RESUMO
The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.
Assuntos
Cerebelo , Evolução Molecular , Mamíferos , Neurogênese , Animais , Humanos , Camundongos , Linhagem da Célula/genética , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Feto/citologia , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Gambás/embriologia , Gambás/crescimento & desenvolvimento , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Análise da Expressão Gênica de Célula Única , Especificidade da Espécie , Transcriptoma , Mamíferos/embriologia , Mamíferos/crescimento & desenvolvimentoRESUMO
Human prefrontal cortex (hPFC) is a complex brain region involved in cognitive and emotional processes and several psychiatric disorders. Here, we present an overview of the distribution of the peptidergic systems in 17 subregions of hPFC and three reference cortices obtained by microdissection and based on RNA sequencing and RNAscope methods integrated with published single-cell transcriptomics data. We detected expression of 60 neuropeptides and 60 neuropeptide receptors in at least one of the hPFC subregions. The results reveal that the peptidergic landscape in PFC consists of closely located and functionally different subregions with unique peptide/transmitter-related profiles. Neuropeptide-rich PFC subregions were identified, encompassing regions from anterior cingulate cortex/orbitofrontal gyrus. Furthermore, marked differences in gene expression exist between different PFC regions (>5-fold; cocaine and amphetamine-regulated transcript peptide) as well as between PFC regions and reference regions, for example, for somatostatin and several receptors. We suggest that the present approach allows definition of, still hypothetical, microcircuits exemplified by glutamatergic neurons expressing a peptide cotransmitter either as an agonist (hypocretin/orexin) or antagonist (galanin). Specific neuropeptide receptors have been identified as possible targets for neuronal afferents and, interestingly, peripheral blood-borne peptide hormones (leptin, adiponectin, gastric inhibitory peptide, glucagon-like peptides, and peptide YY). Together with other recent publications, our results support the view that neuropeptide systems may play an important role in hPFC and underpin the concept that neuropeptide signaling helps stabilize circuit connectivity and fine-tune/modulate PFC functions executed during health and disease.
Assuntos
Neuropeptídeos , Córtex Pré-Frontal , Receptores de Neuropeptídeos , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismoRESUMO
Stress disorders impair sleep and quality of life; however, their pathomechanisms are unknown. Prolactin-releasing peptide (PrRP) is a stress mediator; we therefore hypothesized that PrRP may be involved in the development of stress disorders. PrRP is produced by the medullary A1/A2 noradrenaline (NA) cells, which transmit stress signals to forebrain centers, and by non-NA cells in the hypothalamic dorsomedial nucleus. We found in male rats that both PrRP and PrRP-NA cells innervate melanin-concentrating hormone (MCH) producing neurons in the dorsolateral hypothalamus (DLH). These cells serve as a key hub for regulating sleep and affective states. Ex vivo, PrRP hyperpolarized MCH neurons and further increased the hyperpolarization caused by NA. Following sleep deprivation, intracerebroventricular PrRP injection reduced the number of REM sleep-active MCH cells. PrRP expression in the dorsomedial nucleus was upregulated by sleep deprivation, while downregulated by REM sleep rebound. Both in learned helplessness paradigm and after peripheral inflammation, impaired coping with sustained stress was associated with (1) overactivation of PrRP cells, (2) PrRP protein and receptor depletion in the DLH, and (3) dysregulation of MCH expression. Exposure to stress in the PrRP-insensitive period led to increased passive coping with stress. Normal PrRP signaling, therefore, seems to protect animals against stress-related disorders. PrRP signaling in the DLH is an important component of the PrRP's action, which may be mediated by MCH neurons. Moreover, PrRP receptors were downregulated in the DLH of human suicidal victims. As stress-related mental disorders are the leading cause of suicide, our findings may have particular translational relevance.SIGNIFICANCE STATEMENT Treatment resistance to monoaminergic antidepressants is a major problem. Neuropeptides that modulate the central monoaminergic signaling are promising targets for developing alternative therapeutic strategies. We found that stress-responsive prolactin-releasing peptide (PrRP) cells innervated melanin-concentrating hormone (MCH) neurons that are crucial in the regulation of sleep and mood. PrRP inhibited MCH cell activity and enhanced the inhibitory effect evoked by noradrenaline, a classic monoamine, on MCH neurons. We observed that impaired PrRP signaling led to failure in coping with chronic/repeated stress and was associated with altered MCH expression. We found alterations of the PrRP system also in suicidal human subjects. PrRP dysfunction may underlie stress disorders, and fine-tuning MCH activity by PrRP may be an important part of the mechanism.
Assuntos
Hormônios Hipotalâmicos , Privação do Sono , Ratos , Masculino , Humanos , Animais , Hormônio Liberador de Prolactina/farmacologia , Hormônio Liberador de Prolactina/metabolismo , Privação do Sono/metabolismo , Transtornos do Humor/etiologia , Qualidade de Vida , Ratos Wistar , Hormônios Hipotalâmicos/metabolismo , Sono/fisiologia , Neurônios/fisiologia , Norepinefrina/metabolismoRESUMO
Almost 150 papers about brain lymphatics have been published in the last 150 years. Recently, the information in these papers has been synthesized into a picture of central nervous system (CNS) "glymphatics," but the fine structure of lymphatic elements in the human brain based on imaging specific markers of lymphatic endothelium has not been described. We used LYVE1 and PDPN antibodies to visualize lymphatic marker-positive cells (LMPCs) in postmortem human brain samples, meninges, cavernous sinus (cavum trigeminale), and cranial nerves and bolstered our findings with a VEGFR3 antibody. LMPCs were present in the perivascular space, the walls of small and large arteries and veins, the media of large vessels along smooth muscle cell membranes, and the vascular adventitia. Lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater. There were many LMPCs in the perineurium and endoneurium of cranial nerves. Soluble waste may move from the brain parenchyma via perivascular and paravascular routes to the closest subarachnoid space and then travel along the dura mater and/or cranial nerves. Particulate waste products travel along the laminae of the dura mater toward the jugular fossa, lamina cribrosa, and perineurium of the cranial nerves to enter the cervical lymphatics. CD3-positive T cells appear to be in close proximity to LMPCs in perivascular/perineural spaces throughout the brain. Both immunostaining and qPCR confirmed the presence of adhesion molecules in the CNS known to be involved in T cell migration.
Assuntos
Encéfalo/metabolismo , Sistema Linfático/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Transporte Vesicular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Autopsia , Encéfalo/diagnóstico por imagem , Movimento Celular/genética , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Dura-Máter/diagnóstico por imagem , Dura-Máter/metabolismo , Endotélio Linfático/diagnóstico por imagem , Endotélio Linfático/metabolismo , Feminino , Sistema Glinfático/metabolismo , Humanos , Imuno-Histoquímica/métodos , Sistema Linfático/diagnóstico por imagem , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/metabolismo , Masculino , Glicoproteínas de Membrana/isolamento & purificação , Espaço Subaracnóideo/diagnóstico por imagem , Espaço Subaracnóideo/metabolismo , Linfócitos T/imunologia , Proteínas de Transporte Vesicular/isolamento & purificaçãoRESUMO
The perception of and response to danger is critical for an individual's survival and is encoded by subcortical neurocircuits. The amygdaloid complex is the primary neuronal site that initiates bodily reactions upon external threat with local-circuit interneurons scaling output to effector pathways. Here, we categorize central amygdala neurons that express secretagogin (Scgn), a Ca2+-sensor protein, as a subset of protein kinase Cδ (PKCδ)+ interneurons, likely "off cells." Chemogenetic inactivation of Scgn+/PKCδ+ cells augmented conditioned response to perceived danger in vivo. While Ca2+-sensor proteins are typically implicated in shaping neurotransmitter release presynaptically, Scgn instead localized to postsynaptic compartments. Characterizing its role in the postsynapse, we found that Scgn regulates the cell-surface availability of NMDA receptor 2B subunits (GluN2B) with its genetic deletion leading to reduced cell membrane delivery of GluN2B, at least in vitro. Conclusively, we describe a select cell population, which gates danger avoidance behavior with secretagogin being both a selective marker and regulatory protein in their excitatory postsynaptic machinery.
Assuntos
Tonsila do Cerebelo/metabolismo , Interneurônios/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secretagoginas/metabolismo , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/fisiologia , Animais , Aprendizagem da Esquiva , Linhagem Celular Tumoral , Células Cultivadas , Medo , Feminino , Humanos , Interneurônios/fisiologia , Masculino , Transporte Proteico , Ratos , Ratos Wistar , Secretagoginas/genética , Potenciais SinápticosRESUMO
Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.
Assuntos
Neurônios Adrenérgicos/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Hipotálamo/metabolismo , Locus Cerúleo/metabolismo , Estresse Fisiológico , Neurônios Adrenérgicos/patologia , Animais , Fator Neurotrófico Ciliar/genética , Hipotálamo/patologia , Locus Cerúleo/patologia , Camundongos , Camundongos Knockout , RatosRESUMO
BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1), a cation channel, is expressed predominantly in primary sensory neurons, but its central distribution and role in mood control are not well understood. We investigated whether TRPA1 is expressed in the urocortin 1 (UCN1)-immunoreactive centrally projecting Edinger-Westphal nucleus (EWcp), and we hypothesized that chronic variable mild stress (CVMS) would reduce its expression in mice. We anticipated that TRPA1 mRNA would be present in the human EWcp, and that it would be downregulated in people who died by suicide. METHODS: We exposed Trpa1 knockout and wild-type mice to CVMS or no-stress control conditions. We then performed behavioural tests for depression and anxiety, and we evaluated physical and endocrinological parameters of stress. We assessed EWcp Trpa1 and Ucn1 mRNA expression, as well as UCN1 peptide content, using RNA-scope in situ hybridization and immunofluorescence. We tested human EWcp samples for TRPA1 using reverse transcription polymerase chain reaction. RESULTS: Trpa1 mRNA was colocalized with EWcp/UCN1 neurons. Non-stressed Trpa1 knockout mice expressed higher levels of Ucn1 mRNA, had less body weight gain and showed greater immobility in the forced swim test than wild-type mice. CVMS downregulated EWcp/Trpa1 expression and increased immobility in the forced swim test only in wild-type mice. We confirmed that TRPA1 mRNA expression was downregulated in the human EWcp in people who died by suicide. LIMITATIONS: Developmental compensations and the global lack of TRPA1 may have influenced our findings. Because experimental data came from male brains only, we have no evidence for whether findings would be similar in female brains. Because a TRPA1-specific antibody is lacking, we have provided mRNA data only. Limited access to high-quality human tissues restricted sample size. CONCLUSION: TRPA1 in EWcp/UCN1 neurons might contribute to the regulation of depression-like behaviour and stress adaptation response in mice. In humans, TRPA1 might contribute to mood control via EWcp/UCN1 neurons.
Assuntos
Núcleo de Edinger-Westphal , Suicídio , Animais , Núcleo de Edinger-Westphal/metabolismo , Feminino , Humanos , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Urocortinas/metabolismoRESUMO
Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have been approved for the treatment of type 2 diabetes mellitus (T2DM); however, the brain actions of these drugs are not properly established. We used post mortem microdissected human hypothalamic samples for RT-qPCR and Western blotting. For in situ hybridization histochemistry and immunolabelling, parallel cryosections were prepared from the hypothalamus. We developed in situ hybridization probes for human GLP-1R and oxytocin. In addition, GLP-1 and oxytocin were visualized by immunohistochemistry. Radioactive in situ hybridization histochemistry revealed abundant GLP-1R labelling in the human paraventricular hypothalamic nucleus (PVN), particularly in its magnocellular subdivision (PVNmc). Quantitative analysis of the mRNA signal demonstrated increased GLP-1R expression in the PVNmc in post mortem hypothalamic samples from T2DM subjects as compared to controls, while there was no difference in the expression level of GLP-1R in the other subdivisions of the PVN, the hypothalamic dorsomedial and infundibular nuclei. Our results in the PVN were confirmed by RT-qPCR. Furthermore, we demonstrated by Western blot technique that the GLP-1R protein level was also elevated in the PVN of T2DM patients. GLP-1 fibre terminals were also observed in the PVNmc closely apposing oxytocin neurons using immunohistochemistry. The data suggest that GLP-1 activates GLP-1Rs in the PVNmc and that GLP-1R is elevated in T2DM patients, which may be related to the dysregulation of feeding behaviour and glucose homeostasis in T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , Núcleo Hipotalâmico Paraventricular , Humanos , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ocitocina/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismoRESUMO
The default mode network (DMN) plays an outstanding role in psychiatric disorders. Still, gene expressional changes in its major component, the dorsomedial prefrontal cortex (DMPFC), have not been characterized. We used RNA sequencing in postmortem DMPFC samples to investigate suicide victims compared to control subjects. 1400 genes differed using log2FC > ±1 and adjusted p-value < 0.05 criteria between groups. Genes associated with depressive disorder, schizophrenia and impaired cognition were strongly overexpressed in top differentially expressed genes. Protein−protein interaction and co-expressional networks coupled with gene set enrichment analysis revealed that pathways related to cytokine receptor signaling were enriched in downregulated, while glutamatergic synaptic signaling upregulated genes in suicidal individuals. A validated differentially expressed gene, which is known to be associated with mGluR5, was the N-terminal EF-hand calcium-binding protein 2 (NECAB2). In situ hybridization histochemistry and immunohistochemistry proved that NECAB2 is expressed in two different types of inhibitory neurons located in layers II-IV and VI, respectively. Our results imply extensive gene expressional alterations in the DMPFC related to suicidal behavior. Some of these genes may contribute to the altered mental state and behavior of suicide victims.
Assuntos
Transtorno Depressivo Maior , Suicídio , Transtorno Depressivo Maior/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Córtex Pré-Frontal/metabolismo , Ideação Suicida , TranscriptomaRESUMO
Somatostatin is an important mood and pain-regulating neuropeptide, which exerts analgesic, anti-inflammatory, and antidepressant effects via its Gi protein-coupled receptor subtype 4 (SST4) without endocrine actions. SST4 is suggested to be a unique novel drug target for chronic neuropathic pain, and depression, as a common comorbidity. However, its neuronal expression and cellular mechanism are poorly understood. Therefore, our goals were (i) to elucidate the expression pattern of Sstr4/SSTR4 mRNA, (ii) to characterize neurochemically, and (iii) electrophysiologically the Sstr4/SSTR4-expressing neuronal populations in the mouse and human brains. Here, we describe SST4 expression pattern in the nuclei of the mouse nociceptive and anti-nociceptive pathways as well as in human brain regions, and provide neurochemical and electrophysiological characterization of the SST4-expressing neurons. Intense or moderate SST4 expression was demonstrated predominantly in glutamatergic neurons in the major components of the pain matrix mostly also involved in mood regulation. The SST4 agonist J-2156 significantly decreased the firing rate of layer V pyramidal neurons by augmenting the depolarization-activated, non-inactivating K+ current (M-current) leading to remarkable inhibition. These are the first translational results explaining the mechanisms of action of SST4 agonists as novel analgesic and antidepressant candidates.
Assuntos
Analgésicos/farmacologia , Encéfalo/metabolismo , Neurônios/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Afeto/fisiologia , Animais , Encéfalo/citologia , Butanos/farmacologia , Células CHO , Cricetulus , Eletrofisiologia/métodos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Terapia de Alvo Molecular , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Somatostatina/agonistas , Sulfonas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genéticaRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder. Depositions of amyloid ß peptide (Aß) and tau protein are among the major pathological hallmarks of AD. Aß and tau burden follows predictable spatial patterns during the progression of AD. Nevertheless, it remains obscure why certain brain regions are more vulnerable than others; to investigate this and dysregulated pathways during AD progression, a mass spectrometry-based proteomics study was performed. METHODS: In total 103 tissue samples from regions early (entorhinal and parahippocampal cortices - medial temporal lobe (MTL)) and late affected (temporal and frontal cortices - neocortex) by tau pathology were subjected to label-free quantitative proteomics analysis. RESULTS: Considering dysregulated proteins during AD progression, the majority (625 out of 737 proteins) was region specific, while some proteins were shared between regions (101 proteins altered in two areas and 11 proteins altered in three areas). Analogously, many dysregulated pathways during disease progression were exclusive to certain regions, but a few pathways altered in two or more areas. Changes in protein expression indicate that synapse loss occurred in all analyzed regions, while translation dysregulation was preponderant in entorhinal, parahippocampal and frontal cortices. Oxidative phosphorylation impairment was prominent in MTL. Differential proteomic analysis of brain areas in health state (controls) showed higher metabolism and increased expression of AD-related proteins in the MTL compared to the neocortex. In addition, several proteins that differentiate brain regions in control tissue were dysregulated in AD. CONCLUSIONS: This work provides the comparison of proteomic changes in brain regions affected by tau pathology at different stages of AD. Although we identified commonly regulated proteins and pathways during disease advancement, we found that the dysregulated processes are predominantly region specific. In addition, a distinct proteomic signature was found between MTL and neocortex in healthy subjects that might be related to AD vulnerability. These findings highlight the need for investigating AD's cascade of events throughout the whole brain and studies spanning more brain areas are required to better understand AD etiology and region vulnerability to disease.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteoma , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Encéfalo/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , ProteômicaRESUMO
A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.
Assuntos
Corticosterona/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Secretagoginas/metabolismo , Estresse Fisiológico/fisiologia , Animais , Corticosterona/genética , Hormônio Liberador da Corticotropina/genética , Masculino , Camundongos , Neurônios/citologia , Núcleo Hipotalâmico Paraventricular/citologia , Hipófise/citologia , Hipófise/metabolismo , Interferência de RNA , Secretagoginas/genética , Transcriptoma/fisiologiaRESUMO
Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin (GAL) and its three G protein-coupled receptors, GAL1-3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex-specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL3 antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation.
Assuntos
Transtorno Depressivo Maior/metabolismo , Galanina/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 3 de Galanina/metabolismo , Adulto , Afeto , Idoso , Encéfalo/metabolismo , Encéfalo/patologia , Mapeamento Encefálico , Estudos de Casos e Controles , Metilação de DNA , Transtorno Depressivo Maior/genética , Núcleo Dorsal da Rafe/metabolismo , Feminino , Galanina/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Locus Cerúleo/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor Tipo 1 de Galanina/genética , Receptor Tipo 3 de Galanina/genética , Fatores Sexuais , SuicídioRESUMO
OBJECTIVES: The aim of the study was to understand the effects of suckling on the brain of the pups by mapping their brain activation pattern in response to suckling. METHODS: The c-fos method was applied to identify activated neurons. Fasted rat pups were returned to their mothers for suckling and sacrificed 2 hours later for Fos immunohistochemistry. Double labeling was also performed to characterize some of the activated neurons. For comparison, another group of fasted pups were given dry food before Fos mapping. RESULTS: After suckling, we found an increase in the number of Fos-immunoreactive neurons in the insular and somatosensory cortices, central amygdaloid nucleus (CAm), paraventricular (PVN) and supraoptic hypothalamic nuclei, lateral parabrachial nucleus (LPB), nucleus of the solitary tract (NTS), and the area postrema. Double labeling experiments demonstrated the activation of calcitonin gene-related peptide-ir (CGRP-ir) neurons in the LPB, corticotropin-releasing hormone-ir (CRH-ir) but not oxytocin-ir neurons in the PVN, and noradrenergic neurons in the NTS. In the CAm, Fos-ir neurons did not contain CRH but were apposed to CGRP-ir fiber terminals. Refeeding with dry food-induced Fos activation in all brain areas activated by suckling. The degree of activation was higher following dry food consumption than suckling in the insular cortex, and lower in the supraoptic nucleus and the NTS. Furthermore, the accumbens, arcuate, and dorsomedial hypothalamic nuclei, and the lateral hypothalamic area, which were not activated by suckling, showed activation by dry food. DISCUSSION: Neurons in a number of brain areas are activated during suckling, and may participate in the signaling of satiety, taste perception, reward, food, and salt balance regulation.
Assuntos
Animais Lactentes , Encéfalo/fisiologia , Ingestão de Alimentos/fisiologia , Animais , Animais Recém-Nascidos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Núcleo Central da Amígdala/fisiologia , Hormônio Liberador da Corticotropina/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Ocitocina/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismo , DesmameRESUMO
Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term "heteroreceptor complexes" was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1Aâ»FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1Aâ»FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1Aâ»5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1â»15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1â»GalR2â»5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.
Assuntos
Depressão/metabolismo , Núcleos da Rafe/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Depressão/tratamento farmacológico , Ligação Proteica , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
BACKGROUND: Altered levels of urocortin 1 (Ucn1) in the centrally projecting Edinger-Westphal nucleus (EWcp) of depressed suicide attempters or completers mediate the brain's response to stress, while the mechanism regulating Ucn1 expression is unknown. We tested the hypothesis that microRNAs (miRNAs), which are vital fine-tuners of gene expression during the brain's response to stress, have the capacity to modulate Ucn1 expression. METHODS: Computational analysis revealed that the Ucn1 3' untranslated region contained a conserved binding site for miR-326. We examined miR-326 and Ucn1 levels in the EWcp of depressed suicide completers. In addition, we evaluated miR-326 and Ucn1 levels in the serum and the EWcp of a chronic variable mild stress (CVMS) rat model of behavioural despair and after recovery from CVMS, respectively. Gain and loss of miR-326 function experiments examined the regulation of Ucn1 by this miRNA in cultured midbrain neurons. RESULTS: We found reduced miR-326 levels concomitant with elevated Ucn1 levels in the EWcp of depressed suicide completers as well as in the EWcp of CVMS rats. In CVMS rats fully recovered from stress, both serum and EWcp miR-326 levels rebounded to nonstressed levels. While downregulation of miR-326 levels in primary midbrain neurons enhanced Ucn1 expression levels, miR-326 overexpression selectively reduced the levels of this neuropeptide. LIMITATIONS: This study lacked experiments showing that in vivo alteration of miR-326 levels alleviate depression-like behaviours. We show only correlative data for miR-325 and cocaine- and amphetamine-regulated transcript levels in the EWcp. CONCLUSION: We identified miR-326 dysregulation in depressed suicide completers and characterized this miRNA as an upstream regulator of the Ucn1 neuropeptide expression in midbrain neurons.
Assuntos
Transtorno Depressivo/metabolismo , Mesencéfalo/metabolismo , MicroRNAs/metabolismo , Urocortinas/metabolismo , Adulto , Animais , Sítios de Ligação , Células Cultivadas , Doença Crônica , Simulação por Computador , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Estresse Psicológico , SuicídioRESUMO
Using riboprobe in situ hybridization, we studied the localization of the transcripts for the neuropeptide galanin and its receptors (GalR1-R3), tryptophan hydroxylase 2, tyrosine hydroxylase, and nitric oxide synthase as well as the three vesicular glutamate transporters (VGLUT 1-3) in the locus coeruleus (LC) and the dorsal raphe nucleus (DRN) regions of postmortem human brains. Quantitative real-time PCR (qPCR) was used also. Galanin and GalR3 mRNA were found in many noradrenergic LC neurons, and GalR3 overlapped with serotonin neurons in the DRN. The qPCR analysis at the LC level ranked the transcripts in the following order in the LC: galanin >> GalR3 >> GalR1 > GalR2; in the DRN the ranking was galanin >> GalR3 >> GalR1 = GalR2. In forebrain regions the ranking was GalR1 > galanin > GalR2. VGLUT1 and -2 were strongly expressed in the pontine nuclei but could not be detected in LC or serotonin neurons. VGLUT2 transcripts were found in very small, nonpigmented cells in the LC and in the lateral and dorsal aspects of the periaqueductal central gray. Nitric oxide synthase was not detected in serotonin neurons. These findings show distinct differences between the human brain and rodents, especially rat, in the distribution of the galanin system and some other transmitter systems. For example, GalR3 seems to be the important galanin receptor in both the human LC and DRN versus GalR1 and -2 in the rodent brain. Such knowledge may be important when considering therapeutic principles and drug development.
Assuntos
Encéfalo/metabolismo , Galanina/genética , Galanina/metabolismo , Neurotransmissores/metabolismo , Animais , Humanos , Hibridização In Situ , Locus Cerúleo/metabolismo , Neurotransmissores/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Núcleos da Rafe/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Galanina/genética , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/genética , Receptor Tipo 2 de Galanina/metabolismo , Receptor Tipo 3 de Galanina/genética , Receptor Tipo 3 de Galanina/metabolismo , Especificidade da Espécie , Distribuição Tecidual , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/genética , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
We have recently shown that the ATP-forming SUCLA2 subunit of succinyl-CoA ligase, an enzyme of the citric acid cycle, is exclusively expressed in neurons of the human cerebral cortex; GFAP- and S100-positive astroglial cells did not exhibit immunohistoreactivity or in situ hybridization reactivity for either SUCLA2 or the GTP-forming SUCLG2. However, Western blotting of post mortem samples revealed a minor SUCLG2 immunoreactivity. In the present work we sought to identify the cell type(s) harboring SUCLG2 in paraformaldehyde-fixed, free-floating surgical human cortical tissue samples. Specificity of SUCLG2 antiserum was supported by co-localization with mitotracker orange staining of paraformaldehyde-fixed human fibroblast cultures, delineating the mitochondrial network. In human cortical tissue samples, microglia and oligodendroglia were identified by antibodies directed against Iba1 and myelin basic protein, respectively. Double immunofluorescence for SUCLG2 and Iba1 or myelin basic protein exhibited no co-staining; instead, SUCLG2 appeared to outline the cerebral microvasculature. In accordance to our previous work there was no co-localization of SUCLA2 immunoreactivity with either Iba1 or myelin basic protein. We conclude that SUCLG2 exist only in cells forming the vasculature or its contents in the human brain. The absence of SUCLA2 and SUCLG2 in human glia is in compliance with the presence of alternative pathways occurring in these cells, namely the GABA shunt and ketone body metabolism which do not require succinyl CoA ligase activity, and glutamate dehydrogenase 1, an enzyme exhibiting exquisite sensitivity to inhibition by GTP.
Assuntos
Córtex Cerebral/enzimologia , Neuroglia/enzimologia , Succinato-CoA Ligases/metabolismo , Idoso , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos/enzimologia , Humanos , Masculino , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Proteína Básica da Mielina/metabolismo , Fosforilação/fisiologiaRESUMO
Alzheimer's disease and other age-related neurodegenerative disorders are associated with deterioration of the noradrenergic locus coeruleus (LC), a probable trigger for mood and memory dysfunction. LC noradrenergic neurons exhibit particularly high levels of somatostatin binding sites. This is noteworthy since cortical and hypothalamic somatostatin content is reduced in neurodegenerative pathologies. Yet a possible role of a somatostatin signal deficit in the maintenance of noradrenergic projections remains unknown. Here, we deployed tissue microarrays, immunohistochemistry, quantitative morphometry and mRNA profiling in a cohort of Alzheimer's and age-matched control brains in combination with genetic models of somatostatin receptor deficiency to establish causality between defunct somatostatin signalling and noradrenergic neurodegeneration. In Alzheimer's disease, we found significantly reduced somatostatin protein expression in the temporal cortex, with aberrant clustering and bulging of tyrosine hydroxylase-immunoreactive afferents. As such, somatostatin receptor 2 (SSTR2) mRNA was highly expressed in the human LC, with its levels significantly decreasing from Braak stages III/IV and onwards, i.e., a process preceding advanced Alzheimer's pathology. The loss of SSTR2 transcripts in the LC neurons appeared selective, since tyrosine hydroxylase, dopamine ß-hydroxylase, galanin or galanin receptor 3 mRNAs remained unchanged. We modeled these pathogenic changes in Sstr2(-/-) mice and, unlike in Sstr1(-/-) or Sstr4(-/-) genotypes, they showed selective, global and progressive degeneration of their central noradrenergic projections. However, neuronal perikarya in the LC were found intact until late adulthood (<8 months) in Sstr2(-/-) mice. In contrast, the noradrenergic neurons in the superior cervical ganglion lacked SSTR2 and, as expected, the sympathetic innervation of the head region did not show any signs of degeneration. Our results indicate that SSTR2-mediated signaling is integral to the maintenance of central noradrenergic projections at the system level, and that early loss of somatostatin receptor 2 function may be associated with the selective vulnerability of the noradrenergic system in Alzheimer's disease.