Grafting Embryonic Raphe Neurons Reestablishes Serotonergic Regulation of Sympathetic Activity to Improve Cardiovascular Function after Spinal Cord Injury.

Cardiovascular dysfunction often occurs after high-level spinal cord injury. Disrupting supraspinal vasomotor pathways affects basal hemodynamics and contributes to the development of autonomic dysreflexia (AD). Transplantation of early-stage neurons to the injured cord may reconstruct the descending projections to enhance cardiovascular performance. To determine the specific role of reestablishing serotonergic regulation of hemodynamics, we implanted serotonergic (5-HT+) neuron-enriched embryonic raphe nucleus-derived neural stem cells/progenitors (RN-NSCs) into a complete spinal cord transection lesion site in adult female rats. Grafting embryonic spinal cord-derived NSCs or injury alone served as 2 controls. Ten weeks after injury/grafting, histological analysis revealed well-survived grafts and partial integration with host tissues in the lesion site. Numerous graft-derived serotonergic axons topographically projected to the caudal autonomic regions. Neuronal tracing showed that host supraspinal vasomotor pathways regenerated into the graft, and 5-HT+ neurons within graft and host brainstem neurons were transsynaptically labeled by injecting pseudorabies virus (PRV-614) into the kidney, indicating reconnected serotonergic circuits regulating autonomic activity. Using an implanted telemeter to record cardiovascular parameters, grafting RN-NSCs restored resting mean arterial pressure to normal levels and remarkably alleviated naturally occurring and colorectal distension-induced AD. Subsequent pharmacological blockade of 5-HT2A receptors with ketanserin in RN-NSC-grafted rats reduced resting mean arterial pressure and increased heart rate in all but 2 controls. Furthermore, spinal cord retransection below RN-NSC grafts partially eliminated the recovery in AD. Collectively, these data indicate that RN-NSCs grafted into a spinal cord injury site relay supraspinal control of serotonergic regulation for sympathetic activity to improve cardiovascular function.SIGNIFICANCE STATEMENT Disruption of supraspinal vasomotor pathways results in cardiovascular dysfunction following high-level spinal cord injury. To reestablish the descending regulation of autonomic function, we transplanted serotonergic neuron enriched embryonic raphe nucleus-derived neural stem cells/progenitors into the lesion site of completely transected rat spinal cord. Consequently, grafted raphe nucleus-derived neural stem cells/progenitors acted as a neuronal relay to reconnect supraspinal center and spinal sympathetic neurons below the injury. The reconstituted serotonergic regulation of sympathetic activity led to the improvement of hemodynamic parameters and mitigated autonomic dysreflexia. Based on morphological and physiological results, this study validates the effectiveness of transplanting early-stage serotonergic neurons into the spinal cord for cardiovascular functional recovery after spinal cord injury.

Cocaine Selectively Reorganizes Excitatory Inputs to Substantia Nigra Pars Compacta Dopamine Neurons.

Substantia nigra pars compacta (SNc) dopamine neurons and their targets are involved in addiction and cue-induced relapse. However, afferents onto SNc dopamine neurons themselves appear insensitive to drugs of abuse, such as cocaine, when afferents are collectively stimulated electrically. This contrasts with ventral tegmental area (VTA) dopamine neurons, whose glutamate afferents react robustly to cocaine. We used an optogenetic strategy to isolate identified SNc inputs and determine whether cocaine sensitivity in the mouse SNc circuit is conferred at the level of three glutamate afferents: dorsal raphé nucleus (DR), pedunculopontine nucleus (PPN), and subthalamic nucleus (STN). We found that excitatory afferents to SNc dopamine neurons are sensitive to cocaine in an afferent-specific manner. A single exposure to cocaine in vivo led to PPN-innervated synapses reducing the AMPA-to-NMDA receptor-mediated current ratio. In contrast to work in the VTA, this was due to increased NMDA receptor function with no change in AMPA receptor function. STN synapses showed a decrease in calcium-permeable AMPA receptors after cocaine, but no change in the AMPA-to-NMDA ratio. Cocaine also increased the release probability at DR-innervated and STN-innervated synapses, quantified by decreases in paired-pulse ratios. However, release probability at PPN-innervated synapses remained unaffected. By examining identified inputs, our results demonstrate a functional distribution among excitatory SNc afferent nuclei in response to cocaine, and suggest a compelling architecture for differentiation and separate parsing of inputs within the nigrostriatal system.SIGNIFICANCE STATEMENT Prior studies have established that substantia nigra pars compacta (SNc) dopamine neurons are a key node in the circuitry that drives addiction and relapse, yet cocaine apparently has no effect on electrically stimulated excitatory inputs. Our study is the first to demonstrate the functional impact of a drug of abuse on synaptic mechanisms of identified afferents to the SNc. Optogenetic dissection of inputs originating from dorsal raphé, pedunculopontine, and subthalamic nuclei were tested for synaptic modifications following in vivo cocaine exposure. Our results demonstrate that cocaine differentially induces modifications to SNc synapses depending on input origin. This presents implications for understanding dopamine processing of motivated behavior; most critically, it indicates that dopamine neurons selectively modulate signal reception processed by afferent nuclei.

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex.

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei.SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO2Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.

Cell-Type-Specific Modulation of Sensory Responses in Olfactory Bulb Circuits by Serotonergic Projections from the Raphe Nuclei.

UNLABELLED: Serotonergic neurons in the brainstem raphe nuclei densely innervate the olfactory bulb (OB), where they can modulate the initial representation and processing of olfactory information. Serotonergic modulation of sensory responses among defined OB cell types is poorly characterized in vivo Here, we used cell-type-specific expression of optical reporters to visualize how raphe stimulation alters sensory responses in two classes of GABAergic neurons of the mouse OB glomerular layer, periglomerular (PG) and short axon (SA) cells, as well as mitral/tufted (MT) cells carrying OB output to piriform cortex. In PG and SA cells, brief (1-4 s) raphe stimulation elicited a large increase in the magnitude of responses linked to inhalation of ambient air, as well as modest increases in the magnitude of odorant-evoked responses. Near-identical effects were observed when the optical reporter of glutamatergic transmission iGluSnFR was expressed in PG and SA cells, suggesting enhanced excitatory input to these neurons. In contrast, in MT cells imaged from the dorsal OB, raphe stimulation elicited a strong increase in resting GCaMP fluorescence with only a slight enhancement of inhalation-linked responses to odorant. Finally, optogenetically stimulating raphe serotonergic afferents in the OB had heterogeneous effects on presumptive MT cells recorded extracellularly, with an overall modest increase in resting and odorant-evoked responses during serotonergic afferent stimulation. These results suggest that serotonergic afferents from raphe dynamically modulate olfactory processing through distinct effects on multiple OB targets, and may alter the degree to which OB output is shaped by inhibition during behavior. SIGNIFICANCE STATEMENT: Modulation of the circuits that process sensory information can profoundly impact how information about the external world is represented and perceived. This study investigates how the serotonergic system modulates the initial processing of olfactory information by the olfactory bulb, an obligatory relay between sensory neurons and cortex. We find that serotonergic projections from the raphe nuclei to the olfactory bulb dramatically enhance the responses of two classes of inhibitory interneurons to sensory input, that this effect is mediated by increased glutamatergic drive onto these neurons, and that serotonergic afferent activation alters the responses of olfactory bulb output neurons in vivo These results elucidate pathways by which neuromodulatory systems can dynamically regulate brain circuits during behavior.

A transient placental source of serotonin for the fetal forebrain.

Serotonin (5-hydroxytryptamine or 5-HT) is thought to regulate neurodevelopmental processes through maternal-fetal interactions that have long-term mental health implications. It is thought that beyond fetal 5-HT neurons there are significant maternal contributions to fetal 5-HT during pregnancy but this has not been tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used Pet1(-/-) (also called Fev) mice in which most dorsal raphe neurons lack 5-HT. We detected previously unknown differences in accumulation of 5-HT between the forebrain and hindbrain during early and late fetal stages, through an exogenous source of 5-HT which is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source. We uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor in both mice and humans. This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and indicates that maternal-placental-fetal interactions could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.

Mechanisms of 5-HT1A receptor-mediated transmission in dorsal raphe serotonin neurons.

KEY POINTS: In the dorsal raphe nucleus, it is known that serotonin release activates metabotropic 5-HT1A autoreceptors located on serotonin neurons that leads to an inhibition of firing through the activation of G-protein-coupled inwardly rectifying potassium channels. We found that in mouse brain slices evoked serotonin release produced a 5-HT1A receptor-mediated inhibitory postsynaptic current (IPSC) that resulted in only a transient pause in firing. While spillover activation of receptors contributed to evoked IPSCs, serotonin reuptake transporters prevented pooling of serotonin in the extrasynaptic space from activating 5-HT1A -IPSCs. As a result, the decay of 5-HT1A -IPSCs was independent of the intensity of stimulation or the probability of transmitter release. These results indicate that evoked serotonin transmission in the dorsal raphe nucleus mediated by metabotropic 5-HT1A autoreceptors may occur via point-to-point synapses rather than by paracrine mechanisms. ABSTRACT: In the dorsal raphe nucleus (DRN), feedback activation by Gαi/o -coupled 5-HT1A autoreceptors reduces the excitability of serotoninergic neurons, which decreases serotonin release both locally within the DRN and in projection regions. Serotonin transmission within the DRN is thought to occur via transmitter spillover and paracrine activation of extrasynaptic receptors. Here, we tested the volume transmission hypothesis in mouse DRN brain slices by recording 5-HT1A receptor-mediated inhibitory postsynaptic currents (5-HT1A -IPSCs) generated by the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs). We found that in the DRN of ePET1-EYFP mice, which selectively express enhanced yellow fluorescent protein in serontonergic neurons, the local release of serotonin generated 5-HT1A -IPSCs in serotonin neurons that rose and fell within a second. The transient activation of 5-HT1A autoreceptors resulted in brief pauses in neuron firing that did not alter the overall firing rate. The duration of 5-HT1A -IPSCs was primarily shaped by receptor deactivation due to clearance via serotonin reuptake transporters. Slowing diffusion with dextran prolonged the rise and reduced the amplitude the IPSCs and the effects were potentiated when uptake was inhibited. By examining the decay kinetics of IPSCs, we found that while spillover may allow for the activation of extrasynaptic receptors, efficient uptake by serotonin reuptake transporters (SERTs) prevented the pooling of serotonin from prolonging the duration of transmission when multiple inputs were active. Together the results suggest that the activation of 5-HT1A receptors in the DRN results from the local release of serotonin rather than the extended diffusion throughout the extracellular space.

Presynaptic gating of excitation in the dorsal raphe nucleus by GABA.

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus.

[EXPRESSION OF SEROTONIN TRANSPORTER IN THE DORSAL RAPHE NUCLEUS DURING THE EARLY POSTNATAL PERIOD IN NORMAL STATE AND UNDER PRENATAL DEFICIENCY OF THE SEROTONERGIC SYSTEM IN RATS].

The expression of the serotonin transport membrane protein (5-NTT) in the dorsal raphe nucleus (DNR) was investigated in laboratory Wistar rats during the early postnatal period. The results of the immunocytochemical study using primary antibodies--anti-Serotonin transporter antibody (AbCam, UK)--showed that during the first 3 postnatal weeks the intensity of 5-NTT expression in DNR of control animals changes. At the earliest postnatal times the main part of subnuclear neurons (dorsal, ventral and lateral ones) of the dorsal raphe nucleus (DNR-d, DNR-v, DNR-lat) was shown to intensely express 5-NTT. Sites of 5-NTT localization are found on the membrane surface of neuron bodies and processes in neuropile. The reduction in the number of neurons expressing 5-NTT and of its binding sites was observed on P10. At this time a redistribution of 5-NTT localization sites occurs: they are very few on neuron bodies and dendrites but are located rather densely on the plasma membrane of axons. The number of neurons expressing 5-NTT gradually increases with age and in neuropile the density of 5-NTT localization sites rises. It is shown that during the prenatal development the reduction of serotonin level in all parts of the DNR leads to a reduction in both the number of neurons expressing 5-NTT and sites of its localization in the early postnatal period, this trend continuing with age.

Dorsal raphe serotonin neurons in mice: immature hyperexcitability transitions to adult state during first three postnatal weeks suggesting sensitive period for environmental perturbation.

Trauma during early life is a major risk factor for the development of anxiety disorders and suggests that the developing brain may be particularly sensitive to perturbation. Increased vulnerability most likely involves altering neural circuits involved in emotional regulation. The role of serotonin in emotional regulation is well established, but little is known about the postnatal development of the raphe where serotonin is made. Using whole-cell patch-clamp recording and immunohistochemistry, we tested whether serotonin circuitry in the dorsal and median raphe was functionally mature during the first 3 postnatal weeks in mice. Serotonin neurons at postnatal day 4 (P4) were hyperexcitable. The increased excitability was due to depolarized resting membrane potential, increased resistance, increased firing rate, lack of 5-HT1A autoreceptor response, and lack of GABA synaptic activity. Over the next 2 weeks, membrane resistance decreased and resting membrane potential hyperpolarized due in part to potassium current activation. The 5-HT1A autoreceptor-mediated inhibition did not develop until P21. The frequency of spontaneous inhibitory and excitatory events increased as neurons extended and refined their dendritic arbor. Serotonin colocalized with vGlut3 at P4 as in adulthood, suggesting enhanced release of glutamate alongside enhanced serotonin release. Because serotonin affects circuit development in other brain regions, altering the developmental trajectory of serotonin neuron excitability and release could have many downstream consequences. We conclude that serotonin neuron structure and function change substantially during the first 3 weeks of life during which external stressors could potentially alter circuit formation.

Dorsal raphe neuroinflammation promotes dramatic behavioral stress dysregulation.

Impulsivity, risk-taking behavior, and elevated stress responsivity are prominent symptoms of mania, a behavioral state common to schizophrenia and bipolar disorder. Though inflammatory processes activated within the brain are involved in the pathophysiology of both disorders, the specific mechanisms by which neuroinflammation drives manic behavior are not well understood. Serotonin cell bodies originating within the dorsal raphe (DR) play a major role in the regulation of behavioral features characteristic of mania. Therefore, we hypothesized that the link between neuroinflammation and manic behavior may be mediated by actions on serotonergic neurocircuitry. To examine this, we induced local neuroinflammation in the DR by viral delivery of Cre recombinase into interleukin (IL)-1ß(XAT) transgenic male and female mice, resulting in overexpressing of the proinflammatory cytokine, IL-1ß. For assertion of brain-region specificity of these outcomes, the prefrontal cortex (PFC), as a downstream target of DR serotonergic projections, was also infused. Inflammation within the DR, but not the PFC, resulted in a profound display of manic-like behavior, characterized by increased stress-induced locomotion and responsivity, and reduced risk-aversion/fearfulness. Microarray analysis of the DR revealed a dramatic increase in immune-related genes, and dysregulation of genes important in GABAergic, glutamatergic, and serotonergic neurotransmission. Behavioral and physiological changes were driven by a loss of serotonergic neurons and reduced output as measured by high-performance liquid chromatography, demonstrating inflammation-induced serotonergic hypofunction. Behavioral changes were rescued by acute selective serotonin reuptake inhibitor treatment, supporting the hypothesis that serotonin dysregulation stemming from neuroinflammation in the DR underlies manic-like behaviors.

Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes.

Neuronal connectivity between habenular glutamate-kisspeptin1 co-expressing neurons and the raphe 5-HT system.

The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.

[EXPRESSION OF SEROTONIN TRANSPORT PROTEIN IN THE DORSAL RAPHE NUCLEUS IN THE EARLY POSTNATAL PERIOD IN RATS].

In this work an expression of serotonin transport protein (5-HTT) was studied in the dorsal raphe nucleus (DRN)--in its dorsal, ventral and lateral subnuclei in Wistar rats (n = 15) during the early postnatal period. Histological methods and immunocytochemical staining demonstrating 5-HTT were used in the investigation. It was shown that at postnatal Day 5 major part of neurons of the subnuclei studied intensively expressed 5-HTT. However, by Day 10, the level of its expression decreased dramatically, but with age (by Day 20) the expression was increased once again. This was manifested by the augmentation of size of 5-HTT-positive neuronal population, the increase in the density of plexus network of their processes. The detected changes of 5-HTT expression indicate the varying degrees of functional activity of serotonin in the DRN in the early postnatal period.

Raphe GABAergic neurons mediate the acquisition of avoidance after social defeat.

Serotonin (5-HT) modulates neural responses to socioaffective cues and can bias approach or avoidance behavioral decisions, yet the cellular mechanisms underlying its contribution to the regulation of social experiences remain poorly understood. We hypothesized that GABAergic neurons in the dorsal raphe nucleus (DRN) may participate in socioaffective regulation by controlling serotonergic tone during social interaction. We tested this hypothesis using whole-cell recording techniques in genetically identified DRN GABA and 5-HT neurons in mice exposed to social defeat, a model that induces long-lasting avoidance behaviors in a subset of mice responsive to serotonergic antidepressants. Our results revealed that social defeat engaged DRN GABA neurons and drove GABAergic sensitization that strengthened inhibition of 5-HT neurons in mice that were susceptible, but not resilient to social defeat. Furthermore, optogenetic silencing of DRN GABA neurons disinhibited neighboring 5-HT neurons and prevented the acquisition of social avoidance in mice exposed to a social threat, but did not affect a previously acquired avoidance phenotype. We provide the first characterization of GABA neurons in the DRN that monosynaptically inhibit 5-HT neurons and reveal their key role in neuroplastic processes underlying the development of social avoidance.

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.

Suppression of serotonin neuron firing increases aggression in mice.

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

Serotonin 1A receptors alter expression of movement representations.

Serotonin has a myriad of central functions involving mood, appetite, sleep, and memory and while its release within the spinal cord is particularly important for generating movement, the corresponding role on cortical movement representations (motor maps) is unknown. Using adult rats we determined that pharmacological depletion of serotonin (5-HT) via intracerebroventricular administration of 5,7 dihydroxytryptamine resulted in altered movements of the forelimb in a skilled reaching task as well as higher movement thresholds and smaller maps derived using high-resolution intracortical microstimulation (ICMS). We ruled out the possibility that reduced spinal cord excitability could account for the serotonin depletion-induced changes as we observed an enhanced Hoffman reflex (H-reflex), indicating a hyperexcitable spinal cord. Motor maps derived in 5-HT1A receptor knock-out mice also showed higher movement thresholds and smaller maps compared with wild-type controls. Direct cortical application of the 5-HT1A/7 agonist 8-OH-DPAT lowered movement thresholds in vivo and increased map size in 5-HT-depleted rats. In rats, electrical stimulation of the dorsal raphe lowered movement thresholds and this effect could be blocked by direct cortical application of the 5-HT1A antagonist WAY-100135, indicating that serotonin is primarily acting through the 5-HT1A receptor. Next we developed a novel in vitro ICMS preparation that allowed us to track layer V pyramidal cell excitability. Bath application of WAY-100135 raised the ICMS current intensity to induce action potential firing whereas the agonist 8-OH-DPAT had the opposite effect. Together our results demonstrate that serotonin, acting through 5-HT1A receptors, plays an excitatory role in forelimb motor map expression.

Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling.

BACKGROUND: Genetic and environmental factors are critical elements influencing the etiology of major depression. It is now accepted that neuroinflammatory processes play a major role in neuropsychological disorders. Neuroinflammation results from the dysregulation of the synthesis and/or release of pro- and anti-inflammatory cytokines with central or peripheral origin after various insults. Systemic bacterial lipopolysaccharide (LPS) challenge is commonly used to study inflammation-induced depressive-like behaviors in rodents. In the present study, we investigated immune-to-brain communication in mice by examining the effects of peripheral LPS injection on neuroinflammation encompassing cytokine and chemokine production, microglia and central nervous system (CNS)-associated phagocyte activation, immune cell infiltration and serotonergic neuronal function. METHODS: LPS was administered to C57BL/6 J mice by intraperitoneal injection; brains were collected and pro-inflammatory cytokine mRNA and proteins were measured. To examine the relative contribution of the different populations of brain immune cells to the occurrence of neuroinflammation after acute systemic inflammation, we precisely characterized them by flow cytometry, studied changes in their proportions and level of activation, and measured the amount of cytokines they released by Cytometric Bead Array™ after cell sorting and ex vivo culture. Because of the central role that the chemokine CCL2 seems to play in our paradigm, we studied the effect of CCL2 on the activity of serotonergic neurons of the raphe nucleus using electrophysiological recordings. RESULTS: We report that systemic LPS administration in mice caused a marked increase in pro-inflammatory IL-1ß, IL-6, TNFα and CCL2 (monocyte chemoattractant protein-1) mRNA and protein levels in the brain. Moreover, we found that LPS caused microglia and CNS-associated phagocyte activation characterized by upregulation of CCR2, TLR4/CD14, CD80 and IL-4Rα, associated with overproduction of pro-inflammatory cytokines and chemokines, especially CCL2. LPS also induced a marked and selective increase of CCR2(+) inflammatory monocytes within the brain. Finally, we showed that CCL2 hyperpolarized serotonergic raphe neurons in mouse midbrain slices, thus probably reducing the serotonin tone in projection areas. CONCLUSION: Together, we provide a detailed characterization of the molecular and cellular players involved in the establishment of neuroinflammation after systemic injection of LPS. This highlights the importance of the CCL2/CCR2 signaling and suggests a possible link with depressive disorders.

Involvement of estrogen receptor ß in maintenance of serotonergic neurons of the dorsal raphe.

The serotonergic neurons of the dorsal raphe (DR) nucleus in the CNS are involved in fear, anxiety and depression. Depression and anxiety occur quite frequently in postmenopausal women, but estrogen replacement to correct these CNS disorders is at present not favored because estrogen carries with it an increased risk for breast cancer. Serotonin synthesis, release and reuptake in the DR are targets of pharmaceuticals in the treatment of depression. In the present study we have examined by immunohistochemistry, the expression of two nuclear receptors, that is, the estrogen receptors ERα and ERß. We found that ERß but not ERα is strongly expressed in the DR and there is no sex difference and no change with ageing in the number of tryptophan hydroxylase (TPH)-positive neurons in the DR of wild-type (WT) mice. However, in ovariectomized (OVX) WT and in ERß(-/-) mice, there was a marked reduction in the number of TPH-positive normal-looking neurons and a marked increase in TPH-positive spindle-shaped cells. These neuronal changes were prevented in mice 1-3 weeks (but not 10 weeks) after OVX by the selective ERß agonist, LY3201, given as continuous release pellets for 3 days. The ERß agonist had no effects on glucose homeostasis. Thus, the onset of action of the ERß agonist is rapid but there is a limited window in time after estrogen loss when the drug is useful. We conclude that, rather than estradiol, ERß agonists could be useful pharmaceuticals in maintaining functional DR neurons to treat postmenopausal depression.

The dorsal raphe modulates sensory responsiveness during arousal in zebrafish.

During waking behavior, animals adapt their state of arousal in response to environmental pressures. Sensory processing is regulated in aroused states, and several lines of evidence imply that this is mediated at least partly by the serotonergic system. However, there is little information directly showing that serotonergic function is required for state-dependent modulation of sensory processing. Here we find that zebrafish larvae can maintain a short-term state of arousal during which neurons in the dorsal raphe modulate sensory responsiveness to behaviorally relevant visual cues. After a brief exposure to water flow, larvae show elevated activity and heightened sensitivity to perceived motion. Calcium imaging of neuronal activity after flow revealed increased activity in serotonergic neurons of the dorsal raphe. Genetic ablation of these neurons abolished the increase in visual sensitivity during arousal without affecting baseline visual function or locomotor activity. We traced projections from the dorsal raphe to a major visual area, the optic tectum. Laser ablation of the tectum demonstrated that this structure, like the dorsal raphe, is required for improved visual sensitivity during arousal. These findings reveal that serotonergic neurons of the dorsal raphe have a state-dependent role in matching sensory responsiveness to behavioral context.