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.

ASSESSMENT OF RAT BRAIN MORPHOFUNCTIONAL STATE IN A PARKINSON'S MODEL: INFLUENCE OF THERAPEUTIC AGENTS OF ANIMAL AND SYNTHETIC ORIGINS.

In neurodegenerative diseases, particularly in Parkinson's disease (PD), antinociceptive centers are often implicated in neurodegeneration, leading to persistent pain unresponsive to narcotic substances. This study investigated the periaqueductal gray matter (PAG) and the nucleus raphe magnus (NRM), components of the brain's antinociceptive system. In conditions of rotenone intoxication (an experimental PD model), morphological changes in intracellular structures were observed in PAG and NRM neurons, indicating metabolic disorders characteristic of PD (alterations in the shape and size of neuronal bodies and processes, disruption of acid phosphatase activity in neuron cytoplasm). Under the influence of bacterial melanin and in combination with synoestrol, positive changes in structural properties were observed in PAG and NRM neurons compared to the rotenone model of PD. This included the preservation of the morphological characteristics typical of these brain regions, with cells exhibiting shapes and sizes close to normal. Furthermore, under the influence of these therapeutic agents, an increase in phosphatase activity in cell cytoplasm was detected, indicating an acceleration of metabolic processes (metabolic activation) disrupted by rotenone intoxication. The data obtained suggests that bacterial melanin and synoestrol may act as potential neuroprotective agents against PAG and NRM neurons in the rat brain in the rotenone model of PD. Further research is needed to elucidate the mechanisms of action of therapeutic doses and propose their use in the treatment of PD, either in isolation or combination therapy.

Evidence for a Role of 5-HT-glutamate Co-releasing Neurons in Acute Stress Mechanisms.

A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

5-HT1A receptors within the intermediate lateral septum modulate stress vulnerability in male mice.

Chronic stress is a major risk factor for psychiatric disorders. However, certain individuals may be at higher risk due to greater stress susceptibility. Elucidating the neurobiology of stress resilience and susceptibility may facilitate the development of novel strategies to prevent and treat stress-related disorders such as depression. Mounting evidence suggests that the serotonin (5-HT) system is a major regulator of stress sensitivity. In this study, we assessed the functions of 5-HT1A and 5-HT2A receptors within the lateral septum (LS) in regulating stress vulnerability. Among a group of male mice exposed to chronic social defeat stress (CSDS), 47.2% were classified as stress-susceptible, and these mice employed more passive coping strategies during the defeat and exhibited more severe anxiety- and depression-like behaviors during the following behavioral tests. These stress-susceptible mice also exhibited elevated neuronal activity in the LS as evidenced by greater c-Fos expression, greater activity of 5-HT neurons in both the dorsal and median raphe nucleus, and downregulated expression of the 5-HT1A receptor in the intermediate LS (LSi). Finally, we found the stress-induced social withdrawal symptoms could be rapidly relieved by LSi administration of 8-OH-DPAT, a 5-HT1A receptor agonist. These results indicate that 5-HT1A receptors within the LSi play an important role in stress vulnerability in mice. Therefore, modulation of stress vulnerable via 5-HT1A receptor activation in the LSi is a potential strategy to treat stress-related psychiatric disorders.

The habenula in Parkinson's disease: Anatomy, function, and implications for mood disorders - A narrative review.

Parkinson's disease (PD), a widespread neurodegenerative disorder, often coexists with mood disorders. Degeneration of serotonergic neurons in brainstem raphe nuclei have been linked to depression and anxiety. Additionally, the locus coeruleus and its noradrenergic neurons are among the first areas to degenerate in PD and contribute to stress, emotional memory, motor, sensory, and autonomic symptoms. Another brain region of interest is habenula, which is especially related to anti-reward processing, and its function has recently been linked to PD and to mood-related symptoms. There are several neuroimaging studies that investigated role of the habenula in mood disorders. Differences in habenular size and hemispheric symmetry were found in healthy controls compared to individuals with mood disorders. The lateral habenula, as a link between the dopaminergic and serotonergic systems, is thought to contribute to depressive symptoms in PD. However, there is only one imaging study about role of habenula in mood disorders in PD, although the relationship between PD and mood disorders is known. There is little known about habenula pathology in PD but given these observations, the question arises whether habenular dysfunction could play a role in PD and the development of PD-related mood disorders. In this review, we evaluate neuroimaging techniques and studies that investigated the habenula in the context of PD and mood disorders. Future studies are important to understand habenula's role in PD patients with mood disorders. Thus, new potential diagnostic and treatment opportunities would be found for mood disorders in PD.

Modulatory effects of acupuncture on raphe nucleus-related brain circuits in patients with chronic neck pain: A randomized neuroimaging trial.

OBJECTIVE: Acupuncture has shown promise in treating neck pain. Clinical trials have shown mixed results, possibly due to heterogeneous methodologies and the lack of knowledge regarding underlying brain circuit mechanism of action. In this study, we investigated the specific contribution of the serotonergic system in treating neck pain, and the specific brain circuits involved. METHODS: A total of 99 patients with chronic neck pain (CNP) were randomized to receive true acupuncture (TA) or sham acupuncture (SA) 3 times weekly for 4 weeks. Patients with CNP in each group were assessed for primary outcomes by measuring the Visual Analog Scale (VAS) and the duration of each attack; secondary outcomes were measured using the Neck Disability Index (NDI), Northwick Park Neck Pain Questionnaire (NPQ), McGill Pain Questionnaire (MPQ), Self-rating Anxiety Scale (SAS), Self-rating Depression Scale (SDS) and the 12-item Short Form Quality Life Scale (SF-12); levels of functional circuits connectivity were assessed using resting-state functional magnetic resonance imaging in the dorsal (DR) and median (MR) raphe nucleus, before and after undergoing acupuncture. RESULTS: Patients receiving TA showed more extensive symptom improvement compared with SA. Regarding the primary outcomes, changes observed in the TA group were as follows: VAS = 16.9 mm (p < 0.001) and the duration of each attack = 4.30 h (p < 0.001); changes in the SA group: VAS = 5.41 mm (p = 0.138) and the duration of each attack = 2.06 h (p = 0.058). Regarding the secondary outcomes, changes in the TA group: NDI = 7.99 (p < 0.001), NPQ = 10.82 (p < 0.001), MPQ = 4.23 (p < 0.001), SAS = 5.82 (p < 0.001), SDS = 3.67 (p = 0.003), and SF-12 = 3.04 (p < 0.001); changes in the SA group: NDI = 2.97 (p = 0.138), NPQ = 5.24 (p = 0.035) and MPQ = 2.90 (p = 0.039), SAS = 1.48 (p = 0.433), SDS = 2.39 (p = 0.244), and SF-12 = 2.19 (p = 0.038). The modulatory effect of TA exhibited increased functional connectivity (FC) between the DR and thalamus, between the MR and parahippocampal gyrus, amygdala, and insula, with decreased FC between the DR and lingual gyrus and middle frontal gyrus, between the MR and middle frontal gyrus. Furthermore, changes in the DR-related circuit were specifically associated with the intensity and duration of pain, and the MR-related circuit was correlated with the quality of life with CNP. CONCLUSION: These results demonstrated the effectiveness of TA in treating neck pain and suggested that it regulates CNP by reconfiguring the function of the raphe nucleus-related serotonergic system.

Projection-defined median raphe Pet+ subpopulations are diversely implicated in seizure.

The raphe nuclei, the primary resource of forebrain 5-HT, play an important but heterogeneous role in regulating subcortical excitabilities. Fundamental circuit organizations of different median raphe (MR) subsystems are far from completely understood. In the present study, using cell-specific viral tracing, Ca2+ fiber photometry and epilepsy model, we map out the forebrain efferent and afferent of different MR Pet+ subpopulations and their divergent roles in epilepsy. We found that PetMR neurons send both collateral and parallel innervations to different downstream regions through different subpopulations. Notably, CA3-projecting PetMR subpopulations are largely distinct from habenula (Hb)-projecting PetMR subpopulations in anatomical distribution and topological organization, while majority of the CA3-projecting PetMR subpopulations are overlapped with the medial septum (MS)-projecting PetMR subpopulations. Further, using Ca2+ fiber photometry, we monitor activities of PetMR neurons in hippocampal-kindling seizure, a classical epilepsy model with pathological mechanisms caused by excitation-inhibition imbalance. We found that soma activities of PetMR neurons are heterogeneous during different periods of generalized seizures. These divergent activities are contributed by different projection-defined PetMR subpopulations, manifesting as increased activities in CA3 but decreased activity in Hb resulting from their upstream differences. Together, our findings provide a novel framework of MR subsystems showing that projection-defined MR Pet+ subpopulations are topologically heterogenous with divergent circuit connections and are diversely implicated in seizures. This may help in the understanding of heterogeneous nature of MR 5-HTergic subsystems and the paradox roles of 5-HTergic systems in epilepsy.

Sex-specific modulation of the medial prefrontal cortex by glutamatergic median raphe neurons.

A substantial proportion of raphe neurons are glutamatergic. However, little is known about how these glutamatergic neurons modulate the forebrain. We investigated how glutamatergic median raphe nucleus (MRN) input modulates the medial prefrontal cortex (mPFC), a critical component of fear circuitry. We show that vesicular glutamate transporter 3 (VGLUT3)-expressing MRN neurons activate VGLUT3- and somatostatin-expressing neurons in the mPFC. Consistent with this modulation of mPFC GABAergic neurons, activation of MRN (VGLUT3) neurons enhances GABAergic transmission in mPFC pyramidal neurons and attenuates fear memory in female but not male mice. Serotonin plays a key role in MRN (VGLUT3) neuron-mediated GABAergic plasticity in the mPFC. In agreement with these female-specific effects, we observed sex differences in glutamatergic transmission onto MRN (VGLUT3) neurons and in mPFC (VGLUT3) neuron-mediated dual release of glutamate and GABA. Our results demonstrate a cell type-specific modulation of the mPFC by MRN (VGLUT3) neurons and reveal a sex-specific role of this neuromodulation in mPFC synaptic plasticity.

Serotonergic regulation of appetite and sodium appetite.

Serotonin is a neurotransmitter that is synthesized and released from the brainstem raphe nuclei to affect many brain functions. It is well known that the activity of raphe serotonergic neurons is changed in response to the changes in feeding status to regulate appetite via the serotonin receptors. Likewise, changes in volume status are known to alter the activity of raphe serotonergic neurons and drugs targeting serotonin receptors were shown to affect sodium appetite. Therefore, the central serotonin system appears to regulate ingestion of both food and salt, although neural mechanisms that induce appetite in response to hunger and sodium appetite in response to volume depletion are largely distinct from each other. In this review, we discuss our current knowledge regarding the regulation of ingestion - appetite and sodium appetite - by the central serotonin system.

Sustained and potent analgesia with negligible side effects enabled by adaptive individualized granular stimulation in rat brainstem.

Objectives.To clarify if an adaptive current stimulation protocol, in which current amplitude is modulated during continuous stimulation, provides better efficacy than constant current stimulation protocol with respect to analgesia caused by individualized stimulation in rat periaqueductal gray matter (PAG) /dorsal raphe nuclei (DRN).Approach.Ultrathin microelectrodes adapted for recording (n= 6) and stimulation (n= 16) were implanted in rat primary somatosensory cortex and PAG/DRN, respectively. In each animal included (n= 12), a subset of PAG/DRN microelectrodes (n= 1-3 per animal) was selected that on simultaneous stimulation blocked nociceptive withdrawal reflexes in awake unrestrained animals without noticeable side effects. Analgesic effects were subsequently assessed from both nociceptive withdrawal reflexes and intracortical pain-related responses on CO2laser hind paw stimulation. The analgesic effects of adaptive current PAG/DRN stimulation comprising incremental increases of 5µA/microelectrode (initial median current 30µA/microelectrode) when effects declined were compared to the effects of constant current stimulation. Behavioral effects and brain state related changes were analyzed using quantitative movement analysis and electrocorticography (recorded on top of the dura mater), respectively. Tissue reactions and probe placement in PAG/DRN were assessed with immunohistochemistry.Main results.Powerful and sustained (4 h) analgesia was achieved with the adaptive current protocol within a rather wide area of PAG/DRN. Analgesic after-effects were seen for up to 30 min. Behavioral and brain state related side effects were minimal. Moreover, 6 weeks after implantation, there were no traces of bleedings, only small glial reactions and small but not statistically significant loss of neurons nearby indicating that the microelectrode stimulation employed is biocompatible.Significance.The results indicate that sustained and powerful analgesia with minimal side effects can be achieved by granular and individualized stimulation in PAG/DRN using an adaptive current stimulation protocol. This microelectrode technology and stimulation paradigm thus has the potential of providing a highly efficient and safe pain therapy.

Nucleus incertus projections to rat medial septum and entorhinal cortex: rare collateralization and septal-gating of temporal lobe theta rhythm activity.

Nucleus incertus (NI) neurons in the pontine tegmentum give rise to ascending forebrain projections and express the neuropeptide relaxin-3 (RLN3) which acts via the relaxin-family peptide 3 receptor (RXFP3). Activity in the hippocampus and entorhinal cortex can be driven from the medial septum (MS), and the NI projects to all these centers, where a prominent pattern of activity is theta rhythm, which is related to spatial memory processing. Therefore, we examined the degree of collateralization of NI projections to the MS and the medial temporal lobe (MTL), comprising medial and lateral entorhinal cortex (MEnt, LEnt) and dentate gyrus (DG), and the ability of the MS to drive entorhinal theta in the adult rat. We injected fluorogold and cholera toxin-B into the MS septum and either MEnt, LEnt or DG, to determine the percentage of retrogradely labeled neurons in the NI projecting to both or single targets, and the relative proportion of these neurons that were RLN3-positive ( +). The projection to the MS was threefold stronger than that to the MTL. Moreover, a majority of NI neurons projected independently to either MS or the MTL. However, RLN3 + neurons collateralize significantly more than RLN3-negative (-) neurons. In in vivo studies, electrical stimulation of the NI induced theta activity in the MS and the entorhinal cortex, which was impaired by intraseptal infusion of an RXFP3 antagonist, R3(BΔ23-27)R/I5, particularly at ~ 20 min post-injection. These findings suggest that the MS plays an important relay function in the NI-induced generation of theta within the entorhinal cortex.

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.

Resting-state functional connectivity of the raphe nuclei in major depressive Disorder: A Multi-site study.

Accumulating evidence showed that major depressive disorder (MDD) is characterized by a dysfunction of serotonin neurotransmission. Raphe nuclei are the sources of most serotonergic neurons that project throughout the brain. Incorporating measurements of activity within the raphe nuclei into the analysis of connectivity characteristics may contribute to understanding how neurotransmitter synthesized centers are involved in thepathogenesisof MDD. Here, we analyzed the resting-state functional magnetic resonance imaging (RS-fMRI) dataset from 1,148 MDD patients and 1,079 healthy individuals recruited across nine centers. A seed-based analysis with the dorsal raphe and median raphe nuclei was performed to explore the functional connectivity (FC) alterations. Compared to controls, for dorsal raphe, the significantly decreased FC linking with the right precuneus and median cingulate cortex were found; for median raphe, the increased FC linking with right superior cerebellum (lobules V/VI) was found in MDD patients. In further exploratory analyzes, MDD-related connectivity alterations in dorsal and median raphe nuclei in different clinical factors remained highly similar to the main findings, indicating these abnormal connectivities are a disease-related alteration. Our study highlights a functional dysconnection pattern of raphe nuclei in MDD with multi-site big data. These findings help improve our understanding of the pathophysiology of depression and provide evidence of the theoretical foundation for the development of novel pharmacotherapies.

Hypoechogenicity of the raphe nuclei as a biomarker of migraine: A case-control study, review, and meta-analysis.

BACKGROUND AND PURPOSE: Hypoechogenicity of the raphe nuclei (hR) has been related to major depression. Comorbidity between migraine and depression is bidirectional postulating a common mechanism of serotonergic dysfunction. We aimed to investigate the association between migraine and hR and its role as biomarker of migraine-associated depression and disease severity. METHODS: This is a single-center cross-sectional descriptive study. We included consecutive patients with episodic (EM) and chronic migraine (CM). We collected their comorbidities, analgesic consumption, hospital anxiety and depression scale (HADS), disability, and impact on quality of life associated with migraine. We also included a group of control subjects, matched for age and sex with the patients. In both groups, hR was assessed by means of transcranial sonography. We performed a meta-analysis of the studies investigating the association between migraine and hR. RESULTS: A total of 107 subjects were included (57 cases and 50 controls). hR rate was lower in controls than in migraine patients (22.2% vs. 42.9%, p = .02) with a progressive increase in EM and CM groups respect to the control group (33.3% and 50% vs. 22.2%, respectively; p = .03). Among patients, hR was not associated with depression, higher HADS score, greater migraine-related disability, or higher consumption of analgesic medication. The meta-analysis showed a significant association between migraine and hR (odds ratio = 2.16; 95% confidence interval: 1.42-3.29). CONCLUSION: hR is more prevalent in migraine patients than in controls and, in our population, its prevalence increases in a stepwise manner in patients with EM and CM. These findings support the role of raphe nuclei in migraine pathophysiology.

Stimulation of the center of the lateral reticular nucleus suppresses the swallowing reflex in rats.

Morphological studies have demonstrated that the lateral reticular nucleus (LRt) receives fibers projected from sites that are related to control of the swallowing reflex. Although the LRt may therefore be related to control of the swallowing reflex, the functional role of the LRt in the swallowing reflex remains unknown. The present study examined whether the swallowing reflex is modulated by stimulation of the LRt. These experiments were performed on rats anesthetized by urethane. The swallowing reflex was evoked by repetitive electrical stimulation of the superior laryngeal nerve (SLN) and was identified by electromyographic activities from the mylohyoid muscle. Electrical stimulation was applied to the LRt or glutamate was injected into the LRt. The number of swallows was reduced, and the latency of the onset of the first swallow was increased during electrical stimulation near the middle of the rostrocaudal direction of the LRt. The number of swallows was reduced, and the latency of onset of the first swallow increased after microinjection of glutamate near the rostrocaudal center of the LRt. The present study suggests that the LRt is involved in control of the swallowing reflex.

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.

Neural serotonergic circuits for controlling long-term voluntary alcohol consumption in mice.

Alcohol-use-disorders are chronic relapsing illnesses, often co-morbid with anxiety. We have previously shown using the "drinking-in-the-dark" model in mice that the stimulation of the serotonin receptor 1A (5-HT1A) reduces ethanol binge-drinking behaviour and withdrawal-induced anxiety. The 5-HT1A receptor is located either on Raphe neurons as autoreceptors, or on target neurons as heteroreceptors. By combining a pharmacological approach with biased agonists targeting the 5-HT1A auto- or heteroreceptor and a chemogenetic approach (DREADDs), here we identified that ethanol-binge drinking behaviour is dependent on 5-HT1A autoreceptors and 5-HT neuronal function, with a transition from DRN-dependent regulation of short-term (6 weeks) ethanol intake, to MRN-dependent regulation after longer ethanol exposure (12 weeks). We further identified a serotonergic microcircuit (5-HTMRN→DG) originating from the MRN and projecting to the dentate gyrus (DG) of the hippocampus, that is specifically affected by, and modulates long-term ethanol consumption. The present study indicates that targeting Raphe nuclei 5-HT1A autoreceptors with agonists might represent an innovative pharmacotherapeutic strategy to combat alcohol abuse.

Diabetes mellitus causes changes in the activity and expression of brain tryptophan-5-hydroxylases and in the number of serotonergic neurons, which do not return to normal with insulin treatment.

INTRODUCTION: Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. OBJECTIVES: To determine whether DM-induced changes in brain TPH1 or TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. METHODS: Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expression of TPH1 and TPH2 by West-ern blot, and the number of serotonergic neurons by immunohistochemistry. RESULTS: In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine, and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hy-droxytryptamine, TPH expression, or the number of serotonergic neurons. CONCLUSIONS: DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

INTRODUCCIÓN: La diabetes mellitus (DM) inhibe la biosíntesis de serotonina cerebral mediante cambios en la actividad y expresión de la triptófano-5-hidroxilasa (TPH). OBJETIVOS: Determinar si los cambios en la expresión de TPH1 o TPH2 cerebral y en el número de neuronas serotoninérgicas causados por la DM retornan a la normalidad en las ratas con diabetes tratadas con insulina. MÉTODOS: Ratas con diabetes inducida con estreptozotocina se dividieron en dos grupos: uno tratado con insulina y otro sin tratamiento. En el día 14, se obtuvieron tallos cerebrales para cuantificar niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH. La expresión de TPH1 y TPH2 fue mediante Western blot y el número de neuronas serotoninérgicas por inmu­nohistoquímica. RESULTADOS: En las ratas con diabetes se confirmó disminución de los niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH, así como una menor expresión de TPH1 y 2 y un menor número de neuronas serotoninérgicas. Cuando las ratas diabéticas fueron tratadas con insulina, el L-triptófano regreso a la normalidad, no así la 5-hidroxitriptamina, la expresión de TPH y el número de neuronas serotoninérgicas. CONCLUSIONES: La DM inhibe crónicamente la síntesis de 5-hidroxitriptamina cerebral mediante modificaciones en la expresión de TPH1 y TPH2 y disminución de las neuronas seroto­ninérgicas, que persisten a pesar del tratamiento con insulina.

Catecholaminergic innervation and D2-like dopamine receptor-mediated modulation of brainstem nucleus incertus neurons in the rat.

Nucleus incertus (NI) is a brainstem structure involved in the control of arousal, stress responses and locomotor activity. It was reported recently that NI neurons express the dopamine type 2 (D2) receptor that belongs to the D2-like receptor (D2R) family, and that D2R activation in the NI decreased locomotor activity. In this study, using multiplex in situ hybridization, we observed that GABAergic and glutamatergic NI neurons express D2 receptor mRNA, and that D2 receptor mRNA-positive neurons belong to partially overlapping relaxin-3- and cholecystokinin-positive NI neuronal populations. Our immunohistochemical and viral-based retrograde tract-tracing studies revealed a dense innervation of the NI area by fibers containing the catecholaminergic biosynthesis enzymes, tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH), and indicated the major sources of the catecholaminergic innervation of the NI as the Darkschewitsch, raphe and hypothalamic A13 nuclei. Furthermore, using whole-cell patch clamp recordings, we demonstrated that D2R activation by quinpirole produced excitatory and inhibitory influences on neuronal activity in the NI, and that both effects were postsynaptic in nature. Moreover, the observed effects were cell-type specific, as type I NI neurons were either excited or inhibited, whereas type II NI neurons were mainly excited by D2R activation. Our results reveal that rat NI receives a strong catecholaminergic innervation and suggest that catecholamines acting within the NI are involved in the control of diverse processes, including locomotor activity, social interaction and nociceptive signaling. Our data also strengthen the hypothesis that the NI acts as a hub integrating arousal-related neuronal information.

Serotonergic mechanisms associated with experimental models of hypoxia: A systematic review.

PURPOSE: The aim of this systematic review was to explore and discuss the literature concerning the effects of hypoxia or anoxia during the perinatal period on the serotoninergic network in rodents, through mechanisms that lead to changes in serotonergic neurons, levels, segments of central nervous system affected, 5-HT transporter, and 5-HT receptor. METHODS: Literature searches were performed in Embase, Medline (PubMed), Web of Science, and SCOPUS, from April to July 2021, with a total of 1045 published studies found. Using a predefined protocol, as registered on the CAMARADES website, 10 articles were included in this review. The PRISMA statement was used for reporting this systematic review. The internal validity was assessed using the SYRCLE's risk of bias tool. RESULTS: Our main findings show that hypoxia in the first days of postnatal life led to a disturbance in the serotonergic system with reduced in 5-HT fibers, reduced brain levels of 5-HT and 5-HIAA, reduced SERT protein expression, and reduced receptor 5-HT7 . Putative mechanisms involving damage in the serotoninergic system include retrograde cell death resulting from primary damage mainly in forebrain areas, which impairs remote areas including serotonergic raphe nuclei. Other probable mechanisms associated with the serotoninergic network damage may be triggered by excitotoxic lesion and neuroinflammation. CONCLUSION: Hypoxia at the beginning of an animal's life leads to modification of the serotonergic components associated with putative mechanisms that include cell damage and neuroinflammation.