The Role of Vesicular Glutamate Transporter Type 3 in Social Behavior, with a Focus on the Median Raphe Region.

Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24 h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance.

The Dopaminergic Cells in the Median Raphe Region Regulate Social Behavior in Male Mice.

According to previous studies, the median raphe region (MRR) is known to contribute significantly to social behavior. Besides serotonin, there have also been reports of a small population of dopaminergic neurons in this region. Dopamine is linked to reward and locomotion, but very little is known about its role in the MRR. To address that, we first confirmed the presence of dopaminergic cells in the MRR of mice (immunohistochemistry, RT-PCR), and then also in humans (RT-PCR) using healthy donor samples to prove translational relevance. Next, we used chemogenetic technology in mice containing the Cre enzyme under the promoter of the dopamine transporter. With the help of an adeno-associated virus, designer receptors exclusively activated by designer drugs (DREADDs) were expressed in the dopaminergic cells of the MRR to manipulate their activity. Four weeks later, we performed an extensive behavioral characterization 30 min after the injection of the artificial ligand (Clozapine-N-Oxide). Stimulation of the dopaminergic cells in the MRR decreased social interest without influencing aggression and with an increase in social discrimination. Additionally, inhibition of the same cells increased the friendly social behavior during social interaction test. No behavioral changes were detected in anxiety, memory or locomotion. All in all, dopaminergic cells were present in both the mouse and human samples from the MRR, and the manipulation of the dopaminergic neurons in the MRR elicited a specific social response.

Long-Term Impact of Early-Life Stress on Serotonin Connectivity.

BACKGROUND: Chronic childhood stress is a prominent risk factor for developing affective disorders, yet mechanisms underlying this association remain unclear. Maintenance of optimal serotonin (5-HT) levels during early postnatal development is critical for the maturation of brain circuits. Understanding the long-lasting effects of early-life stress (ELS) on serotonin-modulated brain connectivity is crucial to develop treatments for affective disorders arising from childhood stress. METHODS: Using a mouse model of chronic developmental stress, we determined the long-lasting consequences of ELS on 5-HT circuits and behavior in females and males. Using FosTRAP mice, we cross-correlated regional c-Fos density to determine brain-wide functional connectivity of the raphe nucleus. We next performed in vivo fiber photometry to establish ELS-induced deficits in 5-HT dynamics and optogenetics to stimulate 5-HT release to improve behavior. RESULTS: Adult female and male mice exposed to ELS showed heightened anxiety-like behavior. ELS further enhanced susceptibility to acute stress by disrupting the brain-wide functional connectivity of the raphe nucleus and the activity of 5-HT neuron population, in conjunction with increased orbitofrontal cortex (OFC) activity and disrupted 5-HT release in medial OFC. Optogenetic stimulation of 5-HT terminals in the medial OFC elicited an anxiolytic effect in ELS mice in a sex-dependent manner. CONCLUSIONS: These findings suggest a significant disruption in 5-HT-modulated brain connectivity in response to ELS, with implications for sex-dependent vulnerability. The anxiolytic effect of the raphe-medial OFC circuit stimulation has potential implications for developing targeted stimulation-based treatments for affective disorders that arise from early life adversities.

Essential roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of colorectal motility in rats.

The supraspinal brain regions controlling defecation reflex remain to be elucidated. The purpose of this study was to determine the roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of defecation. For chemogenetic manipulation of specific neurons, we used the double virus vector infection method in rats. hM3Dq or hM4Di was expressed in neurons of the A11 region and/or the raphe nuclei that send output to the lumbosacral defecation center. Immunohistological and functional experiments revealed that both the A11 region and the raphe nuclei directly connected with the lumbosacral spinal cord through descending pathways composed of stimulatory monoaminergic neurons. Stimulation of the hM3Dq-expressing neurons in the A11 region or the raphe nuclei enhanced colorectal motility only when GABAergic transmission in the lumbosacral spinal cord was blocked by bicuculline. Experiments using inhibitory hM4Di-expressing rats revealed that enhancement of colorectal motility caused by noxious stimuli in the colon is mediated by both the A11 region and the raphe nuclei. Furthermore, suppression of the A11 region and/or the raphe nuclei significantly inhibited water avoidance stress-induced defecation. These findings demonstrate that the A11 region and the raphe nuclei play an essential role in the regulation of colorectal motility. This is important because brain regions that mediate both intracolonic noxious stimuli-induced defecation and stress-induced defecation have been clarified for the first time.NEW & NOTEWORTHY The A11 region and the raphe nuclei, constituting descending pain inhibitory pathways, are related to both intracolonic noxious stimuli-induced colorectal motility and stress-induced defecation. Our findings may provide an explanation for the concurrent appearance of abdominal pain and defecation disorders in patients with irritable bowel syndrome. Furthermore, overlap of the pathway controlling colorectal motility with the pathway mediating stress responses may explain why stress exacerbates bowel symptoms.

Reduced midbrain raphe echogenicity in patients with fibromyalgia syndrome.

OBJECTIVES: The pathogenesis of fibromyalgia syndrome (FMS) is unclear. Transcranial ultrasonography revealed anechoic alteration of midbrain raphe in depression and anxiety disorders, suggesting affection of the central serotonergic system. Here, we assessed midbrain raphe echogenicity in FMS. METHODS: Sixty-six patients underwent transcranial sonography, of whom 53 were patients with FMS (27 women, 26 men), 13 patients with major depression and physical pain (all women), and 14 healthy controls (11 women, 3 men). Raphe echogenicity was graded visually as normal or hypoechogenic, and quantified by digitized image analysis, each by investigators blinded to the clinical diagnosis. RESULTS: Quantitative midbrain raphe echogenicity was lower in patients with FMS compared to healthy controls (p<0.05), but not different from that of patients with depression and accompanying physical pain. Pain and FMS symptom burden did not correlate with midbrain raphe echogenicity as well as the presence and severity of depressive symptoms. CONCLUSION: We found reduced echogenicity of the midbrain raphe area in patients with FMS and in patients with depression and physical pain, independent of the presence or severity of pain, FMS, and depressive symptoms. Further exploration of this sonographic finding is necessary before this objective technique may enter diagnostic algorithms in FMS and depression.

Serotonin 5-HT7 receptor overexpression in the raphe nuclei area produces antidepressive effect and affects brain serotonin system in male mice.

Heterodimerization between 5-HT7 and 5-HT1A receptors seems to play an important role in the mechanism of depression and antidepressant drug action. It was suggested that the shift of the ratio between 5-HT1A /5-HT7 hetero- and 5-HT1A /5-HT1A homodimers in presynaptic neurons toward 5-HT1A /5-HT1A homodimers is one of the reasons of depression. Consequently, the artificial elevation of 5-HT7 receptor number in presynaptic terminals might restore physiological homo-/heterodimer ratio resulting in antidepressive effect. Here we showed that adeno-associated virus (AAV)-based 5-HT7 receptor overexpression in the midbrain raphe nuclei area produced antidepressive effect in male mice of both C57Bl/6J and genetically predisposed to depressive-like behavior ASC (antidepressant sensitive cataleptics) strains. These changes were accompanied by the elevation of 5-HT7 receptor mRNA level in the frontal cortex of C57Bl/6J and its reduction in the hippocampus of ASC mice. The presence of engineered 5-HT7 receptor in the midbrain of both mouse strains was further demonstrated. Importantly that 5-HT7 receptor overexpression resulted in the reduction of 5-HT1A receptor level in the membrane protein fraction from the midbrain samples of C57Bl/6J, but not ASC, mice. 5-HT7 receptor overexpression caused an increase of 5-HIAA/5-HT ratio in the midbrain and the frontal cortex of C57Bl/6J and in all investigated brain structures of ASC mice. Thus, 5-HT7 receptor overexpression in the raphe nuclei area affects brain 5-HT system and causes antidepressive effect both in C57Bl/6J and in "depressive" ASC male mice. Obtained results indicate the involvement of 5-HT7 receptor in the mechanisms underlying depressive behavior.

Role of hydrogen sulfide in ventilatory responses to hypercapnia in the medullary raphe of adult rats.

NEW FINDINGS: What is the central question of this study? There is evidence that H2 S plays a role in the control of breathing: what are its actions on the ventilatory and thermoregulatory responses to hypercapnia via effects in the medullary raphe, a brainstem region that participates in the ventilatory adjustments to hypercapnia? What is the main finding and its importance? Hypercapnia increased the endogenous production of H2 S in the medullary raphe. Inhibition of endogenous H2 S attenuated the ventilatory response to hypercapnia in unanaesthetized rats, suggesting its excitatory action via the cystathionine ß-synthase-H2 S pathway in the medullary raphe. ABSTRACT: Hydrogen sulfide (H2 S) has been recently recognized as a gasotransmitter alongside carbon monoxide (CO) and nitric oxide (NO). H2 S seems to modulate the ventilatory and thermoregulatory responses to hypoxia and hypercapnia. However, the action of the H2 S in the medullary raphe (MR) on the ventilatory responses to hypercapnia remains to be elucidated. The present study aimed to assess the role of H2 S in the MR (a brainstem region that contains CO2 -sensitive cells and participates in the ventilatory adjustments to hypercapnia) in the ventilatory responses to hypercapnia in adult unanaesthetized Wistar rats. To do so, aminooxyacetic acid (AOA; a cystathionine ß-synthase (CBS) enzyme inhibitor), propargylglycine (PAG; a cystathionine γ-lyase enzyme inhibitor) and sodium sulfide (Na2 S; an H2 S donor) were microinjected into the MR. Respiratory frequency (fR ), tidal volume (VT ), ventilation ( V̇E ), oxygen consumption ( V̇O2 ) and body temperature (Tb ) were measured under normocapnic (room air) and hypercapnic (7% CO2 ) conditions. H2 S concentration within the MR was determined. Microinjection of the drugs did not affect fR , VT and V̇E during normocapnia when compared to the control group. However, the microinjection of AOA, but not PAG, attenuated fR and V̇E during hypercapnia in comparison to the vehicle group, but had no effects on Tb . In addition, we observed an increase in the endogenous production of H2 S in the MR during hypercapnia. Our findings indicate that endogenously produced H2 S in the MR plays an excitatory role in the ventilatory response to hypercapnia, acting through the CBS-H2 S pathway.

Serotonin neurons in the median raphe nucleus bidirectionally regulate somatic signs of nicotine withdrawal in mice.

In chronic smokers, nicotine withdrawal symptoms during tobacco cessation can lead to smoking relapse. In rodent models, chronic exposure to nicotine elicited physical dependence, whereas acute antagonism of nicotinic acetylcholine receptors (nAChRs) immediately precipitated withdrawal symptoms. Although the central serotonergic system plays an important role in nicotine withdrawal, the exact serotonergic raphe nuclei regulating these symptoms remain unknown. We used transgenic mice expressing archaerhodopsinTP009 or channelrhodopsin-2[C128S] exclusively in the central serotonergic neurons to selectively manipulate serotonergic neurons in each raphe nucleus. Nicotine withdrawal symptoms were precipitated by an acute injection of mecamylamine, a nonspecific nAChR antagonist, following chronic nicotine consumption. Somatic signs were used as measures of nicotine withdrawal symptoms. Acute mecamylamine administration significantly increased ptosis occurrence in nicotine-drinking mice compared with that in control-drinking mice. Optogenetic inhibition of the serotonergic neurons in the median raphe nucleus (MRN), but not of those in the dorsal raphe nucleus (DRN), mimicked the symptoms observed during mecamylamine-precipitated nicotine withdrawal even in nicotine-naïve mice following the administration of acute mecamylamine injection. Optogenetic activation of the serotonergic neurons in the MRN nearly abolished the occurrence of ptosis in nicotine-drinking mice. The serotonergic neurons in the MRN, but not those in the DRN, are necessary for the occurrence of somatic signs, a nicotine withdrawal symptom, and the activation of these neurons may act as a potential therapeutic strategy for preventing the somatic manifestations of nicotine withdrawal.

Pten is a key intrinsic factor regulating raphe 5-HT neuronal plasticity and depressive behaviors in mice.

Serotonin (5-HT)-based antidepressants, selective serotonin reuptake inhibitors (SSRIs) aim to enhance serotonergic activity by blocking its reuptake. We propose PTEN as a target for an alternative approach for regulating 5-HT neuron activity in the brain and depressive behaviors. We show that PTEN is elevated in central 5-HT neurons in the raphe nucleus by chronic stress in mice, and selective deletion of Pten in the 5-HT neurons induces its structural plasticity shown by increases of dendritic branching and density of PSD95-positive puncta in the dendrites. 5-HT levels are elevated and electrical stimulation of raphe neurons evokes more 5-HT release in the brain of condition knockout (cKO) mice with Pten-deficient 5-HT neurons. In addition, the 5-HT neurons remain normal electrophysiological properties but have increased excitatory synaptic inputs. Single-cell RNA sequencing revealed gene transcript alterations that may underlay morphological and functional changes in Pten-deficient 5-HT neurons. Finally, Pten cKO mice and wild-type mice treated with systemic application of PTEN inhibitor display reduced depression-like behaviors. Thus, PTEN is an intrinsic regulator of 5-HT neuron activity, representing a novel therapeutic strategy for producing antidepressant action.

Inhaled Cannabis Suppresses Chemotherapy-Induced Neuropathic Nociception by Decoupling the Raphe Nucleus: A Functional Imaging Study in Rats.

BACKGROUND: Efficacy of inhaled cannabis for treating pain is controversial. Effective treatment for chemotherapy-induced neuropathy represents an unmet medical need. We hypothesized that cannabis reduces neuropathic pain by reducing functional coupling in the raphe nuclei. METHODS: We assessed the impact of inhalation of vaporized cannabis plant (containing 10.3% Δ9-tetrahydrocannabinol/0.05% cannabidiol) or placebo cannabis on brain resting-state blood oxygen level-dependent functional connectivity and pain behavior induced by paclitaxel in rats. Rats received paclitaxel to produce chemotherapy-induced peripheral neuropathy or its vehicle. Behavioral and imaging experiments were performed after neuropathy was established and stable. Images were registered to, and analyzed using, a 3D magnetic resonance imaging rat atlas providing site-specific data on more than 168 different brain areas. RESULTS: Prior to vaporization, paclitaxel produced cold allodynia. Inhaled vaporized cannabis increased cold withdrawal latencies relative to prevaporization or placebo cannabis, consistent with Δ9-tetrahydrocannabinol-induced antinociception. In paclitaxel-treated rats, the midbrain serotonergic system, comprising the dorsal and median raphe, showed hyperconnectivity to cortical, brainstem, and hippocampal areas, consistent with nociceptive processing. Inhalation of vaporized cannabis uncoupled paclitaxel-induced hyperconnectivity patterns. No such changes in connectivity or cold responsiveness were observed following placebo cannabis vaporization. CONCLUSIONS: Inhaled vaporized cannabis plant uncoupled brain resting-state connectivity in the raphe nuclei, normalizing paclitaxel-induced hyperconnectivity to levels observed in vehicle-treated rats. Inhaled vaporized cannabis produced antinociception in both paclitaxel- and vehicle-treated rats. Our study elucidates neural circuitry implicated in the therapeutic effects of Δ9-tetrahydrocannabinol and supports a role for functional imaging studies in animals in guiding indications for future clinical trials.

Induction of aberrant agonistic behavior by a combination of serotonergic and dopaminergic manipulation in rats.

Serotonin (5-HT) and dopamine (DA) are involved in the regulation of social behaviors. However, the effects of their interactions on social behavior are not well understood. In this study, rats received a serotonergic neurotoxin injection into the raphe nuclei and/or systemic administration of L-3, 4-dihydroxyphenylalanine (L-DOPA), and their agonistic behaviors were investigated using the resident-intruder (RI) paradigm. Rats in the DA + /5-HT-group, which were administered both monoaminergic treatments, exhibited intense jump and flight responses to intruders. These behaviors were not observed in rats that received either 5-HT lesions or L-DOPA treatment only. To address the neural basis of these aberrant behaviors, we compared c-Fos immunoreactivity in the brain among the different groups. The DA + /5-HT-group had c-Fos activation in areas related to anti-predatory defensive behaviors, such as the ventromedial hypothalamic nucleus, premammillary nucleus, and periaqueductal gray. Moreover, this group had increased c-Fos expression in the ventroposterior part of the anterior olfactory nucleus (AOVP). To test the involvement of this area in the aberrant behaviors, cytotoxic lesions were performed in the AOVP prior to the monoaminergic treatments, and subsequent behaviors were examined using the RI test. The AOVP-lesioned DA + /5-HT-rats had attenuation of the aberrant behaviors. Together, these results suggest that the AOVP is involved in the generation of the aberrant defensive behaviors, and that 5-HT/DA balance is important in the regulation of social behaviors.

Activation of PPG neurons following acute stressors differentially involves hindbrain serotonin in male rats.

Within the hindbrain, serotonin (5-HT) functions as a modulator of the central glucagon-like peptide-1 (GLP-1) system. This interaction between 5-HT and GLP-1 is achieved via 5-HT2C and 5-HT3 receptors and is relevant for GLP-1-mediated feeding behavior. The central GLP-1 system is activated by various stressors, activates the hypothalamic pituitary adrenocortical (HPA) axis, and contributes to stress-related behaviors. Whether 5-HT modulates GLP-1's role in the stress response in unknown. We hypothesized that the serotonergic modulation of GLP-1-producing neurons (i.e., PPG neurons) is stimuli-specific and that stressed-induced PPG activity is one of the modalities in which 5-HT plays a role. In this study, we investigated the roles of 5-HT2C and 5-HT3 receptors in mediating the activation of PPG neurons in the nucleus tractus solitarius (NTS) following exposure to three different acute stressors: lithium chloride (LiCl), noncontingent cocaine (Coc), and novel restraint stress (RES). Results showed that increased c-Fos expression in PPG neurons following LiCl and RES-but not Coc-is dependent on hindbrain 5-HT2C and 5-HT3 receptor signaling. Additionally, stressors that depend on 5-HT signaling to activate PPG neurons (i.e., LiCl and RES) increased c-Fos expression in 5-HT-expressing neurons within the caudal raphe (CR), specifically in the raphe magnus (RMg). Finally, we showed that RMg neurons innervate NTS PPG neurons and that some of these PPG neurons lie in close proximity to 5-HT axons, suggesting RMg 5-HT-expressing neurons are the source of 5-HT input responsible for engaging NTS PPG neurons. Together, these findings identify a direct RMg to NTS pathway responsible for the modulatory effect of 5-HT on the central GLP-1 system-specifically via activation of 5-HT2C and 5-HT3 receptors-in the facilitation of acute stress responses.

Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice.

Spinal cord injury (SCI) often causes severe and permanent disabilities due to the regenerative failure of severed axons. Here we report significant locomotor recovery of both hindlimbs after a complete spinal cord crush. This is achieved by the unilateral transduction of cortical motoneurons with an AAV expressing hyper-IL-6 (hIL-6), a potent designer cytokine stimulating JAK/STAT3 signaling and axon regeneration. We find collaterals of these AAV-transduced motoneurons projecting to serotonergic neurons in both sides of the raphe nuclei. Hence, the transduction of cortical neurons facilitates the axonal transport and release of hIL-6 at innervated neurons in the brain stem. Therefore, this transneuronal delivery of hIL-6 promotes the regeneration of corticospinal and raphespinal fibers after injury, with the latter being essential for hIL-6-induced functional recovery. Thus, transneuronal delivery enables regenerative stimulation of neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI.

Neuronal mechanisms of adenosine A2A receptors in the loss of consciousness induced by propofol general anesthesia with functional magnetic resonance imaging.

Propofol is the most common intravenous anesthetic agent for induction and maintenance of anesthesia, and has been used clinically for more than 30 years. However, the mechanism by which propofol induces loss of consciousness (LOC) remains largely unknown. The adenosine A2A receptor (A2A R) has been extensively proven to have an effect on physiological sleep. It is, therefore, important to investigate the role of A2A R in the induction of LOC using propofol. In the present study, the administration of the highly selective A2A R agonist (CGS21680) and antagonist (SCH58261) was utilized to investigate the function of A2A R under general anesthesia induced by propofol by means of animal behavior studies, resting-state magnetic resonance imaging and c-Fos immunofluorescence staining approaches. Our results show that CGS21680 significantly prolonged the duration of LOC induced by propofol, increased the c-Fos expression in nucleus accumbens (NAc) and suppressed the functional connectivity of NAc-dorsal raphe nucleus (DR) and NAc-cingulate cortex (CG). However, SCH58261 significantly shortened the duration of LOC induced by propofol, decreased the c-Fos expression in NAc, increased the c-Fos expression in DR, and elevated the functional connectivity of NAc-DR and NAc-CG. Collectively, our findings demonstrate the important roles played by A2A R in the LOC induced by propofol and suggest that the neural circuit between NAc-DR maybe controlled by A2A R in the mechanism of anesthesia induced by propofol.

Kisspeptin-1 regulates forebrain dopaminergic neurons in the zebrafish.

The habenula is a phylogenetically conserved epithalamic structure, which conveys negative information via inhibition of mesolimbic dopamine neurons. We have previously shown the expression of kisspeptin (Kiss1) in the habenula and its role in the modulation of fear responses in the zebrafish. In this study, to investigate whether habenular Kiss1 regulates fear responses via dopamine neurons in the zebrafish, Kiss1 peptides were intracranially administered close to the habenula, and the expression of dopamine-related genes (th1, th2 and dat) were examined in the brain using real-time PCR and dopamine levels using LC-MS/MS. th1 mRNA levels and dopamine levels were significantly increased in the telencephalon 24-h and 30-min after Kiss1 administration, respectively. In fish administered with Kiss1, expression of neural activity marker gene, npas4a and kiss1 gene were significantly decreased in the ventral habenula. Application of neural tracer into the median raphe, site of habenular Kiss1 neural terminal projections showed tracer-labelled projections in the medial forebrain bundle towards the telencephalon where dopamine neurons reside. These results suggest that Kiss1 negatively regulates its own neuronal activity in the ventral habenula via autocrine action. This, in turn affects neurons of the median raphe via interneurons, which project to the telencephalic dopaminergic neurons.

Mapping Cell Types and Efferent Pathways in the Ascending Relaxin-3 System of the Nucleus Incertus.

Relaxin-3 (Rln3) is an insulin-family peptide neurotransmitter expressed primarily in neurons of the nucleus incertus (NI) of the pontine tegmentum, with smaller populations located in the deep mesencephalon (DpMe) and periaqueductal gray (PAG). Here, we have used targeted recombination at the Rln3 gene locus to generate an Rln3Cre transgenic mouse line, and characterize the molecular identity and axonal projections of Rln3-expressing neurons. Expression of Cre recombinase in Rln3Cre mice, and the expression of Cre-mediated reporters, accurately reflect the expression of Rln3 mRNA in all brain regions. In the NI, Rln3 mRNA is expressed in a subset of a larger population of tegmental neurons that express the neuropeptide neuromedin-b (NMB). These Rln3-expressing and NMB-expressing neurons also express the GABAergic marker GAD2 but not the glutamatergic marker Slc17a6 (VGluT2). Cre-mediated anterograde tracing with adeno-associated viruses (AAVs) shows that the efferents of the Rln3-expressing neurons in the DpMe and PAG are largely confined to the brain regions in which they originate, while the NI-Rln3 neurons form an extensive ascending system innervating the limbic cortex, septum, hippocampus, and hypothalamus. Viral anterograde tracing also reveals the potential synaptic targets of NI-Rln3 neurons in several brain regions, and the distinct projections of Rln3-expressing and non-expressing neurons in the pontine tegmentum. Rabies virus (RV)-mediated transsynaptic retrograde tracing demonstrates a probable synaptic link between NI-Rln3 neurons and GABAergic neurons in the septum, with implications for the modulation of neural activity in the septo-hippocampal system. Together, these results form the basis for functional studies of the NI-Rln3 system.

Perinatal Nicotine Reduces Chemosensitivity of Medullary 5-HT Neurons after Maturation in Culture.

Perinatal exposure to nicotine produces ventilatory and chemoreflex deficits in neonatal mammals. Medullary 5-HT neurons are putative central chemoreceptors that innervate respiratory nuclei and promote ventilation, receive cholinergic input and express nicotinic acetylcholine receptors (nAChRs). Perforated patch clamp recordings were made from cultured 5-HT neurons dissociated from the medullary raphé of 0-3 day old mice expressing enhanced yellow fluorescent protein driven by the enhancer region for PET1 (ePet-EYFP). The effect of exposure to low (6 mg kg-1day-1) or high (60 mg kg-1day-1) doses of nicotine in utero (prenatal), in culture (postnatal), or both and the effect of acute nicotine exposure (10 µM), were examined on baseline firing rate (FR at 5% CO2, pH = 7.4) and the change in FR with acidosis (9% CO2, pH 7.2) in young (12-21 days in vitro, DIV) and older (≥22 DIV) acidosis stimulated 5-HT neurons. Nicotine exposed neurons exhibited ∼67% of the response to acidosis recorded in neurons given vehicle (p = 0.005), with older neurons exposed to high dose prenatal and postnatal nicotine, exhibiting only 28% of that recorded in the vehicle neurons (p < 0.01). In neurons exposed to low or high dose prenatal and postnatal nicotine, acute nicotine exposure led to a smaller increase in FR (∼+51% vs +168%, p = 0.026) and response to acidosis (+6% vs +67%, p = 0.014) compared to vehicle. These data show that exposure to nicotine during development reduces chemosensitivity of 5-HT neurons as they mature, an effect that may be related to the abnormal chemoreflexes reported in rodents exposed to nicotine in utero, and may cause a greater risk for sudden infant death syndrome (SIDS).

An optical brain-to-brain interface supports rapid information transmission for precise locomotion control.

Brain-to-brain interfaces (BtBIs) hold exciting potentials for direct communication between individual brains. However, technical challenges often limit their performance in rapid information transfer. Here, we demonstrate an optical brain-to-brain interface that transmits information regarding locomotor speed from one mouse to another and allows precise, real-time control of locomotion across animals with high information transfer rate. We found that the activity of the genetically identified neuromedin B (NMB) neurons within the nucleus incertus (NI) precisely predicts and critically controls locomotor speed. By optically recording Ca2+ signals from the NI of a "Master" mouse and converting them to patterned optogenetic stimulations of the NI of an "Avatar" mouse, the BtBI directed the Avatar mice to closely mimic the locomotion of their Masters with information transfer rate about two orders of magnitude higher than previous BtBIs. These results thus provide proof-of-concept that optical BtBIs can rapidly transmit neural information and control dynamic behaviors across individuals.

Dopamine response gene pathways in dorsal striatum MSNs from a gene expression viewpoint: cAMP-mediated gene networks.

BACKGROUND: Medium spiny neurons (MSNs) comprise the main body (95% in mouse) of the dorsal striatum neurons and represent dopaminoceptive GABAergic neurons. The cAMP (cyclic Adenosine MonoPhosphate)-mediated cascade of excitation and inhibition responses observed in MSN intracellular signal transduction is crucial for neuroscience research due to its involvement in the motor and behavioral functions. In particular, all types of addictions are related to MSNs. Shedding the light on the mechanics of the above-mentioned cascade is of primary importance for this research domain. RESULTS: A mouse model of chronic social conflicts in daily agonistic interactions was used to analyze dorsal striatum neurons genes implicated in cAMP-mediated phosphorylation activation pathways specific for MSNs. Based on expression correlation analysis, we succeeded in dissecting Drd1- and Drd2-dopaminoceptive neurons (D1 and D2, correspondingly) gene pathways. We also found that D1 neurons genes clustering are split into two oppositely correlated states, passive and active ones, the latter apparently corresponding to D1 firing stage upon protein kinase A (PKA) activation. We observed that under defeat stress in chronic social conflicts the loser mice manifest overall depression of dopamine-mediated MSNs activity resulting in previously reported reduced motor activity, while the aggressive mice with positive fighting experience (aggressive mice) feature an increase in both D1-active phase and D2 MSNs genes expression leading to hyperactive behavior pattern corresponded by us before. Based on the alternative transcript isoforms expression analysis, it was assumed that many genes (Drd1, Adora1, Pde10, Ppp1r1b, Gnal), specifically those in D1 neurons, apparently remain transcriptionally repressed via the reversible mechanism of promoter CpG island silencing, resulting in alternative promoter usage following profound reduction in their expression rate. CONCLUSION: Based on the animal stress model dorsal striatum pooled tissue RNA-Seq data restricted to cAMP related genes subset we elucidated MSNs steady states exhaustive projection for the first time. We correspond the existence of D1 active state not explicitly outlined before, and connected with dynamic dopamine neurotransmission cycles. Consequently, we were also able to indicate an oscillated postsynaptic dopamine vs glutamate action pattern in the course of the neurotransmission cycles.

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.