Rat Dorsal Raphe Nucleus Research Articles

The 5-HT7 receptor antagonist SB 269970 reverses prenatal stress-induced changes in synaptic transmission in the rat dorsal raphe nucleus

The 5-HT7 receptor antagonist SB 269970 reverses prenatal stress-induced changes in synaptic transmission in the rat dorsal raphe nucleus

The 5-HT7 receptor antagonist SB 269970 ameliorates corticosterone-induced alterations in 5-HT7 receptor-mediated modulation of GABAergic transmission in the rat dorsal raphe nucleus

RationaleChronic stress and corticosterone have been shown to affect serotonin (5-HT) neurotransmission; however, the influence of stress on the activity of the dorsal raphe nucleus (DRN), the main source of 5-HT in the forebrain, is not well understood. In particular, it is unknown if and how stress modifies DRN 5-HT7 receptors, which are involved in the modulation of the firing of local inhibitory interneurons responsible for regulating the activity of DRN projection cells.ObjectivesOur study aimed to investigate the effect of repeated corticosterone injections on the modulation of the inhibitory transmission within the DRN by 5-HT7 receptors and whether it could be reversed by treatment with a 5-HT7 receptor antagonist.MethodsMale Wistar rats received corticosterone injections repeated twice daily for 14 days. Spontaneous inhibitory postsynaptic currents (sIPSCs) were then recorded from DRN projection cells in ex vivo slice preparations obtained 24 h after the last injection.ResultsRepeated corticosterone administration resulted in decreased frequency, but not amplitude, of sIPSCs in DRN projection cells. There were no changes in the excitability of these cells; however, corticosterone treatment suppressed the 5-HT7 receptor-mediated increase in sIPSC frequency. Administration of the 5-HT7 receptor antagonist SB 269970 for 7 days beginning on the eighth day of corticosterone treatment reversed the detrimental effects of corticosterone on 5-HT7 receptor reactivity and GABAergic transmission in the DRN.ConclusionsElevated corticosterone level reduces DRN 5HT7 receptor reactivity and decreases GABAergic transmission within the DRN, which can be reversed by the 5-HT7 receptor antagonist SB 269970.

Serotonin2B receptors in the rat dorsal raphe nucleus exert a GABA-mediated tonic inhibitory control on serotonin neurons

The central serotonin2B receptor (5-HT2BR) is a well-established modulator of dopamine (DA) neuron activity in the rodent brain. Recent studies in rats have shown that the effect of 5-HT2BR antagonists on accumbal and medial prefrontal cortex (mPFC) DA outflow results from a primary action in the dorsal raphe nucleus (DRN), where they activate 5-HT neurons innervating the mPFC. Although the mechanisms underlying this interaction remain largely unknown, data in the literature suggest the involvement of DRN GABAergic interneurons in the control of 5-HT activity. The present study examined this hypothesis using in vivo (intracerebral microdialysis) and in vitro (immunohistochemistry coupled to reverse transcription-polymerase chain reaction) experimental approaches in rats. Intraperitoneal (0.16 mg/kg) or intra-DRN (1 μM) administration of the selective 5-HT2BR antagonist RS 127445 increased 5-HT outflow in both the DRN and the mPFC, these effects being prevented by the intra-DRN perfusion of the GABAA antagonist bicuculline (100 μM), as well as by the subcutaneous (0.16 mg/kg) or the intra-DRN (0.1 μM) administration of the selective 5-HT1AR antagonist WAY 100635. The increase in DRN 5-HT outflow induced by the intra-DRN administration of the selective 5-HT reuptake inhibitor citalopram (0.1 μM) was potentiated by the intra-DRN administration (0.5 μM) of RS 127445 only in the absence of bicuculline perfusion. Finally, in vitro experiments revealed the presence of the 5-HT2BR mRNA on DRN GABAergic interneurons. Altogether, these results show that, in the rat DRN, 5-HT2BRs are located on GABAergic interneurons, and exert a tonic inhibitory control on 5-HT neurons innervating the mPFC.

The antidepressant-like effect of galanin in the dorsal raphe nucleus of rats involves GAL2 receptors

Galanin is a neuropeptide distributed in human and rat brain regions that are involved with emotional regulation, such as the dorsal raphe nucleus (DRN). Galanin effects in the DRN are mediated by GAL1 and GAL2 receptors. Intracerebral infusion of a GAL2 (AR-M1896) or a GAL1 (M617) agonist induced either antidepressant or depressive-like effect, respectively, in rats exposed to the forced swimming test (FST). However, it is not clear if GAL1 and/or GAL2 receptors present in the DRN would be involved in such effects. Therefore, we investigated the effects induced by intra-DRN infusion of galanin (0.3 nmol), AR-M1896 (1 nmol, GAL2 agonist), or M617 (GAL1 agonist) in rats exposed to the FST. Galanin and AR-M1896 intra-DRN administration induced antidepressant-like effect in the FST. However, M617 did not induce any change in the FST. Neither M617 nor AR-M1896 changed the locomotor activity of rats in the open field test. Intra-DRN pre-treatment with M871 (1 nmol), a selective GAL2 antagonist, counteracted the antidepressant-like effect induced by galanin. These results suggest that galanin signaling through GAL2 receptors in the DRN produces triggers antidepressant-like effect.

Role of nitric oxide and WNK-SPAK/OSR1-KCC2 signaling in daily changes in GABAergic inhibition in the rat dorsal raphe neurons

Serotonergic neurons in the dorsal raphe nucleus (DRN) act as wake-inducing neurons in the sleep-wake cycle and are controlled by gamma-aminobutyric acid (GABA) synaptic inputs. We investigated daily changes in GABAergic inhibition of the rat DRN neurons and the role of nitric oxide (NO) and cation-chloride co-transporters in the GABAergic action. Neuronal NO synthase (nNOS) was co-expressed in 74% of serotonergic DRN neurons and nNOS expression was higher during daytime (the sleep cycle) than that during nighttime (the wake cycle). GABAergic hyperpolarization of DRN neurons produced by GABAA receptor agonist muscimol was greater and the equilibrium potential of muscimol showed a hyperpolarizing shift during daytime compared to that during nighttime. Expression levels of potassium-chloride co-transporter 2 (KCC2) were higher during daytime than that during nighttime, whereas there were no changes in sodium-potassium-chloride co-transporter 1 (NKCC1) expression. With-no-lysine kinase (WNK) isoform 1 was more highly expressed during daytime than that during nighttime. Total Ste20-related proline alanine rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were also higher during daytime than during nighttime, while there were no changes in phosphorylated SPAK and OSR1. Consistent with the findings during the sleep-wake cycle, ex vivo treatment of DRN slices with a NO donor sodium nitroprusside (SNP) increased the expression of KCC2, WNK1, WNK2, WNK3, SPAK, and OSR1, whilst decreasing phosphorylated SPAK. These results suggest that GABAergic synaptic inhibition of DRN serotonergic neurons shows daily changes during the sleep-wake cycle, which might be regulated by daily changes in nNOS-derived NO and WNK-SPAK/OSR1-KCC2 signaling.

Effects of Estrogen Therapy on the Serotonergic System in an Animal Model of Perimenopause Induced by 4-Vinylcyclohexen Diepoxide (VCD).

Chronic exposure to 4-vinylcycloxene diepoxide (VCD) in rodents accelerates the natural process of ovarian follicular atresia modelling perimenopause in women. We investigated why estrogen therapy is beneficial for symptomatic women despite normal or high estrogen levels during perimenopause. Female rats (28 d) were injected daily with VCD or oil for 15 d; 55-65 d after the first injection, pellets of 17β-estradiol or oil were inserted subcutaneously. Around 20 d after, the rats were euthanized (control rats on diestrus and estradiol-treated 21 d after pellets implants). Blood was collected for hormone measurement, the brains were removed and dorsal raphe nucleus (DRN), hippocampus (HPC), and amygdala (AMY) punched out for serotonin (5-HT), estrogen receptor β (ERβ), and progesterone receptor (PR) mRNA level measurements. Another set of rats was perfused for tryptophan hydroxylase (TPH) immunohistochemistry in the DRN. Periestropausal rats exhibited estradiol levels similar to controls and a lower progesterone level, which was restored by estradiol. The DRN of periestropausal rats exhibited lower expression of PR and ERβ mRNA and a lower number of TPH cells. Estradiol restored the ERβ mRNA levels and number of serotonergic cells in the DRN caudal subregion. The 5-HT levels were lower in the AMY and HPC in peristropausal rats, and estradiol treatment increased the 5-HT levels in the HPC and also increased ERβ expression in this area. In conclusion, estradiol may improve perimenopause symptoms by increasing progesterone and boosting serotonin pathway from the caudal DRN to the dorsal HPC potentially through an increment in ERβ expression in the DRN.

Evaluation of food intake and Fos expression in serotonergic neurons of raphe nuclei after intracerebroventricular injection of adrenaline in free-feeding rats

Previous studies indicate that the modification of adrenergic neurotransmission in median raphe nucleus (MRN) enhances or removes an inhibitory influence on food intake, possibly serotonergic, due to a presence of serotonin-producing neurons in that nucleus. Therefore, the aim of this study is evaluated whether the activity of neurons in the MRN and dorsal raphe nucleus (DRN) are affected by intracerebroventricular injection of adrenaline (AD) in free-feeding rats. Male Wistar rats with guide cannulae chronically implanted in the lateral ventricle were injected with AD followed by evaluation of ingestive behavioral parameters. Behavior was monitored and the amount of food ingested was assessed. The highest dose (20 nmol) of AD was the most effective dose in increasing food intake. Subsequently, AD 20 nmol was injected to study neuronal activity indicated by the presence of Fos protein and its co-localization with serotonergic neurons in the MRN and DRN of naive rats with or without access to food during the recording of behavior. The administration of AD 20 nmol increased Fos expression and double labeling with serotonergic neurons in the DRN in rats with access to food, but not in animals without access. No statistically significant changes in Fos expression were observed in the MRN in any of the experimental conditions tested. These results suggest that DRN serotonergic and non-serotonergic neurons are activated by post-prandial signals. In contrast, the absence of Fos expression in the MRN suggests that this nucleus does not participate in the circuit involved in the control of post-prandial satiety.

5-HT in the dorsal raphe nucleus is involved in the effects of 100-Hz electro-acupuncture on the pain-depression dyad in rats.

The pain-depression dyad is becoming widespread in the clinic and is attracting increasing attention. A previous study by our group found that 100-Hz electro-acupuncture (EA), but not 2-, 50- and 2/100-Hz EA, was effective against the reserpine-induced pain-depression dyad. This finding is in contrast to the fact that low-frequency EA is commonly used to treat supraspinal-originating diseases. The present study aimed to investigate the mechanism underlying the effects of 100-Hz EA on the pain-depression dyad. Repeated reserpine injection was found to induce allodynia and depressive behaviors in rats. It decreased 5-hydroxytryptamine (5-HT) levels and immunoreactive expressions in the dorsal raphe nucleus (DRN). 100-Hz EA alleviated the pain-depression dyad and upregulated 5-HT in the DRN of reserpine-injected rats. Intracerebroventricular injection of para-chlorophenylalanine, an inhibitor of 5-HT resynthesis, suppressed the upregulation of 5-HT in the DRN by 100-Hz EA and partially counteracted the analgesic and anti-depressive effects of 100-Hz EA. The present study was the first to demonstrate that 5-HT in the DRN is involved in mediating the analgesic and anti-depressive effects of 100-Hz EA on the pain-depression dyad. This finding provided a scientific basis for high-frequency EA as a potential treatment for the pain-depression dyad.

GluA2-Lacking AMPA Receptors and Nitric Oxide Signaling Gate Spike-Timing-Dependent Potentiation of Glutamate Synapses in the Dorsal Raphe Nucleus.

The dorsal raphe nucleus (DRn) receives glutamatergic inputs from numerous brain areas that control the function of DRn serotonin (5-HT) neurons. By integrating these synaptic inputs, 5-HT neurons modulate a plethora of behaviors and physiological functions. However, it remains unknown whether the excitatory inputs onto DRn 5-HT neurons can undergo activity-dependent change of strength, as well as the mechanisms that control their plasticity. Here, we describe a novel form of spike-timing–dependent long-term potentiation (tLTP) of glutamate synapses onto rat DRn 5-HT neurons. This form of synaptic plasticity is initiated by an increase in postsynaptic intracellular calcium but is maintained by a persistent increase in the probability of glutamate release. The tLTP of glutamate synapses onto DRn 5-HT is independent of NMDA receptors but requires the activation of calcium-permeable AMPA receptors and voltage-dependent calcium channels. The presynaptic expression of the tLTP is mediated by the retrograde messenger nitric oxide (NO) and activation of cGMP/PKG pathways. Collectively, these results indicate that glutamate synapses in the DRn undergo activity-dependent synaptic plasticity gated by NO signaling and unravel a previously unsuspected role of NO in controlling synaptic function and plasticity in the DRn.

Galanin subtype 1 and subtype 2 receptors mediate opposite anxiety-like effects in the rat dorsal raphe nucleus

About 40% of the dorsal raphe nucleus (DRN) neurons co-express serotonin (5-HT) and galanin. Serotonergic pathways from the DRN to the amygdala facilitate learned anxiety, while those from the DRN to the dorsal periaqueductal grey matter (DPAG) impair innate anxiety. Previously, we showed that galanin infusion in the DRN of rats induces anxiolytic effect by impairing inhibitory avoidance without changing escape behaviour in the elevated T-maze (ETM). Here, we evaluated: (1) which galanin receptors would be involved in the anxiolytic effect of galanin in the DRN of rats tested in the ETM; (2) the effects of galanin intra-DRN on panic-like behaviours evoked by electrical stimulation of the DPAG. The activation of DRN GAL1 receptors by M617 (1.0 and 3.0nmol) facilitated inhibitory avoidance, whereas the activation of GAL2 receptors by AR-M1896 (3.0nmol) impaired the inhibitory avoidance in the ETM, suggesting an anxiogenic and an anxiolytic-like effect respectively. Both agonists did not change escape behaviour in the ETM or locomotor activity in the open field. The anxiolytic effect of AR-M1896 was attenuated by the prior administration of WAY100635 (0.18nmol), a 5-HT1A antagonist. Galanin (0.3nmol) administered in the DRN increased discreetly flight behaviours induced by electrical stimulation of the DPAG, suggesting a panicolytic effect. Together, our results showed that galanin mediates opposite anxiety responses in the DRN by activation of GAL1 and GAL2 receptors. The anxiolytic effect induced by activation of Gal2 receptors may depend on serotonergic tone. Finally, the role of galanin in panic related behaviours remains uncertain.

Lateral habenula as a link between thyroid and serotoninergic system modiates depressive symptoms in hypothyroidism rats.

Depression-like behavior is observed in both rats and people with hypothyroidism, which suggests that altered thyroid hormone levels are closely associated with mental illness. Furthermore, decreased serotonin (5-hydroxytryptamine, 5-HT) levels are found in some brain regions of hypothyroid rats with depression-like behavior. However, the mechanism underlying the effects of hypothyroidism on the central serotonin system is unclear. The lateral habenula (LHb) is related to both the serotonin and thyroid systems and also plays an important role in the pathogenesis of depression. Our study aimed to disclose the role of the LHb in the onset of depression-like behavior in thyroidectomy (TD) rats. Forced swimming (FST) and open-field tests (OFT) were performed to measure behavioral changes in TD rats. The expression of β calmodulin-dependent protein kinase type II (β CaMKII) in the LHb, cytochrome C oxidase (COX) activity in the LHb and dorsal raphe nucleus (DRN), and 5-HT levels in the DRN were assayed. We found that TD rats exhibited depression-like behavior in the FST and OFT. Compared with the sham group, neural activity and the expression of β CaMKII in TD rats were higher in the LHb, and neural activity and 5-HT levels were lower in the DRN. Depressive behavior and decreased 5-HT levels in the DRN in TD rats were reversed by LHb lesioning. Our study indicates that depression-like behavior in TD rats can be attributed to decreased 5-HT levels in the DRN resulting from inhibition by an overactive LHb. The LHb mediates the effect of the thyroid system on 5-HT function in the DRN.

Ethanol withdrawal induces anxiety-like effects: Role of nitric oxide synthase in the dorsal raphe nucleus of rats.

Nitric oxide (NO) mediated transmission in the dorsal raphe nucleus (DRN) has been shown to be involved in the modulation of anxiety-like behaviors. We investigated whether inhibition of nitric oxide synthase (NOS) in the DRN would prevent anxiety-like behavior induced by ethanol withdrawal. Male Wistar rats were treated with ethanol 2-6% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48h after ethanol discontinuation. Rats with a guide cannula aimed at the DRN received intra-DRN injections of the non-selective NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME), selective neuronal NOS (nNOS) inhibitor N(ω)-propyl-l-arginine (NPLA), or selective inhibitor of inducible NOS (iNOS) N-([3-(aminomethyl)phenyl] methyl) ethanimidamidedihydrochloride (1400W). Five minutes later, the animals were tested in the elevated plus maze (EPM). Plasma ethanol levels were determined by gas chromatography. There was a reduction in plasma ethanol levels 48h after ethanol withdrawal. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the EPM with no change in the exploration of enclosed arms. Intra-DRN treatment with l-NAME (100nmoles/0.2μL) and 1400W (1nmol/0.2μL), but not NPLA (10nmoles/0.2μL) in the DRN attenuated the decrease in the exploration of the open arms of the EPM induced by ethanol withdrawal. The major new finding of the present study is that iNOS in the DRN plays a role in the anxiety-like behavior induced by ethanol withdrawal.

The Dorsal Raphe Nucleus Receives Afferents From Alpha-Like Retinal Ganglion Cells and Intrinsically Photosensitive Retinal Ganglion Cells in the Rat.

A retinal projection into the dorsal raphe nucleus (DRN), namely, the retino-raphe projection, exists in many species. The rat is one of several species in which a retino-raphe projection has been described; however, the retinal ganglion cell (RGC) types that contribute to this pathway are unknown. Retrograde tracing via cholera toxin subunit B (CTB) was used to reveal DRN-projecting RGCs in rats, combined with intracellular injection in vitro, melanopsin immunostaining in whole-mounted retinas, and serotonin immunostaining to define the DRN. We modified methods of CTB injection into DRN used previously in order to avoid possible contamination with other retinorecipient regions, particularly the superior colliculus (SC). The majority of DRN-projecting RGCs showed alpha-like morphology, and some CTB-positive RGCs were colabeled with melanopsin. Approximately 80% of the total population of CTB-labeled DRN-projecting RGCs was alpha-like cells including ON alpha cells and OFF alpha cells; these alpha-like cells were melanopsin immunonegative. Approximately 10% of the remaining DRN-projecting RGCs were melanopsin immunopositive, in which the M1 subtype of intrinsically photosensitive retinal ganglion cell (ipRGC) provided the dominant projection of ipRGCs into DRN, with only few non-M1 ipRGCs involved. The DRN-projecting ipRGCs could be retrogradely labeled following tracer injection into all rostrocaudal aspects of the DRN. Both conventional RGCs with alpha-like morphology and melanopsin-expressing ipRGCs project into the rat DRN. Approximately 10% of DRN-projecting RGCs were colabeled with melanopsin, and the majority of these were the M1 subtype of ipRGCs. An ipRGC component of the retino-raphe projection may contribute to a sustained light-mediated modulation of DRN serotonin release.

Melanin-concentrating hormone projections to the dorsal raphe nucleus: An immunofluorescence and in vivo microdialysis study

Melanin-concentrating hormone (MCH)-containing neurons are localized in the lateral hypothalamus and incerto-hypothalamic areas, and project to several brain regions including the dorsal raphe nucleus (DRN). The MCHergic system has been involved in the regulation of emotional states and we have demonstrated that MCH microinjections into the rat DRN promote a depressive-like state. To understand the MCHergic transmission into the DRN, in the present study we characterized the distribution and density of the MCHergic fibers along the rostro-caudal axis of the rat DRN and their anatomical relationship with the 5-HT- and GABA-containing neurons. Additionally, a functional in vivo microdialysis study was carried out in order to evaluate the MCH effects on the 5-HT extracellular levels.Immunolabeling studies showed that MCHergic fibers were widely distributed throughout the rostro-caudal DRN extent and a reduced density at the most caudal level was observed. Interestingly, MCHergic fibers appeared in close apposition to 5-HT and GABA-containing neurons. Microdialysis studies evidenced an opposite effect of two concentrations of MCH on 5-HT levels: the lower concentration (30μM) produced a significant and long-lasting (up to 120min) decrease while the higher (100μM) induced a slight and brief (20min) increase.Morphological and functional results strongly suggest that both 5-HT- and GABA-containing neurons of the DRN are modulated by MCH. A different sensitivity of these neurons to MCH may explain the dose-response effect on 5-HT release. The decrease in extracellular 5-HT levels may account for the depressive-like effect induced by MCH reported in our previous studies.

Phosphorylation of CaMKII in the rat dorsal raphe nucleus plays an important role in sleep-wake regulation.

The Ca(2+) modulation in the dorsal raphe nucleus (DRN) plays an important role in sleep-wake regulation. Calmodulin-dependent kinase II (CaMKII) is an important signal-transducing molecule that is activated by Ca(2+) . This study investigated the effects of intracellular Ca(2+) /CaMKII signaling in the DRN on sleep-wake states in rats. Maximum and minimum CaMKII phosphorylation was detected at Zeitgeber time 21 (ZT 21; wakefulness state) and ZT 3 (sleep state), respectively, across the light-dark rhythm in the DRN in rats. Six-hour sleep deprivation significantly reduced CaMKII phosphorylation in the DRN. Microinjection of the CAMKII activation inhibitor KN-93 (5 or 10 nmol) into the DRN suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REM sleep (NREMS). Application of a high dose of KN-93 (10 nmol) increased slow-wave sleep (SWS) time, SWS bouts, the mean duration of SWS, the percentage of SWS relative to total sleep, and delta power density during NREMS. Microinjection of CaCl2 (50 nmol) in the DRN increased CaMKII phosphorylation and decreased NREMS, SWS, and REMS. KN-93 abolished the inhibitory effects of CaCl2 on NREMS, SWS, and REMS. These data indicate a novel wake-promoting and sleep-suppressing role for the Ca(2+) /CaMKII signaling pathway in DRN neurons. We propose that the intracellular Ca(2+) /CaMKII signaling in the dorsal raphe nucleus (DRN) plays wake-promoting and sleep-suppressing role in rats. Intra-DRN application of KN-93 (CaMKII activation inhibitor) suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Intra-DRN application of CaCl2 attenuated REMS and NREMS. We think these findings should provide a novel cellular and molecular mechanism of sleep-wake regulation.

Dorsal raphe nucleus acetylcholine-mediated neurotransmission modulates post-ictal antinociception: The role of muscarinic and nicotinic cholinergic receptors

The dorsal raphe nucleus (DRN) is a key structure of the endogenous pain inhibitory system. Although the DRN is rich in serotoninergic neurons, cholinergic neurons are also found in that nucleus. Both ictal and inter-ictal states are followed by post-ictal analgesia. The present study investigated the role of cholinergic mechanisms in postictal antinociceptive processes using microinjections of atropine and mecamylamine, muscarinic and nicotinic cholinergic receptor antagonists, respectively, in the DRN of rats. Intraperitoneal injection of pentylenetetrazole (PTZ) (at 64mg/kg) caused tonic and tonic-clonic seizures. The convulsive motor reactions were followed by an increase in pain thresholds, a phenomenon known as post-ictal analgesia. Pre-treatment of the DRN with atropine or mecamylamine at 1µg, 3µg and 5µg/0.2µL decreased the post-ictal antinociceptive phenomenon. The present results showed that the post-ictal analgesia was mediated by muscarinic and nicotinic cholinergic receptors in the DRN, a structure crucially involved in the neural network that organises post-ictal hypoalgesia.

5-HT7 receptor modulates GABAergic transmission in the rat dorsal raphe nucleus and controls cortical release of serotonin.

The 5-HT7 receptor is one of the several serotonin (5-HT) receptor subtypes that are expressed in the dorsal raphe nucleus (DRN). Some earlier findings suggested that 5-HT7 receptors in the DRN were localized on GABAergic interneurons modulating the activity of 5-HT projection neurons. The aim of the present study was to find out how the 5-HT7 receptor modulates the GABAergic synaptic input to putative 5-HT DRN neurons, and whether blockade of the 5-HT7 receptor would affect the release of 5-HT in the target structure. Male Wistar rats with microdialysis probes implanted in the prefrontal cortex (PFC) received injections of the 5-HT7 receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB 269970), which induced an increase in the levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the PFC. In another set of experiments whole-cell recordings from presumed projection neurons were carried out using DRN slices. SB 269970 application resulted in depolarization and in an increase in the firing frequency of the cells. In order to activate 5-HT7 receptors, 5-carboxamidotryptamine (5-CT) was applied in the presence of N-[2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY100635). Hyperpolarization of cells and a decrease in the firing frequency were observed after activation of the 5-HT7 receptor. Blockade of 5-HT7 receptors caused a decrease in the mean frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), while its activation induced an increase. The mechanism of these effects appears to involve tonically-active 5-HT7 receptors modulating firing and/or GABA release from inhibitory interneurons which regulate the activity of DRN serotonergic projection neurons.

PKC in rat dorsal raphe nucleus plays a key role in sleep-wake regulation.

Studies suggest a tight relationship between protein kinase C (PKC) and circadian clock. However, the role of PKC in sleep-wake regulation remains unclear. The present study was conducted to investigate the role of PKC signaling in sleep-wake regulation in the rat. Our results showed that the phosphorylation level of PKC in dorsal raphe nucleus (DRN) was decreased after 6h sleep deprivation, while no alterations were found in ventrolateral preoptic nucleus (VLPO) or locus coeruleus (LC). Microinjection of a pan-PKC inhibitor, chelerythrine chloride (CHEL, 5 or 10nmol), into DRN of freely moving rats promoted non rapid eye movement sleep (NREMS) without influences on rapid eye movement sleep (REMS). Especially, CHEL application at 5nmol increased light sleep (LS) time while CHEL application at 10nmol increased slow wave sleep (SWS) time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased the phosphorylation level of PKC, but also reduced NREMS time, especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These data provide the first direct evidence that inhibition of intracellular PKC signaling in DRN could increase NREMS time including SWS time and percentage, while activation of PKC could suppress NREMS and reduce SWS time and percentage. These novel findings further our understanding of the basic cellular and molecular mechanisms of sleep-wake regulation.

Corticotropin-releasing Factor in the Rat Dorsal Raphe Nucleus Promotes Different Forms of Behavioral Flexibility Depending on Social Stress History.

The stress-related neuropeptide, corticotropin-releasing factor (CRF) regulates the dorsal raphe nucleus-serotonin (DRN-5-HT) system during stress and this may underlie affective and cognitive dysfunctions that characterize stress-related psychiatric disorders. CRF acts on both CRF1 and CRF2 receptor subtypes in the DRN that exert opposing inhibitory and excitatory effects on DRN-5-HT neuronal activity and 5-HT forebrain release, respectively. The current study first assessed the cognitive effects of intra-DRN microinfusion of CRF or the selective CRF2 agonist, urocortin II in stress-naive rats on performance of an operant strategy set-shifting task that is mediated by the medial prefrontal cortex (mPFC). CRF (30 ng) facilitated strategy set-shifting performance, whereas higher doses of CRF and urocortin II that would interact with CRF2 were without effect, consistent with a CRF1-mediated action. This dose decreased 5-HT extracellular levels in the mPFC, further supporting a role for CRF1. The effects of CRF were then assessed in rats exposed to repeated social stress using the resident-intruder model. Repeated social stress shifted the CRF effect from facilitation of strategy set shifting to facilitation of reversal learning and this was most prominent in a subpopulation of rats that resist defeat. Notably, in this subpopulation of rats 5-HT neuronal responses to CRF have been demonstrated to shift from CRF1-mediated inhibition to CRF2-mediated excitation. Because 5-HT facilitates reversal learning, the present results suggest that stress-induced changes in the cellular effects of CRF in the DRN translate to changes in cognitive effects of CRF. Together, the results underscore the potential for stress history to shift cognitive processing through changes in CRF neurotransmission in the DRN and the association of this effect with coping strategy.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus

Disorders caused by the malfunction of the serotonergic system in the central nervous system show sex-specific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX+low estradiol (E2), OVX+high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons.