Rat Dorsal Raphe Nucleus Research Articles

Estrogen decreases 5-HT1B autoreceptor mRNA in selective subregion of rat dorsal raphe nucleus: Inverse association between gene expression and anxiety behavior in the open field

We have recently shown that estrogen decreases anxiety and increases expression of tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme for 5-HT synthesis. However, the effects of estrogen on 5-HT release and reuptake may also affect the overall availability of 5-HT in the forebrain. Estrogen has been previously shown to have no effect on the inhibitory 5-HT 1A autoreceptor (5-HT1A) in the rat dorsal raphe nuclei (DRN); however the regulation of the inhibitory 5-HT 1B autoreceptor (5-HT1B) in the midbrain raphe by estrogen has not yet been investigated. Therefore, we examined the effects of estrogen on 5-HT1B mRNA in the rat DRN, focusing on specific subregions, and whether 5-HT1B mRNA levels correlated with TPH2 mRNA levels and with anxiety-like behavior. Ovariectomized rats were treated for 2 weeks with estrogen or placebo, exposed to the open field test, and 5-HT1A and 5-HT1B mRNA was quantified by in situ hybridization histochemistry. Estrogen had no effect on 5HT1A mRNA in any of the DRN subregions examined, confirming a previous report. In contrast, estrogen selectively decreased 5-HT1B mRNA in the mid-ventromedial subregion of the DRN, where 5-HT1B mRNA was associated with higher anxiety-like behavior and inversely correlated with TPH2 mRNA levels. These results suggest that estrogen may reduce 5-HT1B autoreceptor and increase TPH2 synthesis in a coordinated fashion, thereby increasing the capacity for 5-HT synthesis and release in distinct forebrain regions that modulate specific components of anxiety behavior.

Effects of the β 3-adrenoceptor (Adrb3) agonist SR58611A (amibegron) on serotonergic and noradrenergic transmission in the rodent: Relevance to its antidepressant/anxiolytic-like profile

SR58611A is a selective β 3-adrenoceptor (Adrb3) agonist which has demonstrated antidepressant and anxiolytic properties in rodents. The present study confirmed the detection of Adrb3 mRNA transcript in rodent brain sub-regions and evaluated the effect of SR58611A on serotonergic and noradrenergic transmission in rats and mice in an attempt to elucidate the mechanism(s) underlying these properties. SR58611A (3 and 10 mg/kg, p.o.) increased the synthesis of 5-HT and tryptophan (Trp) levels in several rodent brain areas (cortex, hippocampus, hypothalamus, striatum). Moreover, SR58611A (10 mg/kg, p.o.) increased the release of 5-HT assessed by in vivo microdialysis in rat prefrontal cortex. Systemic (3 mg/kg, i.v.) or chronic administration of SR58611A (10 mg/kg, p.o.), in contrast to fluoxetine (15 mg/kg, p.o.), did not modify the activity of serotonergic neurons in the rat dorsal raphe nucleus. The increase in 5-HT synthesis induced by SR58611A was not observed in Adrb3s knockout mice, suggesting a selective involvement of Adrb3s in this effect. SR58611A (3 and 10 mg/kg, p.o.) did not modify norepinephrine synthesis and metabolism but increased its release in rat brain. Repeated administration of SR58611A (10 mg/kg, p.o.) did not modify basal norepinephrine release in rat prefrontal cortex whereas it prevented its tail-pinch stress-induced enhancement similarly to reboxetine (15 mg/kg, p.o.). Finally SR58611A increased the firing rate of noradrenergic neurons in the rat locus coeruleus following systemic (3 mg/kg, i.v.) or local (0.01 and 1 μM) but not chronic (10 mg/kg, p.o.) administration. These results suggest that the anxiolytic- and antidepressant-like activities of SR58611A involve an increase of brain serotonergic and noradrenergic neurotransmissions, triggered by activation of Adrb3s.

Corticotropin‐releasing factor in the dorsal raphe nucleus increases medial prefrontal cortical serotonin via type 2 receptors and median raphe nucleus activity

Interactions between central corticotropin-releasing factor (CRF) and serotonergic systems are believed to be important for mediating fear and anxiety behaviors. Recently we demonstrated that infusions of CRF into the rat dorsal raphe nucleus result in a delayed increase in serotonin release within the medial prefrontal cortex that coincided with a reduction in fear behavior. The current studies were designed to study the CRF receptor mechanisms and pathways involved in this serotonergic response. Infusions of CRF (0.5 microg/0.5 microL) were made into the dorsal raphe nucleus of urethane-anesthetized rats following either inactivation of the median raphe nucleus by muscimol (25 ng/0.25 microL) or antagonism of CRF receptor type 1 or CRF receptor type 2 in the dorsal raphe nucleus with antalarmin (25-50 ng/0.5 microL) or antisauvagine-30 (2 microg/0.5 microL), respectively. Medial prefrontal cortex serotonin levels were measured using in-vivo microdialysis and high-performance liquid chromatography with electrochemical detection. Increased medial prefrontal cortex serotonin release elicited by CRF infusion into the dorsal raphe nucleus was abolished by inactivation of the median raphe nucleus. Furthermore, antagonism of CRF receptor type 2 but not CRF receptor type 1 in the dorsal raphe nucleus abolished CRF-induced increases in medial prefrontal cortex serotonin. Follow-up studies involved electrical stimulation of the central nucleus of the amygdala, a source of CRF afferents to the dorsal raphe nucleus. Activation of the central nucleus increased medial prefrontal cortex serotonin release. This response was blocked by CRF receptor type 2 antagonism in the dorsal raphe. Overall, these results highlight complex CRF modulation of medial prefrontal cortex serotonergic activity at the level of the raphe nuclei.

Projections from the vestibular nuclei and nucleus prepositus hypoglossi to dorsal raphe nucleus in rats

The serotonergic system regulates processing in components of the vestibular nuclear complex, including the medial vestibular nucleus (MVe) and nucleus prepositus hypoglossi (PH). Recent studies using anterograde and retrograde tracers have shown that vestibular nuclei are targeted by regionally selective projections from the serotonergic dorsal raphe nucleus. The objective of the present investigation was to determine whether the DRN is targeted by projections from the vestibular nuclear complex in rats, using the anterograde tracer biotinylated dextran amine (BDA). After injection of BDA into PH or the caudal parvicellular division of MVe, labeled fibers and terminals were observed in the ventromedial and lateral subdivisions of DRN. These findings indicate that projections from the vestibular nuclei and PH are organized to modulate processing within specific functional domains of the DRN.

3,4-Methylenedioxymethamphetamine induces differential regulation of tryptophan hydroxylase 2 protein and mRNA levels in the rat dorsal raphe nucleus

Previous investigations with 3,4-methylenedioxymethamphetamine (MDMA) have suggested that administration of this drug results in a degeneration of 5-HT nerve terminals and subsequent alterations in 5-HT neurotransmission. However, only limited investigations have examined the effects of MDMA on the dorsal raphe nucleus. The present study was designed to assess the effect of MDMA on the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), by measuring TPH2 protein and mRNA levels in rat dorsal raphe (DR) nucleus. Rats were administered MDMA (20 mg/kg, s.c.) or saline twice daily for 4 days and killed 14 days later. Tissue sections of the DR were processed for quantitative immunoautoradiography and in situ hybridization histochemistry for measurements of the levels of TPH2-immunoreactivity (IR) and TPH2 mRNA. To assess 5-HT axon terminal integrity after MDMA treatment, the density of 5-HT transporter (SERT) binding sites was measured by quantitative autoradiography using [125I]RTI-55 ((-)-2beta-carbomethoxy-3 beta-(4-iodophenyl) tropane) ( 125I-RTI-55) as a ligand. TPH2-IR levels were significantly decreased by 45% in the mid DR and by 40% in the caudal DR in the MDMA-treated rats compared with saline-injected rats. In contrast, TPH2 mRNA levels were significantly increased by 24% in the mid DR and by 12% in the caudal DR. MDMA treatment significantly decreased 125I-RTI-55 labeled SERT binding sites in the striatum, nucleus accumbens and cingulate cortex demonstrating a loss of 5-HT terminals. The increase in TPH2 mRNA levels in both the mid DR and caudal DR of MDMA-treated rats may reflect a compensatory mechanism in the injured 5-HT neurons to increase TPH2 protein synthesis. Taken together, our results suggest that a serious defect occurs in the biosynthesis of TPH2 in the DR following MDMA administration.

Alpha4 containing nicotinic receptors are positioned to mediate postsynaptic effects on 5-HT neurons in the rat dorsal raphe nucleus

Nicotinic acetylcholine receptors containing the alpha4 and beta2 subunits constitute the most abundant high-affinity binding site of nicotine in the brain and are critical for the addictive qualities of nicotine. 5-HT neurotransmission is thought to be an important contributor to nicotine addiction. Therefore in this study it was examined how alpha4-containing receptors are positioned to modulate the function of 5-HT neurons using ultrastructural analysis of immunolabeling for the alpha4 receptor subunit in the dorsal raphe nucleus (DR), a primary source of forebrain 5-HT in the rat. Of 150 profiles labeled for the alpha4 subunit, 140 or 93% consisted of either soma or dendrites, these were often small-caliber (distal) dendrites <1.5 μm in diameter (63/150 or 42%). The majority (107/150 or 71%) of profiles containing labeling for alpha4 were dually labeled for the synthetic enzyme for 5-HT, tryptophan hydroxylase (TPH). Within dendrites immunogold labeling for alpha4 was present on the plasma membrane or near postsynaptic densities. However, labeling for alpha4 was commonly localized to the cytoplasmic compartment often associated with smooth endoplasmic reticulum, plausibly representing receptors in transit to or from the plasma membrane. Previous studies have suggested that nicotine presynaptically regulates activity onto 5-HT neurons, however alpha4 immunolabeling was detected in only 10 axons in the DR or 7% of profiles sampled. This finding suggest that alpha4 containing receptors are minor contributors to presynaptic regulation of synaptic activity onto 5-HT neurons, but rather alpha4 containing receptors are positioned to influence 5-HT neurons directly at postsynaptic sites.

Stress upregulates TPH1 but not TPH2 mRNA in the rat dorsal raphe nucleus: identification of two TPH2 mRNA splice variants.

Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3'-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3'-untranslated regions (UTRs). TPH2b (with a short 3'-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3'-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.

In vivo electrophysiological assessment of the putative antidepressant Wf-516 in the rat raphe dorsalis, locus coeruleus and hippocampus

Wf-516 is a potential novel antidepressant. It has high affinity for serotonin (5-hydroxytryptamine; 5-HT) transporters, 5-HT(1A) and 5-HT(2A) receptors. In the present study, the pharmacologic properties of Wf-516 were thus assessed using in vivo electrophysiology in the rat dorsal raphe nucleus (DRN), locus coeruleus (LC) and hippocampus. Glass microelectrodes were lowered into the DRN, LC or hippocampus, and neurons were recorded and tested using systemic or microiontophoretic injections of drugs. In the DRN, cumulative doses of 0.5 mg/kg of Wf-516 were injected intravenously and total inhibition of 5-HT neurons firing was obtained with 2.8 +/- 0.3 mg/kg. The administration of 1 mg/kg of Wf-516, which by itself did not induce a change in the firing of 5-HT neurons, markedly attenuated the inhibitory effect of the 5-HT(1A) autoreceptor agonist LSD, indicating that Wf-516 is a 5-HT(1A) autoreceptor antagonist. In the LC, 1 mg/kg of Wf-516 dampened the inhibitory effect of the preferential 5-HT(2A) agonist DOI on norepinephrine (NE) neurons, indicating that Wf-516 is also a 5-HT(2A) receptor antagonist. In the hippocampus, cumulative intravenous doses of Wf-516 significantly increased the recovery time of firing activity of CA(3) pyramidal neurons after 5-HT applications, indicating an inhibitory effect on 5-HT reuptake. Unlike the 5-HT(1A) antagonist WAY100635, Wf-516 did not block the inhibitory effect of microiontophoretic application of 5-HT, indicating that this drug is devoid of 5-HT(1A) receptor antagonistic activity in this postsynaptic structure. These properties of WF-516 define the transporter/receptorial profile of an antidepressant with superior effectiveness.

Lateral habenula lesions improve the behavioral response in depressed rats via increasing the serotonin level in dorsal raphe nucleus

The dorsal raphe nucleus (DRN)–serotonin (5-HT) system plays a key role in stress-related psychiatric disorders such as anxiety and depression. The habenular nucleus (Hb) is closely connected with the DRN both morphologically and functionally. Here, we used two types of depressive animal models by exposing rats to chronic mild stress (CMS) and by chronically administering the tricyclic antidepressant clomipramine (CLI) in the rat during the neonatal state of life to produce adult depressed rats. We investigated the effects of lateral habenular nucleus (LHb) lesions on the behavioral response and on the level of 5-HT in DRN in the depressed rats. Forced-swimming test (FST) showed that the immobility time decreased, and the climbing time increased after lesioning LHb of depressed rats. Microdialysis results indicated that the 5-HT level in DRN in depressed rats was lower than that of the control group. Lesion of the LHb was followed by an increased 5-HT turnover in the DRN. Our results suggested that the lesion of the LHb could improve the behavioral response of the depressed rats and the 5-HT level of the DRN increased by LHb lesions could be involved in the effects.

Increased extracellular 5‐HT but no change in sleep after perfusion of a 5‐HT1A antagonist into the dorsal raphe nucleus of rats

The 5-HT(1A) receptor antagonist 4-Iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (p-MPPI) (10 microM) was perfused into the dorsal raphe nucleus (DRN) to study simultaneously the effects of the drug on the DRN and frontal cortex extracellular serotonin (5-hydroxytryptamine, 5-HT) levels and concurring behavioural states. Waking, slow wave sleep and rapid eye movement sleep were determined by polygraphic recordings during microdialysis perfusion and extracellular sample collection. The samples were analysed by microbore high-performance liquid chromatography coupled with electrochemical detection for analysis of 5-HT. p-MPPI perfusion into the DRN (n = 6) produced a sixfold 5-HT increase in the DRN during all behavioural states. The increased 5-HT level was most likely related to the blockage of 5-HT(1A) receptors in the DRN by p-MPPI. No significant effect was seen on sleep. Despite the dramatic increase in DRN extracellular 5-HT produced by p-MPPI, only a transient and nonsignificant effect on sleep was recorded. It is suggested that the usual coupling between 5-HT level and behavioural state may be lost when an excessive serotonergic output is pharmacologically achieved.

Morphological and physiological properties of serotonergic neurons in dissociated cultures from the postnatal rat dorsal raphe nucleus

We have developed dissociated primary cultures of the dorsal raphe nucleus from postnatal 9–12-day-old rats. The nucleus was dissected out from brain slices, dissociated, and cultured over a glial feeder layer. Serotonin immunocytochemistry revealed that 62% of cultured neurons were serotonergic. There was no significant difference in diameters between serotonergic and non-serotonergic neurons. With the whole-cell patch-clamp method, cultured neurons were tested for responses to 8-hydroxydipropylaminotetraline (8-OH-DPAT, a selective agonist for 5-HT 1A), and then treated with serotonin immunocytochemistry. Ninety-two percent of neurons responding to 8-OH-DPAT were serotonergic. These results were used to identify serotonergic neurons. In most cases, serotonergic neurons did not show spontaneous firings of action potentials. Constant current depolarizations elicited trains of action potentials that usually did not show marked adaptation. Application of 8-OH-DPAT inhibited action potential firing. The current–voltage relation of the 8-OH-DPAT-induced current indicated an inward rectification with its reversal potential near E K. Serotonergic neurons were depolarized by phenylephrine, bombesin, and gastrin-releasing peptide. This culture system will serve as a useful tool for elucidating the cellular, physiological, and molecular properties of brain serotonergic neurons.

Co-localization of corticotropin-releasing factor and vesicular glutamate transporters within axon terminals of the rat dorsal raphe nucleus

Electrophysiological, microdialysis and behavioral studies support a modulatory role for corticotropin-releasing factor (CRF) in regulating the dorsal raphe nucleus (DRN)–serotonin (5-HT) system. CRF and 5-HT are implicated in the pathophysiology of depression, thus neuroanatomical substrates of CRF–DRN–5-HT interactions are of interest. Identification of co-transmitters within DRN CRF axon terminals is important for elucidating the complex effects underlying CRF afferent regulation of DRN neurons. This study investigated whether CRF-labeled axon terminals within the DRN contain immunoreactivity for vesicular glutamate transporters (isoforms vGlut1 and vGlut2) indicative of the excitatory neurotransmitter glutamate. Dual immunohistochemistry for CRF and either vGlut1 or vGlut2 was conducted within the same tissue section and immunofluorescence results indicated patterns of immunoreactivity consistent with previous reports. Abundant vGlut1- and vGlut2-immunoreactivity was found in puncta exhibiting a largely uniform distribution, whereas CRF-immunoreactivity was localized to topographically distributed varicose processes within the DRN. Profiles containing both CRF- and either vGlut1- or vGlut2-immunoreactivity were apparent in the DRN. Electron microscopy confirmed that immunoreactivity for CRF and vGlut1 was localized primarily to separate axon terminals in the DRN, with a subset co-localizing CRF and vGlut1. Examination of CRF and vGlut2 immunoreactivities in the DRN indicated that CRF and vGlut2 were found within the same axon terminal more frequently than CRF and vGlut1. Overall, these anatomical findings suggest that CRF may function, in part, with the excitatory neurotransmitter glutamate in the modulation of neuronal activity in the DRN.

The synaptic and nonsynaptic glycine transporter type-1 inhibitors Org-24461 and NFPS alter single neuron firing rate in the rat dorsal raphe nucleus: Further evidence for a glutamatergic–serotonergic interaction and its role in antipsychotic action

Single neuron firing rate was recorded from dorsal raphe nucleus of anesthetized rats. The firing rate of raphe neurons varied from 4 to 8 discharge per second before drug administration and this neuronal activity was decreased by L-701,324 (2mg/kg i.v. injection), a competitive antagonist of glycineB binding site of N-methyl-d-aspartate (NMDA) receptors. The glycine transporter type-1 (GlyT1) antagonists Org-24461 (10mg/kg i.v.) and NFPS (3mg/kg i.v.) reversed the inhibitory effect of L-701,324 on single neuron activity recorded from dorsal raphe nucleus of the rat. Org-24461 and NFPS both tended to increase the raphe neuronal firing rate also when given alone but their effect was not significant. This finding serves further evidence that glutamate released from axon terminals of the cortico-striatal projection neurons stimulates serotonergic neurons in the raphe nuclei and this effect is mediated at least in part by postsynaptic NMDA receptors. Thus, GlyT1 inhibitors are able to reverse the hypofunctional state of NMDA receptors, suggesting that these drugs may have beneficial therapeutic effects in neurological and psychiatric disorders characterized with impaired NMDA receptor-mediated transmission.

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

Corticotropin releasing factor (CRF) family peptides play key roles in integrating neural responses to stress. Both major CRF receptors have been pharmacologically identified in the dorsal raphe nucleus (DRN), a stress sensitive and internally heterogeneous nucleus supplying many forebrain regions with serotonergic input. Despite the involvement of chronic stress and serotonergic dysfunction in human mood and anxiety disorders, little is known about the effects of chronic CRF receptor activation on the DRN. We infused ovine CRF (1 ng/h), urocortin II (UCNII, 1 ng/h), or vehicle alone into rat DRN over 6 days. During infusion, animals were allowed to freely explore an open field for 15 min on each of 2 days, with the addition of a novel object on the second day. Following behavioral testing, 5-HT 1A, 5-HT 1B, 5-HT transporter (SERT), and tryptophan hydroxylase-2 ( Tph2) expression was examined through the DRN by in situ hybridization. Ovine CRF infusion resulted in significantly decreased novel object touches, climbs, as well as increased latency to first novel object contact. UCNII had a similar but less dramatic effect, decreasing only climbing behavior. Both ovine CRF and UCNII blunted the decrease in corner time expected on re-exposure to the open field. Both peptides also produced regionally specific changes in gene expression: 5-HT 1A expression was increased 30% in the mid-rostral ventromedial DRN, while SERT was decreased by 30% in the mid-caudal shell dorsomedial DRN. There also appeared to be a shift in the relative level of Tph2 expression between the ventromedial and core dorsomedial DRN at the mid-rostral level. Changes in 5-HT 1A, SERT, and relative Tph2 mRNA abundance were correlated with novel object exploration. These findings suggest chronic intra-DRN administration of CRF agonists decreases exploratory behavior, while producing subregionally limited changes in serotonergic gene expression. These studies may be relevant to mechanisms underlying behavioral changes after chronic stress.

Fos-like immunoreactivity in rat dorsal raphe nuclei induced by alkaloid extract of Mitragyna speciosa

Mitragyna speciosa (MS) has been traditionally used for medicinal purposes especially in southern Thailand. Previously, an alkaloid extract of this plant was demonstrated to mediate antinociception, partly, through the descending serotonergic system. The present study investigated the stimulatory effect of the MS extract on the dorsal raphe nucleus and its antidepressant-like activity. The MS extract containing approximately 60% mitragynine as a major indole alkaloid was used to treat the animals. The stimulatory effect of the MS extract was determined by detecting the expression of the immediate early gene, cfos, in the dorsal raphe nucleus of male Wistar rats. The immunohistochemistry was used to detect Fos protein, the protein product of cfos gene. The present data show that a significant increase in Fos expression was observed following long-term administration of the MS extract (40 mg/kg) for 60 consecutive days. In addition, the antidepressant-like activity of the MS extract was determined by using the forced swimming test (FST) in male mice. The results show that a single injection (either 60 or 90 mg/kg doses) significantly decreased immobility time in the FST. These findings indicate that the MS extract has a stimulatory effect on the dorsal raphe nucleus and an antidepressant-like activity. Stimulation of this brain area has been known to cause antinociception. These findings suggest that the MS extract might produce antinociceptive and/or antidepressive actions partly through activation of the dorsal raphe nucleus. Moreover, the dorsal raphe nucleus may be one of site of MS action in the central nervous system.

Involvement of local orphanin FQ in the development of analgesic tolerance induced by morphine microinjections into the dorsal raphe nucleus of rats

It is well known that dorsal raphe nucleus (DRN) is one of the key structures for the development of opioid analgesia and tolerance. An increased activity of ‘antiopioids’ like orphanin-FQ (OFQ) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of DRN-located OFQ in the opioid analgesic tolerance induced by repeated microinjections of morphine (MOR) into DRN. Male rats were implanted with chronic guide cannulae aimed at the DRN. Microinjection of MOR (0.5 μg in 0.5 μl) into DRN caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the OFQ receptor antagonist nocistatin (NST) (1 ng in 0.5 μl) into the same DRN site, the microinjections of MOR always produced antinociception and did not induce tolerance. If NST microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single NST microinjection into the same DRN site was enough to restore the antinociceptive effect of MOR. On the other hand, if OFQ (1 ng in 0.5 μl) was microinjected into DRN, then MOR microinjection administered 15 min later into the same DRN site did not elicit antinociception. Finally, opioid tolerance induced by repeated systemic MOR injections (5 mg/kg, ip) was reversed by a single microinjection of NST into DRN. This emphasizes the central importance of DRN-located OFQ in the MOR analgesic tolerance.

Increased serotonin efflux in the dorsal raphe nucleus of rats working for delayed rewards

Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRNVgat and DRNVGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding—DRNVgat neurons increase, whereas DRNVGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance.

Neurochemical identification of stereotypic burst‐firing neurons in the rat dorsal raphe nucleus using juxtacellular labelling methods

Recent electrophysiological studies have discovered evidence of heterogeneity of 5-hydroxytryptamine (5-HT) neurons in the mesencephalic raphe nuclei. Of particular interest is a subpopulation of putative 5-HT neurons that display many of the electrophysiological properties of presumed 5-HT-containing neurons (regular and slow firing of single spikes with a broad waveform) but fire spikes in short, stereotyped bursts. In the present study we investigated the chemical identity of these neurons in rats utilizing in vivo juxtacellular labelling methods. Of ten dorsal raphe nucleus (DRN) neurons firing short stereotyped bursts within an otherwise regular firing pattern, all exhibited immunoreactivity for either 5-HT (n = 6) or the 5-HT synthesizing enzyme, tryptophan hydroxylase (TRH; n = 2) or both (n = 2). Supporting pharmacological experiments demonstrated that the burst firing DRN neurons demonstrated equal sensitivity to 5-HT(1A) agonism and alpha(1)-adrenoceptor antagonism to single spiking DRN neurons that we have previously identified as 5-HT-containing. Collectively these data provide direct evidence that DRN neurons that exhibit stereotyped burst firing activity are 5-HT containing. The presence of multiple types of electrophysiologically distinct midbrain 5-HT neurons is discussed.

Effect of chronic citalopram on serotonin-related and stress-regulated genes in the dorsal raphe nucleus of the rat

Using a model of depression in which chronic social stress induces depressive-like symptoms, we investigated effects of the selective serotonin-reuptake inhibitor (SSRI) citalopram on gene expression in the dorsal raphe nucleus of male rats. Expression of tryptophan hydroxylase (TPH) protein was found to be upregulated by the stress and normalized by citalopram, while mRNAs for genes TPH 1 and 2 were differentially affected. Citalopram had no effect on serotonin transporter mRNA but reduced serotonin-1A autoreceptor mRNA in stressed animals. The SSRI prevented the stress-induced upregulation of mRNA for CREB binding protein, synaptic vesicle glycoprotein 2b and the glial N-myc downstream-regulated gene 2, but increased mRNA for neuron-specific enolase (NSE) in both stressed and unstressed animals having no effect on stress-induced upregulation of NSE protein. These findings demonstrate that in the dorsal raphe nucleus of chronically stressed rats, citalopram normalizes TPH expression and blocks stress effects on distinct genes related to neurotransmitter release and neuroplasticity.

Acute restraint increases NADPH-diaphorase staining in distinct subregions of the rat dorsal raphe nucleus: Implications for raphe serotonergic and nitrergic transmission

The brainstem dorsal raphe nucleus (DRN) maintains a rough topographic cell ordering with respect to biological function. This study examined the influence of acute restraint on nitric oxide (NO) synthase (NOS) neurons in distinct DRN subregions. NADPH diaphorase staining (NOS index) intensity was higher in the DRN dorsomedial, ventromedial and lateral wings subregions of restrained vs. control rats. The mean number of cells was not significantly different between both groups of animals. The restrained-induced NADPH-diaphorase activity was significantly higher in the rostral ventromedial and caudal lateral wings than the corresponding caudal and rostral subregions but no significant difference was observed between rostral and caudal dorsomedial subregions. These observations suggest that restraint stress differentially activates NO-producing neurons in distinct DRN subregions.