudal axis of the DRN. Densitometric analysis of TPH2 expression in the DRN subnuclei showed that higher expression levels were observed at posterior anatomical levels of depressed suicides (121% of control in the caudal subnucleus). Higher TPH2 expression in depressed suicides may explain more TPH2 protein and reflect a homeostatic response to deficient serotonin levels in the brains of depressed suicides. Localized changes in TPH2 expression in specific subnuclei of the DRN suggest that the serotonergic compensatory mechanism in depression and suicide is specifically regulated within the DRN and has implications for regions innervated by this subnucleus.

Previous reports establishing raphe cultures typically yield less than 1% serotonin (5-HT)-positive neurons and are impractical for transcriptional studies. In this study, we have established primary cultures enriched in 5-HT neurons and quantified the proportion of cells expressing serotonergic and non-serotonergic markers. We have also shown the feasibility of using the multiplex real-time PCR technique to measure the relative amounts of RNA for some of these markers. Rostral raphe cells derived from E13-15 rat embryos were cultured for 7 days and analyzed by quantitative immunofluorescence and western blot analysis. In these cultures, approximately 8% of neurons were immunopositive for serotonergic markers (5-HT or tryptophan hydroxylase (TPH)). The percentage of cells labeled for GFAP (glial marker), tyrosine hydroxylase (catecholaminergic), and GAD65/67 (GABAergic) was 5, 1, and 54%, respectively. Transcription factors REST/NRSF and Deaf-1 were present in 9 and 98% of cells, respectively. Multiplex quantitative RT-PCR (Q-PCR) analysis was done for TPH2, 5-HT1A receptor or Deaf-1 RNAs paired with GAPDH RNA as control. Using this approach, standard curves for each RNA were obtained over 200-fold concentration range of dilution with r2 values >0.99. The relative abundances determined by Q-PCR are consistent with the expression of TPH2>Deaf-1>5-HT1A receptor RNA in serotonergic raphe cells. The standard error of TPH2 RNA levels between cultures was <20%, indicating a consistent purity of 5-HT neurons. Thus, we have generated a highly consistent and reproducible model system that is enriched in 5-HT neurons and that will be valuable in future investigation of serotonergic regulation.

Background and Purpose: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503., Experimental Approach: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907., Key Results: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity., Conclusions and Implications: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.

The present studies sought to investigate the effect of tryptophan alone or coadministration of tryptophan and ethanol on the interaction of central frontal cortex and dorsal raphe nucleus serotonergic functional activities by utilizing in vivo microdialysis. Tryptophan (50 mg/kg, i.p.) led to a significant increase in the levels of 5-HIAA, a metabolite of serotonin (5-HT), in the dorsal raphe nucleus, but not in the frontal cortex. Coadministration of tryptophan and ethanol caused very marked increases in 5-hydroxyindoleacetic acid (5-HIAA) levels in both the frontal cortex and the dorsal raphe nucleus, although ethanol (1.25 g/kg) did not change 5-HIAA levels in both areas. Moreover, the application of WAY100635 (10 muM), 5-HT(1A) antagonist, into the frontal cortex after coadministration caused a marked increase in 5-HIAA levels in the frontal cortex and a decrease in the levels in the dorsal raphe nucleus, although WAY100635 alone had no effect on these levels. This may suggest that WAY100635-induced increase of 5-HIAA levels in the frontal cortex resulted from negative feedback following the blockade of serotonergic 5-HT(1A) autoreceptors, and that this increase in 5-HIAA levels decreased 5-HIAA levels in the dorsal raphe nucleus by preventing the activation of dorsal raphe 5-HT(1A) autoreceptors. WAY100635 into the dorsal raphe nucleus did not significantly change 5-HIAA levels in both areas. This may indicate that the blockade of dorsal raphe 5-HT(1A) autoreceptors by WAY100635 resulted in unchanged 5-HIAA levels in the frontal cortex. Behavioral sign of teeth-chattering was markedly observed following the coadministration and in combination with WAY100635. These results may suggest that the increased 5-HIAA levels in both areas after coadministration are indicative of the interrelation via activation of serotonergic neurons, and that the increased levels are partly responsible for behavioral activation of rats.

Most glutamatergic neurons in the brain express one of two vesicular glutamate transporters, vGlut1 or vGlut2. Cortical glutamatergic neurons highly express vGlut1, whereas vGlut2 predominates in subcortical areas. In this study immunohistochemical detection of vGlut1 or vGlut2 was used in combination with tryptophan hydroxylase (TPH) to characterize glutamatergic innervation of the dorsal raphe nucleus (DRN) of the rat. Immunofluorescence labeling of both vGlut1 and vGlut2 was punctate and homogenously distributed throughout the DRN. Puncta labeled for vGlut2 appeared more numerous then those labeled for vGlut1. Ultrastructural analysis revealed axon terminals containing vGlut1 and vGlut2 formed asymmetric-type synapses 80% and 95% of the time, respectively. Postsynaptic targets of vGlut1- and vGlut2-containing axons differed in morphology. vGlut1-labeled axon terminals synapsed predominantly on small-caliber (distal) dendrites (42%, 46/110) or dendritic spines (46%, 50/110). In contrast, vGlut2-containing axons synapsed on larger caliber (proximal) dendritic shafts (> 0.5 microm diameter; 48%, 78/161). A fraction of both vGlut1- or vGlut2-labeled axons synapsed onto TPH-containing dendrites (14% and 34%, respectively). These observations reveal that different populations of glutamate-containing axons innervate selective dendritic domains of serotonergic and non-serotonergic neurons, suggesting they play different functional roles in modulating excitation within the DRN.

Research on Substance P (SP) has, until recently, focused on its role in pain and inflammation. However, a report that NK(1) receptor antagonists have utility in the treatment of depression has stimulated research into the function of SP and the NK(1)receptor in anxiety and depression. The distribution of SP and the NK(1) receptor in brain areas implicated in anxiety and depression is initially reviewed. This is followed by evaluation of the preclinical data obtained for SP and NK(1) receptor antagonists in behavioral models of depression as well as the phenotype of genetically modified animals lacking the genes encoding for the NK(1) receptor or for SP. The weight of the evidence supports antidepressant and anxiolytic activity of NK(1) receptor antagonists. However, many of the studies do not control for nonspecific effects of the compounds, and when enantiomers that lack activity at the NK(1) receptor are included, the results, in some cases, suggest that blockade of NK(1) receptors does not account for the observed behavioral activity. Finally, clinical studies in depressed patients assessing SP levels in plasma and cerebrospinal fluid as well as the effect of NK(1) receptor antagonists are reviewed. The clinical studies are a mixture of positive, failed and negative studies on the antidepressant activity of NK(1) receptor antagonists, not unlike the early clinical results obtained with selective serotonin reuptake inhibitors.

Zucker rats, lean and obese, treated with low dose intraperitoneal injections of streptozocin become hyperglycemic within 24h. Insulin levels fall, although the obese animal remains hyperinsulinemic. Associated with these changes in glucose and insulin there are transient decreases in caloric intake. Macronutrient selection studies show that protein consumption decreases. There is a trend for fat intake to decrease. The levels of hypothalamic neurotransmitters in the lean animals are not altered by streptozocin. The levels of 5-hydroxyindoleacetic acid increases in the streptozocin-treated obese animal in the paraventricular region, ventromedial region and the raphe. Serotonin is also significantly increased in the paraventricular region of the obese rat. These results suggest that acutely, treatment with streptozocin injures pancreatic islets, causing, in turn, decreases in insulin levels so that hyperglycemia ensues in both phenotypes. Associated with these perturbations are decreases in caloric intake. The magnitude of change in insulin levels is much greater in the obese rat. It is hypothesized that in the obese Zucker rat decrements in food intake are mediated by increase in serotonin turnover in the hypothalamus and these changes are related to changes of insulin levels. These data support the concept that circulating insulin affects hypothalamic neurotransmitters.

The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either 5HT-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR., (Copyright 2004 Wiley-Liss, Inc.)

Pancreatic glucokinase (GK)-like immunoreactivities are located in ependymocytes and serotonergic neurons of the rat brain. The present study investigated in vitro changes in intracellular calcium concentrations ([Ca(2+)](i)) in response to low (2 mm) or high (20 mm) extracellular glucose concentrations in isolated cells from the wall of the central canal (CC), raphe obscurus nucleus (ROb), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LHA) in male rats. An increase in [Ca(2+)](i) was found in cells from the CC (21.1% or 9.8% of ependymocytes), ROb (10.9% or 14.5% of serotonergic neurons), VMH (7.8% and 25.2% of neurons), and LHA (20% or 15.7% of neurons), when extracellular glucose levels were changed from 10 to either 2 or 20 mm, respectively. Most of the ependymocytes and serotonergic neurons responding to the glucose changes were immunoreactive to the anti-GK in the CC (96.8% for low glucose and 100% for high glucose) and ROb (100% for low and high glucose). The [Ca(2+)](i) increase was blocked with calcium-free medium or L-type calcium channel blocker. Cells with an increase in [Ca(2+)](i) in response to low glucose did not respond to high glucose and vice versa. Inhibition of GK activity with acute alloxan treatment blocked low or high glucose-induced [Ca(2+)](i) increases in most GK-immunoreactive cells from the CC or ROb. The glucose-sensitive [Ca(2+)](i) increase in neurons of the VMH and LHA was also alloxan-sensitive, but no cells taken from the VMH and LHA were immunoreactive to the antibody used. The present study further indicates that ependymocytes of the CC and serotonergic neurons in the ROb are also sensitive to the changes in extracellular glucose in a GK-dependent manner, but that the subtype of GK in these cells could be different from that in the VMH and LHA.

The dorsal (DR) and median raphe (MR) nuclei contain 5-hydroxytryptamine (serotonin, 5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain limbic areas that control emotional behavior. In the past, the electrophysiological identification of neurochemically identified 5-HT neurons has been limited. Recent technical developments have made it possible to re-examine the electrophysiological characteristics of identified 5-HT- and non-5-HT-containing neurons. Visualized whole cell electrophysiological techniques in combination with fluorescence immunohistochemistry for 5-HT were used. In the DR, both 5-HT- and non-5-HT-containing neurons exhibited similar characteristics that have historically been attributed to putative 5-HT neurons. In contrast, in the MR, the 5-HT-and non-5-HT-containing neurons had very different characteristics. Interestingly, the MR 5-HT-containing neurons had a shorter time constant and larger afterhyperpolarization (AHP) amplitude than DR 5-HT-containing neurons. The 5-HT(1A) receptor-mediated response was also measured. The efficacy of the response elicited by 5-HT(1A) receptor activation was greater in 5-HT-containing neurons in the DR than the MR, whereas the potency was similar, implicating greater autoinhibition in the DR. Non-5-HT-containing neurons in the DR were responsive to 5-HT(1A) receptor activation, whereas the non-5-HT-containing neurons in the MR were not. These differences in the cellular characteristics and 5-HT(1A) receptor-mediated responses between the MR and DR neurons may be extremely important in understanding the role of these two 5-HT circuits in normal physiological processes and in the etiology and treatment of pathophysiological states.

Gender differences in serotonin (5-HT) metabolism, synthesis, and release suggest that the gonadal steroids estrogen (E) and progesterone (P) influence 5-HT neurotransmission. Based on the effects of ovarian steroids in forebrain sites, this might involve changes in the strength of GABA afferent tone in the dorsal raphe nucleus (DRN). To test this hypothesis, the present study used in vivo microdialysis to measure basal extracellular 5-HT in the rat DRN across the estrous cycle and changes in 5-HT in response to the GABA(A) receptor antagonists bicuculline (50 microM) and picrotoxin (50 microM). During proestrus and estrus, baseline 5-HT levels were significantly higher compared to ovariectomized (OVX) rats. Mean baseline levels across experiments were 8.4 +/- 1.3 pg/ 30 microl in proestrus, 5.2 +/- 0.8 pg/30 mul in estrus, and 3.1 +/- 0.5 pg/30 microl in the DRN of OVX rats. Bicuculline and picrotoxin produced significantly greater increases in 5-HT during proestrus compared to estrus. Moreover, in the DRN of OVX rats, bicuculline and picrotoxin produced negligible increases in 5-HT. These data provide evidence of decreased 5-HT efflux and GABA tone in the rat DRN associated with low circulating E and P.

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. We report an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls (p = 0.0017 and 0.0006 for G/G genotype and G allele distribution, respectively), and in completed suicide cases the G(-1019) allele was enriched fourfold (p = 0.002 and 0.00008 for G/G genotype and G allele distribution, respectively). The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. Our data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. We suggest a novel transcriptional model in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide.

(1) Using in vivo intracerebral microdialysis in conscious, freely moving rats, we examined the effect of flibanserin, a potential antidepressant drug with high affinity for human 5-HT(1A) receptors and four-50-fold lower affinity for 5-HT(2A) and D(4) receptors, on basal extracellular concentrations of serotonin (5-hydroxytryptamine, 5-HT), dopamine (DA) and noradrenaline (NA) in selected regions of the rat brain. (2) Flibanserin at 3 and 10 mg kg(-1) significantly reduced extracellular 5-HT in the prefrontal cortex (by 30 and 45%) and dorsal raphe (35 and 44%), but had no effect on extracellular 5-HT in the ventral hippocampus. The 3 and 10 mg kg(-1) doses raised extracellular NA to a similar extent in the prefrontal cortex (47 and 50%). In all, 10 mg kg(-1) raised extracellular DA in the prefrontal cortex (63%) whereas 3 mg kg(-1) had no significant effect. (3) Pretreatment with the selective 5-HT(1A) receptor antagonist WAY100,635 (0.3 mg kg(-1)) 30 min before 10 mg kg(-1) flibanserin completely antagonized the latter's effects on extracellular 5-HT, DA and NA in the prefrontal cortex. WAY100,635 by itself had no effect on cortical extracellular monoamines. (4) The results show that the stimulation of 5-HT(1A) receptors plays a major role in the effect of flibanserin on brain extracellular 5-HT, DA and NA.

Injections of rhodamine-B into the dorsal raphe nucleus (DRN) and Fluoro-Gold into the lateral geniculate nucleus (LGN) revealed double-labeled retinal ganglion cells (DL RGCs) projecting to both nuclei. The soma-size distribution of DL RGCs was compared with three other distributions: DRN-projecting RGCs, LGN-projecting RGCs, and a large sample of RGCs labeled via the optic nerve with DiI. DL RGC soma diameters fell primarily within the mid-to-upper size range of all three distributions. DL RGCs may provide information to both nuclei concerning comparable aspects of light and visual stimulation via collateralized axons.

The serotonergic system arising from the dorsal raphe nucleus (DR) has long been implicated in psychiatric disorders, and is considered one site of action of classical anxiolytic and antidepressant agents. Recent studies implicate the DR as a site of action of novel anxiolytic and antidepressant agents that target neuropeptide systems, such as corticotropin-releasing factor (CRF) and neurokinin 1 (NK1) antagonists. The present study identified unique characteristics of the dorsomedial DR that implicate this particular subregion as a key component of a circuit, which may be targeted by these diverse psychotherapeutic agents. First, it was observed that a cluster of CRF-containing cell bodies was present in the dorsomedial DR of colchicine-treated rats. Dual-labeling immunohistochemistry revealed that almost all CRF-containing neurons were serotonergic, implicating CRF as a cotransmitter with serotonin in this subpopulation of DR neurons. Moreover, dendrites laden with immunoreactivity for NK1 had a striking topographic distribution surrounding and extending into the dorsomedial subregion of the DR, suggesting that NK1 receptor ligands may selectively impact the dorsomedial DR. Finally, anterograde tract tracing from the dorsomedial DR combined with CRF immunohistochemistry revealed that CRF-containing axons from this subregion project to CRF-containing neurons of the central nucleus of the amygdala. Taken together, the present results reveal a circuit whereby NK1 receptor activation in the dorsomedial DR can impact on limbic sources of CRF that have been implicated in emotional responses. This circuit may be relevant for understanding the mechanism of action of novel psychotherapeutic agents that act through NK1 or CRF receptors.

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.

GABA neurones in the dorsal raphe nucleus (DRN) influence ascending 5-hydroxytryptamine (5-HT) neurones but are not physiologically or anatomically characterised. Here, in vivo juxtacellular labelling methods in urethane-anaesthetised rats were used to establish the neurochemical and morphological identity of a fast-firing population of DRN neurones, which recent data suggest may be GABAergic. Slow-firing, putative 5-HT DRN neurones were also identified for the first time using this approach. Fast-firing, DRN neurones were successfully labelled with neurobiotin (n=10) and the majority (n=8/10) were immunoreactive for the GABA synthetic enzyme glutamic acid decarboxylase. These neurones were located in the DRN (mainly lateral regions), and consistently fired spikes with short width (1.1+/-0.1 ms) and high frequency (12.1+/-2.0 Hz). In most cases spike trains were regular but displayed low frequency oscillations (1-2 Hz). These neurones were morphologically heterogeneous but commonly had branching axons with varicosities and dendrites that extended across DRN subregions and the midline. Slow-firing DRN neurones were also successfully labelled with neurobiotin (n=24). These neurones comprised a population of neurones immunopositive for 5-HT and/or tryptophan hydroxylase (n=12) that fired broad spikes (2.2+/-0.2 ms) with high regularity and low frequency (1.7+/-0.2 Hz). However, a slow-firing, less regular population of neurones immunonegative for 5-HT/tryptophan hydroxylase (n=12) was also apparent. In summary, this study chemically identifies fast- and slow-firing neurones in the DRN and establishes for the first time that fast-firing DRN neurones are GABAergic. The electrophysiological and morphological properties of these neurones suggest a novel function involving co-ordination between GABA and 5-HT neurones dispersed across DRN subregions.

The authors immunocytochemically identified mesopontine cholinergic and rostral raphe serotonergic neurons in brains obtained at autopsy from four patients with multiple system atrophy (MSA) and four matched controls. There was a severe depletion of cholinergic neurons in the pedunculopontine (20 +/- 2 vs 81 +/- 10 cells/section, p< 0.001) and laterodorsal tegmental nucleus (18 +/- 3 vs 47 +/- 4 cells/section, p < 0.001) in MSA. Whereas there was also depletion of locus ceruleus neurons, there was a striking preservation of rostral raphe neurons in MSA.

The alpha 7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium. Although the expression of the alpha 7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing alpha 7 remains to a large extent unknown. Using an antibody against the alpha 7 nAChR subunit, immunohistochemical staining was observed in rat dorsal raphe nucleus (DRN) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DRN and LC, there appeared to be two histologically distinct alpha 7-expressing cell types as distinguished by size, i.e. large versus small diameter. In rats treated with either a serotonergic (5,7-dihydroxytryptamine) or noradrenergic (anti-dopamine-beta-hydroxylase saporin) neurotoxin, tryptophan hydroxylase and tyrosine hydroxylase immunostaining was abolished, respectively. Similarly, the alpha 7-positive large-diameter cells were no longer detectable, suggesting that these cells were serotonergic DRN and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large cell types coexpression of alpha 7 with tryptophan hydroxylase in the DRN and with tyrosine hydroxylase in the LC of saline-treated rats. In contrast to the large-diameter cells, the alpha 7-positive small-diameter cells were neither serotonergic nor adrenergic, and were still detected in both the DRN and LC of lesioned rats. Moreover, cell counts revealed an increase number of these cells in lesioned rats with expression of alpha 7 in somal processes not seen in non-lesioned controls. Double labeling revealed coexpression of alpha 7 and GABA within the majority, but not all, of the toxin-resistant cells. The results of these studies suggest that both serotonergic and noradrenergic neurons express alpha 7 nAChRs. In addition, there appears to be a small-diameter cell-type in both the DRN and LC, possibly a GABAergic interneuron, expressing alpha 7 that may be regulated by neurotoxic injury.

Interactions are known to occur in the brain between serotonin (5-HT) and substance P (SP). To investigate whether SP can directly influence serotonergic neurons, double immunohistochemical labelings were performed on rat brain sections with NK1 or NK3 affinity-purified antibodies and a 5-HT monoclonal antibody. It was found that the vast majority of serotonergic cell bodies do not colocalize NK1 or NK3 labeling. Only in the central linear nucleus and ventral part of the dorsal raphe nucleus were a few serotonergic neurons double-labeled for NK1 receptors (15 and 0.8% of serotonergic neurons, respectively). It is suggested that serotonergic neurons are not major direct targets for SP in the rat brain.

Recombinant rabbit tryptophan hydroxylase (TPH) was expressed in Escherichia coli and purified from inclusion bodies by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A mouse monoclonal antibody and rabbit and sheep polyclonal antibodies were generated. In immunohistochemical studies of formaldehyde-fixed primate brain, the monoclonal strongly labeled not only cell bodies in the raphe nuclei but also fibers in the cerebral cortex. Truncation mutants and peptide pre-competition were used to localize the epitope to E103SVPWFP109. Although the primary sequences of TPH encoded by mRNAs from brain and pineal gland are identical, differences in the immunoreactivity of TPH protein from these two sources were observed in blot immunolabeling studies. TPH immunoreactivity migrated as an M(r) approximately equal 56000 band in each of the tissues except human pineal glands, in which the TPH reactivity was approximately 3 kDa lower. In addition, the relative intensities of TPH immunolabeling across the four tissues differed among these antibodies and a previously described monoclonal antibody against phenylalanine hydroxylase (PH8), which cross-reacts with TPH. Whereas PH8 exhibited roughly equivalent TPH reactivity per protein in both tissues from both species, TPH from human and rat raphe nuclei was preferentially recognized by the present monoclonal. By contrast, the affinity-purified sheep polyclonal antibody reacted preferentially with TPH from human and rat pineal gland, and the affinity-purified rabbit polyclonal antibody appeared to selectively recognize TPH from human pineal gland.

Female ddY mice were administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gavage for 8 weeks prior to pregnancy. In the male breast-fed offspring born to the TCDD-exposed mice, serotonergic neurons in the brainstem were examined using an immunocytochemical method at 42 days of age. In all offspring, a marked decrease in the intensity of immunostaining occurred in all raphe nuclei compared with the control offspring. The number of serotonin-immunoreactive neurons in each raphe nucleus was measured by computer-assisted analysis. Approximately a quarter to half of immunoreactive neurons were detected in the TCDD-exposed offspring raphe nuclei compared with the control offspring. The present findings suggest that in utero and/or lactational TCDD exposure cause a long-lasting change in the serotonergic system in the raphe nuclei of offspring.

We evaluated serotonergic hindbrain groups of cells for their involvement in the generation and inhibition of sodium appetite. For that purpose, we analyzed the number of Fos-immunoreactive (Fos-ir) cells and double-labeled Fos-serotonin (5-HT)-ir neurons within different nuclei of the hindbrain raphe system and the area postrema (AP). Sodium depletion and sodium appetite were induced by peritoneal dialysis. Twenty-four hours after peritoneal dialysis, a 2% NaCl solution intake test was given to peritoneal dialyzed animals [PD-with access (PD-A) group] and to control dialyzed animals [CD-with access (CD-A) group]. Two additional groups of animals received either peritoneal dialysis or control dialysis but were not given access to the 2% NaCl [CD-no access (CD-NA) group or PD-no access (PD-NA) group]. The number of Fos-ir neurons within different nuclei of the raphe system was increased in spontaneous and induced sodium ingestion of CD-A and PD-A groups compared with the CD-NA and PD-NA groups. The PD-NA group had significantly fewer double-labeled cells along the raphe system compared with the animals in near-normal sodium balance (CD-NA and CD-A) or in the process of restoring sodium balance by consuming NaCl (PD-A). The AP of the PD-A group showed a significant increase in the number of Fos-ir and Fos-5-HT-ir cells compared with the PD-NA and CD groups. Our results suggest that serotonergic pathways with cell bodies in the AP and the raphe system are involved in the control of sodium appetite.

Introduction: The raphe nuclei are involved in numerous mechanisms, included the antinociceptives. In the raphe nuclei of the cat, the distribution of neuropeptides is not very studied. Aim. To know the distribution of peptidergic fibers and cell bodies in the raphe nuclei of the cat. We studied a total of fifteen neuropeptides., Material and Methods: We used four control cats (without colchicine) and six with colchicine (administered into the Sylvian aqueduct). We used an indirect immunocytochemical technique. The histologic controls carried out confirm the specificity of the primary and secondary antibodies used., Results: We observed in the fibers and/or the cell bodies located in the dorsal raphe nucleus a total of 14 neuropeptides, 12 in the raphe pallidus, 11 in the medial raphe, 10 in the raphe magnus, 8 in the raphe pontis and 7 in the raphe obscurus. We observed immunoreactive cell bodies in the raphe pallidus (with neurokinin A/leucine enkephalin), in the medial raphe (beta endorphin/alpha neo endorphin), in the raphe magnus (leucine enkephalin) and in the dorsal raphe (beta endorphin/alpha neo endorphin/methionine enkephalin Arg6 Gly7 Leu8/leucine enkephalin/neurokinin A/neurotensin)., Conclusions: 1. There are differences on the distribution of the peptidergic fibers/cell bodies observed in the raphe nuclei of the rat, the cat and the man; 2. The raphe nuclei could receive peptidergic afferences containing dynorphin A, galanin, neuropeptide Y, somatostatin ; 3. The cell bodies located in the medial raphe and containing beta endorphin or alpha neo endorphin could be projecting neurons; 4. There is a great functional complexity in the raphe nuclei due to the great number of neuropeptides observed in them; 5. The neuropeptides could interact between them, and 6. The neuropeptides located in the raphe nuclei could be involved in the control of the nociceptive information.

Orexins (hypocretins) are neuropeptides synthesized in the central nervous system exclusively by neurons of the lateral hypothalamus. Orexin-containing neurons have widespread projections and have been implicated in complex physiological functions including feeding behavior, sleep states, neuroendocrine function, and autonomic control. Two orexin receptors (OX(1)R and OX(2)R) have been identified, with distinct expression patterns throughout the brain, but a systematic examination of orexin receptor expression in the brain has not appeared. We used in situ hybridization histochemistry to examine the patterns of expression of mRNA for both orexin receptors throughout the brain. OX(1)R mRNA was observed in many brain regions including the prefrontal and infralimbic cortex, hippocampus, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. OX(2)R mRNA was prominent in a complementary distribution including the cerebral cortex, septal nuclei, hippocampus, medial thalamic groups, raphe nuclei, and many hypothalamic nuclei including the tuberomammillary nucleus, dorsomedial nucleus, paraventricular nucleus, and ventral premammillary nucleus. The differential distribution of orexin receptors is consistent with the proposed multifaceted roles of orexin in regulating homeostasis and may explain the unique role of the OX(2)R receptor in regulating sleep state stability., (Copyright 2001 Wiley-Liss, Inc.)

The morphology and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus (DRN) of the rat were examined in frontal brain slices. Biocytin was injected intracellularly into the intracellularly recorded neurons. Then the morphology of the recorded neurons was observed after histochemical visualization of biocytin. The recorded neurons extending their main axons outside the DRN were considered as projection neurons. Subsequently, serotonergic nature of the neurons was examined by serotonin (5-HT) immunohistochemistry. The general form of the dendritic trees is radiant and poorly branching in both 5-HT- and non-5-HT neurons. However, the dendrites of the 5-HT neurons were spiny, whereas those of the non-5-HT neurons were aspiny. The main axons of both 5-HT- and non-5-HT neurons were observed to send richly branching axon collaterals to the DRN, ventrolateral part of the periaqueductal gray and the midbrain tegmentum. In response to weak, long depolarizing current pulses, the 5-HT neurons displayed a slow and regular firing activity. The non-5-HT neurons fired at higher frequencies even when stronger current was injected. Some other differences in electrophysiological properties were also observed between the 5-HT-immunoreactive spiny projection neurons and the 5-HT-immunonegative aspiny projection neurons.

The hypocretins (also known as orexins) are hypothalamic peptides that have been implicated in feeding and sleep regulation. Previous reports have described the distribution of the mRNAs encoding two hypocretin receptors (HCRT-R), but the pattern of protein expression has not been investigated. Here we examine the distribution of the mRNA and protein for the HCRT receptor 1 (HCRT-R1) and HCRT receptor 2 (HCRT-R2) in the pontine brainstem and demonstrate that they are present in many pontine nuclei including those associated with REM sleep. Immunohistochemistry indicates that one or both of the receptor subtypes are expressed in the dorsal raphe, the lateral dorsal tegmental (LDT), the pedunculo pontine (PPT), the locus coeruleus (LC), the locus subcoeruleus, pontis oralis, Barrington's, the trigeminal complex (mesencephalic trigeminal and motor nucleus of the trigeminal nerve), the dorsal tegmental nucleus of Gudden (DTG), the ventral cochlear nucleus (VCA), trapezoid nucleus (TZ), pontine raphe nucleus and the pontine reticular formation. These regions have been shown to be involved in mastication, bladder control, gastrointestinal function and in arousal. Given these projection sites and the functions associated with these sites, we suggest that HCRT may play a role in maintaining alertness and vigilance while the animal is engaged in consummatory behavior.

Sleep disorders increase with ageing. The serotonergic system has been linked with sleep regulation. In fatal familial insomnia, a prion disease with insomnia as one major clinical feature, we recently observed a disturbance in the serotonergic system as likely substrate of typical symptoms. Using immunohistochemistry for the serotonin synthesizing enzyme, tryptophan hydroxylase, we investigated the serotonergic median raphe nuclei (dorsal raphe nucleus, superior central nucleus, and raphe obscurus nucleus) in brains of an older (n = 12; age range 62-84 years) and a younger group (n = 10; age range 5-29 years). We found no significant difference between age groups in the percentage of neurons able to synthesize serotonin. Other changes might relate to sleep disturbances in the elderly.

Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.

We have previously demonstrated that treatment of pregnant C57BL mice from gestation days 8 to 14 with alcohol with 20% ethanol-derived calories (EDC) reduced the number of serotonin (5-HT) neurons and retarded their migration in the fetal brains. In the present study, we obtained similar results with the use of 25% EDC and extended our previous findings by demonstrating that besides the alteration of the number of 5-HT neurons, prenatal alcohol exposure also affects their projecting fibers in their early development. Pregnant C57BL mice were divided into an alcohol-exposed (ALC) group given 25% EDC (4.49%, v/v), a pair-fed group to the ethanol-fed group (PF) and a chow-fed group (Chow). The PF and Chow groups served as controls. Our results showed that in the ALC group, when compared with the control groups, prenatal alcohol exposure with 25% EDC reduced the number of 5-HT-immunoreactive neurons in both the median and dorsal raphe, and the amount of 5-HT-immunoreactive fibers in the medial forebrain bundle (MFB). The diameter of the 5-HT-immunoreactive MFB was also reduced as a result of treatment. No significant differences of the above parameters were found between the PF and Chow groups. The previous and present work confirmed that alcohol reduces the normal formation and growth of 5-HT neurons in the midbrain. Furthermore, the projection of 5-HT fibers, in density as well as in distribution, is reduced in the major trajectory bundle. This may affect the amount of 5-HT fibers available to the forebrain. In light of the importance of the 5-HT system in brain development, alcohol may affect the growth of the forebrain through its effect on 5-HT signaling., (Copyright 2001 National Science Council, ROC and S. Karger AG, Basel)

Preclinical studies in rodents suggest that augmentation of serotonin reuptake inhibitors (SSRIs) therapy by the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agent pindolol might reduce the delay between initiation of treatment and antidepressant response. This hypothesis is based on the ability of pindolol to potentiate the increase in serotonin (5-HT) transmission induced by SSRIs, an effect achieved by blockade of the 5-HT(1A) autoreceptors in the dorsal raphe nuclei (DRN). However, placebo-controlled clinical studies of pindolol augmentation of antidepressant therapy have reported inconsistent results. Here, we evaluated the occupancy of 5-HT(1A) receptors following treatment with controlled release pindolol in nine healthy volunteers with positron-emission tomography (PET). Each subject was studied four times: at baseline (scan 1), following 1 week of oral administration of pindolol CR (7.5 mg/day) at peak level, 4 h after the dose (scan 2), and at 10 h following the dose (scan 3), and following one dose of pindolol CR (30 mg) (at peak level, 4 h) (scan 4). Pindolol occupancy of 5-HT(1A) receptors was evaluated in the DRN and cortical regions as the decrease in binding potential (BP) of the radiolabelled selective 5-HT(1A) antagonist [carbonyl-(11)C]WAY-100635 or [carbonyl-(11)C] N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexa necarboxamide abbreviated as [(11)C]WAY-100635. Pindolol dose-dependently decreased [(11)C]WAY-100635 BP. Combining all the regions, occupancy was 20 +/- 8% at scan 2, 14 +/- 8% at scan 3, and 44 +/- 8% at scan 4. The results of this study suggest that at doses used in clinical studies of augmentation of the SSRI effect by pindolol (2.5 mg t.i.d.), the occupancy of 5-HT(1A) receptors is moderate and highly variable between subjects. This factor might explain the variable results obtained in clinical studies. On the other hand, at each dose tested, pindolol occupancy of 5-HT(1A) receptors was higher in the DRN compared to cortical regions, demonstrating a significant in vivo selectivity for DRN 5-HT(1A) autoreceptors relative to cortico-limbic postsynaptic receptors. This selectivity is necessary for the potentiation of 5-HT transmission, and this finding represents an important proof of concept in the development of 5-HT(1A) agents for this application. Early evaluation of new drugs with PET imaging will enable rapid screening of compounds based on DRN selectivity and more appropriate determination of doses for clinical trials.

Serotonin release from dorsal raphe projections in the forebrain is regulated by terminal 5-HT(1B) autoreceptors; dysregulation of these receptors may be involved in the pathophysiology of clinical depression. Using in situ hybridization, we have previously reported that fluoxetine reduces 5-HT(1B) mRNA in rat dorsal raphe nucleus (DRN) in a time-dependent and reversible manner. In this study we examined longer term treatment (8 weeks) with several different serotonin-selective reuptake inhibitors (SSRIs) or a tricyclic antidepressant on 5-HT(1B) mRNA regulation in DRN and hippocampus, and evaluated the stability of these drugs' effects after drug discontinuation. Fluoxetine (5 mg/kg/d), paroxetine (5 mg/kg/d), sertraline (10 mg/kg/d) or nortriptyline (10 mg/kg/d) was administered to rats via subcutaneous osmotic minipumps. Paroxetine and fluoxetine reduced DRN 5-HT(1B) mRNA by 36% and 27%, respectively whereas sertraline had a no significant effect. After 3-14 days of drug washout, DRN 5-HT(1B) mRNA levels in SSRI treated rats were no longer different from control. 5-HT(1B) mRNA levels in hippocampus were not affected by SSRI drugs at any timepoint. Nortriptyline had no significant effect on 5-HT(1B) mRNA in either DRN or hippocampus. These results confirm that SSRI antidepressants reduce presynaptic 5-HT(1B) mRNA selectively, and that this effect is maintained for at least 8 weeks of antidepressant treatment but reverses rapidly after discontinuation. Furthermore, it is possible that washout after chronic antidepressant treatment, that is routinely used in functional assays of autoreceptor action in animal models, may lead to more rapid reversal of biological effects than has previously been thought., (Copyright 2000 Wiley-Liss, Inc.)

This laboratory previously showed that in utero ethanol exposure severely impairs the development of the cell bodies and projections of serotonin (5-HT) neurons, and that maternal treatment with a 5-HT(1A) agonist prevents many of these abnormalities. Others demonstrated that stimulation of fetal astroglial 5-HT(1A) receptors increases production and release of S100B, a glial trophic factor that is essential for the development of 5-HT neurons. The present study investigated a potential mechanism by which ethanol hinders development of 5-HT neurons, and by which maternal 5-HT(1A) agonist treatment prevents this damage. This study tested the hypothesis that in utero ethanol exposure reduces the number of S100B immunopositive glia and that maternal 5-HT(1A) agonist treatment prevents ethanol-associated changes in S100B. To test our hypothesis, we determined the effects of in utero ethanol exposure and maternal treatments with the 5-HT(1A) agonists ipsapirone and buspirone on S100B immunopositive glial cells. On gestation day 20 (G20), S100B immunopositive cells were quantified in the midline raphe glial structure (MRGS), a large transient structure that contains substantial numbers of S100B-positive glial cells and that spans the dorsal raphe, median raphe, and B9 complex of 5-HT neurons. S100B immunopositive glial cells were also determined in an area proximal to the dorsal raphe in postnatal day 2 (PN2) rats. In utero ethanol exposure significantly reduced S100B immunopositive glial cells in the MRGS at G20 and in the dorsal raphe at PN2. In addition, treatment of pregnant rats with a 5-HT(1A) agonist between G13 and G20 prevented the ethanol-associated reduction in S100B immunopositive glial cells. These studies demonstrated that part of ethanol's damaging effects on developing 5-HT neurons is mediated by a reduction of S100B and that some of the protective effects of maternal 5-HT(1A) agonist treatment are related to the actions of these drugs on glial cells.