Neurons In Dorsal Raphe Nucleus Research Articles

Dorsal Raphe Neurons Signal Reward through 5-HT and Glutamate

The dorsal raphe nucleus (DRN) in the midbrain is a key center for serotonin (5-hydroxytryptamine; 5-HT)-expressing neurons. Serotonergic neurons in the DRN have been theorized to encode punishment by opposing the reward signaling of dopamine neurons. Here, we show that DRN neurons encode reward, but not punishment, through 5-HT and glutamate. Optogenetic stimulation of DRN Pet-1 neurons reinforces mice to explore the stimulation-coupled spatial region, shifts sucrose preference, drives optical self-stimulation, and directs sensory discrimination learning. DRN Pet-1 neurons increase their firing activity during reward tasks, and this activation can be used to rapidly change neuronal activity patterns in the cortex. Although DRN Pet-1 neurons are often associated with 5-HT, they also release glutamate, and both neurotransmitters contribute to reward signaling. These experiments demonstrate the ability of DRN neurons to organize reward behaviors and might provide insights into the underlying mechanisms of learning facilitation and anhedonia treatment.

Neural Circuit Interactions between the Dorsal Raphe Nucleus and the Lateral Hypothalamus: An Experimental and Computational Study

Orexinergic/hypocretinergic (Ox) neurotransmission plays an important role in regulating sleep, as well as in anxiety and depression, for which the serotonergic (5-HT) system is also involved in. However, little is known regarding the direct and indirect interactions between 5-HT in the dorsal raphe nucleus (DRN) and Ox neurons in the lateral hypothalamus (LHA). In this study, we report the additional presence of 5-HT1BR, 5-HT2AR, 5-HT2CR and fast ligand-gated 5-HT3AR subtypes on the Ox neurons of transgenic Ox-enhanced green fluorescent protein (Ox-EGFP) and wild type C57Bl/6 mice using single and double immunofluorescence (IF) staining, respectively, and quantify the colocalization for each 5-HT receptor subtype. We further reveal the presence of 5-HT3AR and 5-HT1AR on GABAergic neurons in LHA. We also identify NMDAR1, OX1R and OX2R on Ox neurons, but none on adjacent GABAergic neurons. This suggests a one-way relationship between LHA’s GABAergic and Ox neurons, wherein GABAergic neurons exerts an inhibitory effect on Ox neurons under partial DRN’s 5-HT control. We also show that Ox axonal projections receive glutamatergic (PSD-95 immunopositive) and GABAergic (Gephyrin immunopositive) inputs in the DRN. We consider these and other available findings into our computational model to explore possible effects of neural circuit connection types and timescales on the DRN-LHA system’s dynamics. We find that if the connections from 5-HT to LHA’s GABAergic neurons are weakly excitatory or inhibitory, the network exhibits slow oscillations; not observed when the connection is strongly excitatory. Furthermore, if Ox directly excites 5-HT neurons at a fast timescale, phasic Ox activation can lead to an increase in 5-HT activity; no significant effect with slower timescale. Overall, our experimental and computational approaches provide insights towards a more complete understanding of the complex relationship between 5-HT in the DRN and Ox in the LHA.

Distribution of VGLUT3 in Highly Collateralized Axons from the Rat Dorsal Raphe Nucleus as Revealed by Single-Neuron Reconstructions

This study aimed at providing the first detailed morphological description, at the single-cell level, of the rat dorsal raphe nucleus neurons, including the distribution of the VGLUT3 protein within their axons. Electrophysiological guidance procedures were used to label dorsal raphe nucleus neurons with biotinylated dextran amine. The somatodendritic and axonal arborization domains of labeled neurons were reconstructed entirely from serial sagittal sections using a computerized image analysis system. Under anaesthesia, dorsal raphe nucleus neurons display highly regular (1.72±0.50 Hz) spontaneous firing patterns. They have a medium size cell body (9.8±1.7 µm) with 2–4 primary dendrites mainly oriented anteroposteriorly. The ascending axons of dorsal raphe nucleus are all highly collateralized and widely distributed (total axonal length up to 18.7 cm), so that they can contact, in various combinations, forebrain structures as diverse as the striatum, the prefrontal cortex and the amygdala. Their morphological features and VGLUT3 content vary significantly according to their target sites. For example, high-resolution confocal analysis of the distribution of VGLUT3 within individually labeled-axons reveals that serotonin axon varicosities displaying VGLUT3 are larger (0.74±0.03 µm) than those devoid of this protein (0.55±0.03 µm). Furthermore, the percentage of axon varicosities that contain VGLUT3 is higher in the striatum (93%) than in the motor cortex (75%), suggesting that a complex trafficking mechanism of the VGLUT3 protein is at play within highly collateralized axons of the dorsal raphe nucleus neurons. Our results provide the first direct evidence that the dorsal raphe nucleus ascending projections are composed of widely distributed neuronal systems, whose capacity to co-release serotonin and glutamate varies from one forebrain locus to the other.

Neurobiologic Mechanisms of Sleep and Wakefulness

Neurobiologic Mechanisms of Sleep and Wakefulness

Analysis of sleep disorders under pain using an optogenetic tool: possible involvement of the activation of dorsal raphe nucleus-serotonergic neurons

BackgroundSeveral etiological reports have shown that chronic pain significantly interferes with sleep. Inadequate sleep due to chronic pain may contribute to the stressful negative consequences of living with pain. However, the neurophysiological mechanism by which chronic pain affects sleep-arousal patterns is as yet unknown. Although serotonin (5-HT) was proposed to be responsible for sleep regulation, whether the activity of 5-HTergic neurons in the dorsal raphe nucleus (DRN) is affected by chronic pain has been studied only infrequently. On the other hand, the recent development of optogenetic tools has provided a valuable opportunity to regulate the activity in genetically targeted neural populations with high spatial and temporal precision. In the present study, we investigated whether chronic pain could induce sleep dysregulation while changing the activity of DRN-5-HTergic neurons. Furthermore, we sought to physiologically activate the DRN with channelrhodopsin-2 (ChR2) to identify a causal role for the DRN-5-HT system in promoting and maintaining wakefulness using optogenetics.ResultsWe produced a sciatic nerve ligation model by tying a tight ligature around approximately one-third to one-half the diameter of the sciatic nerve. In mice with nerve ligation, we confirmed an increase in wakefulness and a decrease in non-rapid eye movement (NREM) sleep as monitored by electroencephalogram (EEG). Microinjection of the retrograde tracer fluoro-gold (FG) into the prefrontal cortex (PFC) revealed several retrogradely labeled-cells in the DRN. The key finding of the present study was that the levels of 5-HT released in the PFC by the electrical stimulation of DRN neurons were significantly increased in mice with sciatic nerve ligation. Using optogenetic tools in mice, we found a causal relationship among DRN neuron firing, cortical activity and sleep-to-wake transitions. In particular, the activation of DRN-5-HTergic neurons produced a significant increase in wakefulness and a significant decrease in NREM sleep. The duration of NREM sleep episodes was significantly decreased during photostimulation in these mice.ConclusionsThese results suggest that neuropathic pain accelerates the activity of DRN-5-HTergic neurons. Although further loss-of-function experiments are required, we hypothesize that this activation in DRN neurons may, at least in part, correlate with sleep dysregulation under a neuropathic pain-like state.

Body Sodium Overload Modulates the Firing Rate and Fos Immunoreactivity of Serotonergic Cells of Dorsal Raphe Nucleus

In order to determine whether serotonergic (5HT) dorsal raphe nucleus (DRN) cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT) plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO), animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the “in vivo” electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an “in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, possibly to modulate sodium and water intake/excretion and avoid extracellular volume expansion.

Prior cold water swim stress alters immobility in the forced swim test and associated activation of serotonergic neurons in the rat dorsal raphe nucleus

Prior adverse experience alters behavioral responses to subsequent stressors. For example, exposure to a brief swim increases immobility in a subsequent swim test 24h later. In order to determine if qualitative differences (e.g. 19°C versus 25°C) in an initial stressor (15-min swim) impact behavioral, physiological, and associated neural responses in a 5-min, 25°C swim test 24h later, rats were surgically implanted with biotelemetry devices 1week prior to experimentation then randomly assigned to one of six conditions (Day 1 (15min)/Day 2 (5min)): (1) home cage (HC)/HC, (2) HC/25°C swim, (3) 19°C swim/HC, (4) 19°C swim/25°C swim, (5) 25°C swim/HC, (6) 25°C swim/25°C swim. Core body temperature (Tb) was measured on Days 1 and 2 using biotelemetry; behavior was measured on Day 2. Rats were transcardially perfused with fixative 2h following the onset of the swim on Day 2 for analysis of c-Fos expression in midbrain serotonergic neurons. Cold water (19°C) swim on Day 1 reduced Tb, compared to both 25°C swim and HC groups on Day 1, and, relative to rats exposed to HC conditions on Day 1, reduced the hypothermic response to the 25°C swim on Day 2. The 19°C swim on Day 1, relative to HC exposure on Day 1, increased immobility during the 5-min swim on Day 2. Also, 19°C swim, relative to HC conditions, on Day 1 reduced swim (25°C)-induced increases in c-Fos expression in serotonergic neurons within the dorsal and interfascicular parts of the dorsal raphe nucleus. These results suggest that exposure to a 5-min 19°C cold water swim, but not exposure to a 5-min 25°C swim alters physiological, behavioral and serotonergic responses to a subsequent stressor.

Electrophysiological evidence for rapid 5-HT1A autoreceptor inhibition by vilazodone, a 5-HT1A receptor partial agonist and 5-HT reuptake inhibitor

This study examined the effect of vilazodone, a combined serotonin (5-HT) reuptake inhibitor and 5-HT1A receptor partial agonist, paroxetine and fluoxetine on the sensitivity of 5-HT1A autoreceptors of serotonergic dorsal raphe nucleus neurons in rats. These effects were assessed by determining the intravenous dose of (±)-8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) required to suppress the basal firing rate of these neurons by 50% (ID50) in anesthetized rats using in vivo electrophysiology. 5-HT uptake inhibition was determined by the ability of the compounds to reverse (±)-p-chloroamphetamine (PCA)-induced rat hypothalamic 5-HT depletion ex vivo. Acute vilazodone administration (0.63 and 2.1µmol/kg, s.c.), compared with vehicle, significantly increased (2–3-fold) the ID50 of 8-OH-DPAT at 4h, but not 24h after administration. Subchronic administration (3 days) significantly increased the ID50 value at 4h (3–4-fold) and at 24h (~2-fold). In contrast, paroxetine and fluoxetine at doses that were supramaximal for 5-HT uptake inhibition did not significantly alter the ID50 value of 8-OH-DPAT after acute or subchronic administration. Vilazodone antagonized the action of PCA 3.5h and 5h after a single dose (ID50 1.49 and 0.46µmol/kg, s.c., respectively), but was inactive 18h post-administration, corroborating the electrophysiological results at 24h following acute administration. The results are consistent with the concept of rapid and, following repeated treatment, prolonged inhibition of 5-HT1A autoreceptors by vilazodone. This effect could occur by either direct interaction with, or desensitization of, these receptors, an effect which cannot be ascribed to vilazodone's 5-HT reuptake inhibiting properties.

Infusion of galanin into the mid-caudal portion of the dorsal raphe nucleus has an anxiolytic effect on rats in the elevated T-maze

Galanin and 5-HT coexist in dorsal raphe nucleus (DRN) neurons. Microinjection of galanin into the DRN reduces the firing rate of serotonin neurons. Serotonergic neurons projecting from the DRN to the amygdala facilitate learned anxiety producing an anxiogenic effect, while those projecting from the periaqueductal grey affect innate anxiety producing a panicolytic effect. We tested the hypothesis that injection of galanin into rat DRN would induce anxiolytic/panicogenic effects in the elevated T-maze (ETM), a model that allows for the evaluation of both of these effects. Galanin infusion into the mid-caudal DRN, but not into the rostral DRN, impaired inhibitory avoidance, suggesting an anxiolytic effect. The effective dose of galanin (0.3nmol) did not modify locomotor activity in the open field. Contrary to expectations, microinjection of galanin into the DRN did not facilitate the latency of one-way escape in the ETM. Pretreatment with a galanin antagonist, M40, attenuated galanin-induced impairment of inhibitory avoidance. The results show that microinjection of a low dose of galanin only into the mid-caudal DRN has an anxiolytic effect. This effect seems to be mediated, at least in part, by galanin receptors. Further investigation is necessary to identify the receptor subtypes and the DRN subregion involved in the anxiolytic effect of galanin.

Acute and subchronic treatments with selective serotonin reuptake inhibitors increase Nociceptin/Orphanin FQ (NOP) receptor density in the rat dorsal raphe nucleus; interactions between nociceptin/NOP system and serotonin

Nociceptin/Orphanin FQ is the endogenous ligand of NOP receptor, formerly referred to as the Opioid Receptor-Like 1 receptor. We have previously shown that NOP receptors were located on serotonergic neurons in the rat dorsal raphe nucleus, suggesting possible direct interactions between nociceptin and serotonin in this region, which is a target for antidepressant action. In the present study, we investigated further the link between Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant treatments and the nociceptin/NOP receptor system. Intraperitoneal administration of the SSRI citalopram induced an increase in NOP-receptor density, measured by autoradiographic [3H] nociceptin binding, in the rat dorsal raphe nucleus, from the first to the 21st day of treatment. This effect was also observed with other SSRIs (sertraline, fluoxetine), but not with two tricyclic antidepressants (imipramine, clomipramine) and was abolished by pre-treatment with para-chlorophenylalanine, an inhibitor of serotonin synthesis. Using microdialysis experiments, we demonstrated that NOP-receptor activation by infusion of nociceptin 10−6M or 10−5M increased the level of extracellular serotonin in the dorsal raphe nucleus. This effect was abolished by co-infusion of the NOP-receptor antagonist UFP 101. These results confirm the existence of reciprocal interactions between serotonin and nociceptin/NOP transmissions in the dorsal raphe nucleus.

Identification and distribution of projections from monoaminergic and cholinergic nuclei to functionally differentiated subregions of prefrontal cortex

The prefrontal cortex (PFC) is implicated in a variety of cognitive and executive functions and is composed of several distinct networks, including anterior cingulate cortex (ACC), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC). These regions serve dissociable cognitive functions, and are heavily innervated by acetylcholine, dopamine, serotonin and norepinephrine systems. In this study, fluorescently labeled retrograde tracers were injected into the ACC, mPFC, and OFC, and labeled cells were identified in the nucleus basalis (NB), ventral tegmental area (VTA), dorsal raphe nucleus (DRN) and locus coeruleus (LC). DRN and LC showed similar distributions of retrogradely labeled neurons such that most were single labeled and the largest population projected to mPFC. VTA showed a slightly greater proportion of double and triple labeled neurons, with the largest population projecting to OFC. NB, on the other hand, showed mostly double and triple labeled neurons projecting to multiple subregions. Therefore, subsets of VTA, DRN and LC neurons may be capable of modulating individual prefrontal subregions independently, whereas NB cells may exert a more unified influence on the three areas simultaneously. These findings emphasize the unique aspects of the cholinergic and monoaminergic projections to functionally and anatomically distinct subregions of PFC.

Association with reward negatively modulates short latency phasic conditioned responses of dorsal raphe nucleus neurons in freely moving rats.

The dorsal raphe nucleus (DRN) is implicated in mood regulation, control of impulsive behavior, and in processing aversive and reward-related signals. DRN neurons show phasic responses to sensory stimuli, but whether association with reward modulates these responses is unknown. We recorded DRN neurons from rats in a contextual conditioned approach paradigm in which an auditory cue was either followed or not followed by reward, depending on a global context signal. Conditioned approach (licking) occurred after cues in the reward context, but was suppressed in the no-reward context. Many DRN neurons showed short-latency phasic activations in response to the cues. There was striking contextual bias, with more and stronger excitations in the no-reward context than in the reward context. Therefore, DRN activity scaled inversely with cue salience and with the probability of subsequent conditioned approach. Tonic changes were similarly discriminatory, with increases being dominant after cues in the no-reward context, when licking was suppressed, and tonic decreases in rate dominant after reward-predictive cues during expression of conditioned licking. Phasic and tonic DRN responses thus provide signals of consistent valence but over different timescales. The tonic changes in activity are consistent with previous data and hypotheses relating DRN activity to response suppression and impulse control. Phasic responses could contribute to this via online modulation of attention allocation through projections to sensory-processing regions.

Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus

Cholinergic signaling mediated by nicotinic receptors has been associated to a large number of physiological and behavioral processes such as learning, memory, attention, food-intake and mood disorders. Although it is well established that many nicotinic actions are mediated through an increase in serotonin (5-HT) release, the physiological mechanisms by which nicotine produces these effects are still unclear. The dorsal raphe nucleus (DRN) contains the major amount of 5-HT neurons projecting to different parts of the brain. DRN also contains nicotinic acetylcholine receptors (nAChRs) located at somatic and presynaptic elements. Nicotine produces both inhibitory and excitatory effects on different subpopulations of 5-HT DRN neurons. In this review, we describe the presynaptic and postsynaptic mechanisms by which nicotine increases the excitability of DRN neurons as well as the subtypes of nAChRs involved. We also describe the inhibitory effects of nicotine and the role of 5-HT1A receptors in this effect. These nicotinic actions modulate the activity of different neuronal subpopulations in the DRN, changing the 5-HT tone in the brain areas where these groups of neurons project. Some of the physiological implications of nicotine-induced 5-HT release are discussed.

Serotonin and norepinephrine depletions can promote anxiety and depression in a rat model of Parkinson�s disease: an electrophysiological and behavioural study.

Event Abstract Back to Event Serotonin and norepinephrine depletions can promote anxiety and depression in a rat model of Parkinson’s disease: an electrophysiological and behavioural study. Claire Delaville1*, Jonathan Chetrit2, Sylvia Navailles2, Philippe De Deurwaerdère2 and Abdelhamid Benazzouz2 1 National Institutes of Health, NINDS, United States 2 CNRS, Neurodegenerative Diseases Institute UMR 5293, France The loss of dopamine (DA) neurons has been the pathophysiological focus of the devastating motor conditions of Parkinson’s disease (PD). Beyond DA, PD is a multi-system disorder characterized also by the loss of serotonin (5-HT) neurons from the dorsal raphe nucleus and norepinephrine (NE) neurons from the locus coeruleus. 5-HT and NE are widely recognized in the development of depression and anxiety and both symptoms are reported with a high prevalence in PD patients. However, a specific role for each neurotransmitter in the pathophysiology of PD is not clearly determined. Here, we investigated, in rats, the respective influence of DA, 5-HT and NE depletions on motor and non-motor behaviors as well as on the neuronal activity measured in vivo by single cell extracellular recordings in subthalamic (STN), globus pallidus (GP) and substantia nigra pars reticulata (SNr). DA, 5-HT and NE depletions were achieved by using classic protocols with 6-hydroxydopamine/desipramine, parachlorophenylalanine and DSP-4, respectively. We showed that NE or DA, but not 5-HT depletion significantly decreased locomotor activity and enhanced the proportion of bursty and irregular STN neurons. Anxiety-like states required DA depletion plus the depletion of 5-HT or NE. Anhedonia and “depressive-like” behavior emerged only from the combined depletion of all three monoamines, an effect paralleled by an increase in the firing rate and the proportion of bursty and irregular STN neurons. As for the STN, DA depletion increased the proportion of bursty neurons in GP and SNr. 5-HT, but not NE depletion modified GP and SNr neuronal activity. Thus, our data show that 5-HT and NE modulate specifically the basal ganglia activity and provides evidence for the exacerbation of behavioral deficits when 5-HT and/or NE depletions are combined with DA depletion. These data bring up new insight into the influence of 5-HT system in non-motor symptoms of PD. Keywords: Parkinson Disease, Norepinephrine, Serotonin, non-motor symptoms, Motor Activity Conference: 4th Conference of the Mediterrarnean Neuroscience Society, Istanbul, Turkey, 30 Sep - 3 Oct, 2012. Presentation Type: Symposium Topic: Abstracts Citation: Delaville C, Chetrit J, Navailles S, De Deurwaerdère P and Benazzouz A (2013). Serotonin and norepinephrine depletions can promote anxiety and depression in a rat model of Parkinson’s disease: an electrophysiological and behavioural study.. Conference Abstract: 4th Conference of the Mediterrarnean Neuroscience Society. doi: 10.3389/conf.fnhum.2013.210.00022 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jan 2013; Published Online: 11 Apr 2013. * Correspondence: Dr. Claire Delaville, National Institutes of Health, NINDS, Bethesda, Maryland, MD 20892-3702, United States, cdelaville@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claire Delaville Jonathan Chetrit Sylvia Navailles Philippe De Deurwaerdère Abdelhamid Benazzouz Google Claire Delaville Jonathan Chetrit Sylvia Navailles Philippe De Deurwaerdère Abdelhamid Benazzouz Google Scholar Claire Delaville Jonathan Chetrit Sylvia Navailles Philippe De Deurwaerdère Abdelhamid Benazzouz PubMed Claire Delaville Jonathan Chetrit Sylvia Navailles Philippe De Deurwaerdère Abdelhamid Benazzouz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Dorsal raphe neuronal activities are modulated by methylphenidate.

This study investigated the electrophysiological properties of the dorsal raphe nucleus (DR) neurons in response to the acute and repetitive administration of methylphenidate (MPH). Activities of DR neurons were recorded from non-anesthetized, freely behaving rats previously implanted bilaterally with permanent semi microelectrodes. The main findings were: (1) after initial (acute) administration of MPH (2.5mg/kg i.p.) on experimental day one (ED1), 56% of DR units significantly changed their firing rates. The majority of the responsive units (88%) exhibited increased firing rate; (2) daily MPH injections were given on ED2 through ED6 followed by 3 washout days. On ED10, 83% of the DR units significantly changed their baseline activity compared to the baseline activity on ED1; (3) after rechallenge MPH administration on ED10, 63% of DR units exhibited significant change in their firing rate; the majority of the responsive units (76%) exhibited a significant increase in their firing rate; (4) The effect of rechallenge MPH administration on ED10 was compared to the effect of initial MPH on ED1, 47% DR units exhibited a further significant increase in their firing rate while 53% DR units exhibited decrease or non-change in their firing rate which can be interpreted as electrophysiological sensitization or tolerance. In conclusion, this study demonstrated that acute MPH administration modulated the DR neuronal activities. Repetitive MPH administration modulated the baseline activities of DR units and elicited neurophysiological sensitization or tolerance. The results indicated that MPH affects DR neuronal activity.

Single-prolonged stress induces apoptosis in dorsal raphe nucleus in the rat model of posttraumatic stress disorder.

IntroductionPost-traumatic stress disorder (PTSD) is an anxiety disorder that develops after exposure to a life-threatening traumatic experience. Meta-analyses of the brainstem showed that midsagittal area of the pons was significantly reduced in patients with PTSD, suggesting a potential apoptosis in dorsal raphe nucleus after single-prolonged stress (SPS). The aim of this study is to investigate whether SPS induces apoptosis in dorsal raphe nucleus in PTSD rats, which may be a possible mechanism of reduced volume of pons and density of gray matter.MethodsIn this study, rats were randomly divided into 1d, 7d and 14d groups after SPS along with the control group. The apoptosis rate was determined using annexin V-FITC/PI double-labeled flow cytometry (FCM). Levels of Cytochrome c (Cyt-C) was examined by Western blotting. Expression of Cyt-C on mitochondria in the dorsal raphe nucleus neuron was determined by enzymohistochemistry under transmission electron microscopy (TEM). The change of thiamine monophosphatase (TMP) levels was assessed by enzymohistochemistry under light microscope and TEM. Morphological changes of the ultrastructure of the dorsal raphe nucleus neuron were determined by TEM.ResultsApoptotic morphological alterations were observed in dorsal raphe nucleus neuron for all SPS-stimulate groups of rats. The apoptosis rates were significantly increased in dorsal raphe nucleus neuron of SPS rats, along with increased release of cytochrome c from the mitochondria into the cytoplasm, increased expression of Cyt-C and TMP levels in the cytoplasm, which reached to the peak of increase 7 days of SPS.ConclusionsThe results indicate that SPS induced Cyt-C released from mitochondria into cytosol and apoptosis in dorsal raphe nucleus neuron of rats. Increased TMP in cytoplasm facilitated the clearance of apoptotic cells. We propose that this presents one of the mechanisms that lead to reduced volume of pons and gray matter associated with PTSD.

Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

Distinct Neurochemical and Functional Properties of GAD67-Containing 5-HT Neurons in the Rat Dorsal Raphe Nucleus

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.

Impaired Chemosensitivity of Mouse Dorsal Raphe Serotonergic Neurons Overexpressing Serotonin 1A (Htr1a) Receptors

BackgroundSerotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1aRO mice). Htr1aRO mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1aRO mice.Principal findingsLoose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1aRO mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO2 produced an increase in firing rate of serotonergic neurons (0.260±0.041 Hz, n = 20, p = 0.0001) and application of 3% CO2 decreased their firing rate (−0.142±0.025 Hz, n = 17, p = 0.0008). In contrast, in Htr1aRO mice, firing rate of serotonergic neurons was not significantly changed by 9% CO2 (0.021±0.034 Hz, n = 16, p = 0.49) and by 3% CO2 (0.012±0.046 Hz, n = 12, p = 0.97).ConclusionsOur findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.

Repeated Stress Dysregulates κ-Opioid Receptor Signaling in the Dorsal Raphe through a p38α MAPK-Dependent Mechanism

Repeated stress releases dynorphins and causes subsequent activation of κ-opioid receptors (KORs) in limbic brain regions. The serotonergic dorsal raphe nucleus (DRN) has previously been found to be an important site of action for the dysphoric effects of dynorphin-κ-opioid receptor system activation during stress-evoked behaviors, and KOR-induced activation of p38α mitogen-activated protein kinase (MAPK) in serotonergic neurons was found to be a critical mediator of the aversive properties of stress. Yet, how dynorphins and KORs functionally regulate the excitability of serotonergic DRN neurons both in adaptive and pathological stress states is poorly understood. Here we report that acute KOR activation by the selective agonist U69,593 [(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide] inhibits serotonergic neuronal excitability within the DRN through both presynaptic inhibition of excitatory synaptic transmission and postsynaptic activation of G-protein-gated inwardly rectifying potassium channels (GIRKs) electrophysiologically recorded in brain slices. C57BL/6 mice subjected to repeated swim, stress sessions had significantly reduced KOR-mediated GIRK currents recorded in serotonergic neurons in DRN postsynaptically, without significantly affecting presynaptic KOR-mediated regulation of excitatory transmission. This effect was blocked by genetic excision of p38α MAPK selectively from serotonergic neurons. An increase in phospho-immunoreactivity suggests that this functional dysregulation may be a consequence of tyrosine phosphorylation of GIRK (K(IR)3.1) channels. These data elucidate a mechanism for stress-induced dysregulation of the excitability of neurons in the DRN and identify a functional target of stress-induced p38α MAPK activation that may underlie some of the negative effects of pathological stress exposure.