RESUMO
The neurobiological mechanisms underlying the induction and remission of depressive episodes over time are not well understood. Through repeated longitudinal imaging of medial prefrontal microcircuits in the living brain, we found that prefrontal spinogenesis plays a critical role in sustaining specific antidepressant behavioral effects and maintaining long-term behavioral remission. Depression-related behavior was associated with targeted, branch-specific elimination of postsynaptic dendritic spines on prefrontal projection neurons. Antidepressant-dose ketamine reversed these effects by selectively rescuing eliminated spines and restoring coordinated activity in multicellular ensembles that predict motivated escape behavior. Prefrontal spinogenesis was required for the long-term maintenance of antidepressant effects on motivated escape behavior but not for their initial induction.
Assuntos
Antidepressivos/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Transtorno Depressivo/fisiopatologia , Ketamina/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Estresse Psicológico/fisiopatologia , Sinapses/efeitos dos fármacos , Animais , Antidepressivos/uso terapêutico , Corticosterona/farmacologia , Espinhas Dendríticas/patologia , Espinhas Dendríticas/fisiologia , Transtorno Depressivo/induzido quimicamente , Transtorno Depressivo/tratamento farmacológico , Modelos Animais de Doenças , Reação de Fuga/efeitos dos fármacos , Ketamina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade Neuronal/efeitos dos fármacos , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Estresse Psicológico/induzido quimicamente , Sinapses/fisiologiaRESUMO
Mutations in coiled-coil-helix-coiled-coil-helix-domain containing 10 (CHCHD10), a mitochondrial twin CX9C protein whose function is still unknown, cause myopathy, motor neuron disease, frontotemporal dementia, and Parkinson's disease. Here, we investigate CHCHD10 topology and its protein interactome, as well as the effects of CHCHD10 depletion or expression of disease-associated mutations in wild-type cells. We find that CHCHD10 associates with membranes in the mitochondrial intermembrane space, where it interacts with a closely related protein, CHCHD2. Furthermore, both CHCHD10 and CHCHD2 interact with p32/GC1QR, a protein with various intra and extra-mitochondrial functions. CHCHD10 and CHCHD2 have short half-lives, suggesting regulatory rather than structural functions. Cell lines with CHCHD10 knockdown do not display bioenergetic defects, but, unexpectedly, accumulate excessive intramitochondrial iron. In mice, CHCHD10 is expressed in many tissues, most abundantly in heart, skeletal muscle, liver, and in specific CNS regions, notably the dopaminergic neurons of the substantia nigra and spinal cord neurons, which is consistent with the pathology associated with CHCHD10 mutations. Homozygote CHCHD10 knockout mice are viable, have no gross phenotypes, no bioenergetic defects or ultrastructural mitochondrial abnormalities in brain, heart or skeletal muscle, indicating that functional redundancy or compensatory mechanisms for CHCHD10 loss occur in vivo. Instead, cells expressing S59L or R15L mutant versions of CHCHD10, but not WT, have impaired mitochondrial energy metabolism. Taken together, the evidence obtained from our in vitro and in vivo studies suggest that CHCHD10 mutants cause disease through a gain of toxic function mechanism, rather than a loss of function.
Assuntos
Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Transporte , Proteínas de Ligação a DNA , Demência Frontotemporal/genética , Estudos de Associação Genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Elementos Estruturais de Proteínas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
There are profound, yet incompletely understood, sex differences in the neurogenic regulation of blood pressure. Both corticotropin signaling and glutamate receptor plasticity, which differ between males and females, are known to play important roles in the neural regulation of blood pressure. However, the relationship between hypertension and glutamate plasticity in corticotropin-releasing factor (CRF)-receptive neurons in brain cardiovascular regulatory areas, including the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), is not understood. In the present study, we used dual-label immuno-electron microscopy to analyze sex differences in slow-pressor angiotensin II (AngII) hypertension with respect to the subcellular distribution of the obligatory NMDA glutamate receptor subunit 1 (GluN1) subunit of the N-methyl-D-aspartate receptor (NMDAR) in the RVLM and PVN. Studies were conducted in mice expressing the enhanced green fluorescence protein (EGFP) under the control of the CRF type 1 receptor (CRF1) promoter (i.e., CRF1-EGFP reporter mice). By light microscopy, GluN1-immunoreactivity (ir) was found in CRF1-EGFP neurons of the RVLM and PVN. Moreover, in both regions tyrosine hydroxylase (TH) was found in CRF1-EGFP neurons. In response to AngII, male mice showed an elevation in blood pressure that was associated with an increase in the proportion of GluN1 on presumably functional areas of the plasma membrane (PM) in CRF1-EGFP dendritic profiles in the RVLM. In female mice, AngII was neither associated with an increase in blood pressure nor an increase in PM GluN1 in the RVLM. Unlike the RVLM, AngII-mediated hypertension had no effect on GluN1 localization in CRF1-EGFP dendrites in the PVN of either male or female mice. These studies provide an anatomical mechanism for sex-differences in the convergent modulation of RVLM catecholaminergic neurons by CRF and glutamate. Moreover, these results suggest that sexual dimorphism in AngII-induced hypertension is reflected by NMDA receptor trafficking in presumptive sympathoexcitatory neurons in the RVLM.
Assuntos
Hipertensão/patologia , Bulbo/citologia , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Caracteres Sexuais , Angiotensina II/toxicidade , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/genética , Masculino , Bulbo/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , RNA Mensageiro/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de N-Metil-D-Aspartato/genética , Estilbamidinas/metabolismo , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Brain-derived neurotrophic factor (BDNF) is a secreted protein that has been linked to numerous aspects of plasticity in the central nervous system (CNS). Stress-induced remodeling of the hippocampus, prefrontal cortex and amygdala is coincident with changes in the levels of BDNF, which has been shown to act as a trophic factor facilitating the survival of existing and newly born neurons. Initially, hippocampal atrophy after chronic stress was associated with reduced BDNF, leading to the hypothesis that stress-related learning deficits resulted from suppressed hippocampal neurogenesis. However, recent evidence suggests that BDNF also plays a rapid and essential role in regulating synaptic plasticity, providing another mechanism through which BDNF can modulate learning and memory after a stressful event. Numerous reports have shown BDNF levels are highly dynamic in response to stress, and not only vary across brain regions but also fluctuate rapidly, both immediately after a stressor and over the course of a chronic stress paradigm. Yet, BDNF alone is not sufficient to effect many of the changes observed after stress. Glucocorticoids and other molecules have been shown to act in conjunction with BDNF to facilitate both the morphological and molecular changes that occur, particularly changes in spine density and gene expression. This review briefly summarizes the evidence supporting BDNF's role as a trophic factor modulating neuronal survival, and will primarily focus on the interactions between BDNF and other systems within the brain to facilitate synaptic plasticity. This growing body of evidence suggests a more nuanced role for BDNF in stress-related learning and memory, where it acts primarily as a facilitator of plasticity and is dependent upon the coactivation of glucocorticoids and other factors as the determinants of the final cellular response.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/metabolismo , Glucocorticoides/metabolismo , Plasticidade Neuronal/fisiologia , Animais , Humanos , Fatores de Crescimento Neural/metabolismo , Estresse Psicológico/metabolismoRESUMO
In the central nervous system, angiotensin II (AngII) binds to angiotensin type 1 receptors (AT(1)Rs) to affect autonomic and endocrine functions as well as learning and memory. However, understanding the function of cells containing AT(1)Rs has been restricted by limited availability of specific antisera, difficulties discriminating AT(1)R-immunoreactive cells in many brain regions and, the identification of AT(1)R-containing neurons for physiological and molecular studies. Here, we demonstrate that an Agtr1a bacterial artificial chromosome (BAC) transgenic mouse line that expresses type A AT(1)Rs (AT1aRs) identified by enhanced green fluorescent protein (EGFP) overcomes these shortcomings. Throughout the brain, AT1aR-EGFP was detected in the nuclei and cytoplasm of cells, most of which were neurons. EGFP often extended into dendritic processes and could be identified either natively or with immunolabeling of GFP. The distribution of AT1aR-EGFP cells in brain closely corresponded to that reported for AngII binding and AT1aR protein and mRNA. In particular, AT1aR-EGFP cells were in autonomic regions (e.g., hypothalamic paraventricular nucleus, central nucleus of the amygdala, parabrachial nucleus, nuclei of the solitary tract and rostral ventrolateral medulla) and in regions involved in electrolyte and fluid balance (i.e., subfornical organ) and learning and memory (i.e., cerebral cortex and hippocampus). Additionally, dual label electron microscopic studies in select brain areas demonstrate that cells containing AT1aR-EGFP colocalize with AT(1)R-immunoreactivity. Assessment of AngII-induced free radical production in isolated EGFP cells demonstrated feasibility of studies investigating AT1aR signaling ex vivo. These findings support the utility of Agtr1a BAC transgenic reporter mice for future studies understanding the role of AT(1)R-containing cells in brain function.
Assuntos
Química Encefálica/genética , Encéfalo/citologia , Cromossomos Artificiais Bacterianos/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Arginina Vasopressina/imunologia , Arginina Vasopressina/metabolismo , Sistema Nervoso Autônomo/citologia , Sistema Nervoso Autônomo/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia Imunoeletrônica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Equilíbrio Hidroeletrolítico/genética , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here, we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.
Assuntos
Axônios/fisiologia , Marcação de Genes/métodos , Vetores Genéticos , Sindbis virus/genética , Adenilil Ciclases/genética , Animais , Axônios/metabolismo , Camundongos , Regeneração Nervosa , Ribossomos/virologia , Medula EspinalRESUMO
Estradiol affects hippocampal-dependent spatial memory and underlying structural and electrical synaptic plasticity in female mice and rats. Using estrogen receptor (ER) alpha and beta knockout mice and wild-type littermates, we investigated the role of ERs in estradiol effects on multiple pathways important for hippocampal plasticity and learning. Six hours of estradiol administration increased immunoreactivity for phosphorylated Akt throughout the hippocampal formation, whereas 48 h of estradiol increased immunoreactivity for phosphorylated TrkB receptor. Estradiol effects on phosphorylated Akt and TrkB immunoreactivities were abolished in ER alpha and ER beta knockout mice. Estradiol also had distinct effects on immunoreactivity for post-synaptic density 95 (PSD-95) and brain derived-neurotrophic factor (BDNF) mRNA in ER alpha and beta knockout mice. Thus, estradiol acts through both ERs alpha and beta in several subregions of the hippocampal formation. The different effects of estradiol at 6 and 48 h indicate that several mechanisms of estrogen receptor signaling contribute to this female hormone's influence on hippocampal synaptic plasticity. By further delineating these mechanisms, we will better understand and predict the effects of endogenous and exogenous ovarian steroids on mood, cognition, and other hippocampal-dependent behaviors.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Interpretação Estatística de Dados , Densitometria , Proteína 4 Homóloga a Disks-Large , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/fisiologia , Feminino , Guanilato Quinases/metabolismo , Hipocampo/citologia , Terapia de Reposição Hormonal , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovariectomia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkB/biossíntese , Receptor trkB/genéticaRESUMO
The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect, likely playing a critical role in the interaction between stress and drug addiction. Prior study findings suggest that the stress-related neuropeptide corticotropin releasing factor (CRF) and the delta opioid receptor (DOR) may localize to similar neuronal populations within HF lamina. Here, hippocampal sections of male and cycling female adult Sprague-Dawley rats were processed for immunolabeling using antisera directed against the DOR and CRF peptide, as well as interneuron subtype markers somatostatin or parvalbumin, and analyzed by fluorescence and electron microscopy. Both DOR- and CRF-labeling was observed in interneurons in the CA1, CA3, and dentate hilus. Males and normal cycling females displayed a similar number of CRF immunoreactive neurons co-labeled with DOR and a similar average number of CRF-labeled neurons in the dentate hilus and stratum oriens of CA1 and CA3. In addition, 70% of DOR/CRF dual-labeled neurons in the hilar region co-labeled with somatostatin, suggesting a role for these interneurons in regulating perforant path input to dentate granule cells. Ultrastructural analysis of CRF-labeled axon terminals within the hilar region revealed that proestrus females have a similar number of CRF-labeled axon terminals that contain DORs compared to males but an increased number of CRF-labeled axon terminals without DORs. Taken together, these findings suggest that while DORs are anatomically positioned to modulate CRF immunoreactive interneuron activity and CRF peptide release, their ability to exert such regulatory activity may be compromised in females when estrogen levels are high.
Assuntos
Hormônio Liberador da Corticotropina/análise , Hipocampo/metabolismo , Interneurônios/química , Receptores Opioides delta/análise , Animais , Hormônio Liberador da Corticotropina/biossíntese , Feminino , Imunofluorescência , Imuno-Histoquímica , Interneurônios/metabolismo , Interneurônios/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Proestro/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/biossíntese , Caracteres SexuaisRESUMO
The rostral ventrolateral medulla (RVLM), a region critical for the tonic and reflex control of arterial pressure, contains a group of adrenergic (C1) neurons that project to the spinal cord and directly modulate pre-ganglionic sympathetic neurons. Epidemiological data suggest that there are gender differences in the regulation of blood pressure. One factor that could be involved is angiotensin II signaling and the associated production of reactive oxygen species (ROS) by NADPH oxidase, which is emerging as an important molecular substrate for central autonomic regulation and dysregulation. In this study dual electron microscopic immunolabeling was used to examine the subcellular distribution of the angiotensin type 1 (AT(1)) receptor and two NADPH oxidase subunits (p47 and p22) in C1 dendritic processes, in tissue from male, proestrus (high estrogen) and diestrus (low estrogen) female rats. Female dendrites displayed significantly more AT(1) labeling and significantly less p47 labeling than males. While elevations in AT(1) labeling primarily resulted from higher levels of receptor on the plasma membrane, p47 labeling was reduced both on the plasma membrane and in the cytoplasm. Across the estrous cycle, proestrus females displayed significantly higher levels of AT(1) labeling than diestrus females, which resulted exclusively from plasma membrane density differences. In contrast, p47 labeling did not change across the estrous cycle, indicating that ROS production might reflect AT(1) receptor membrane density. No significant differences in p22 labeling were observed. These findings demonstrate that both sex and hormonal levels can selectively affect the expression and subcellular distribution of components of the angiotensin II signaling pathway within C1 RVLM neurons. Such effects could reflect differences in the capacity for ROS production, potentially influencing short term excitability and long term gene expression in a cell group which is critically involved in blood pressure regulation, potentially contributing to gender differences in the risk of cardiovascular disease.
Assuntos
Dendritos/metabolismo , Bulbo/metabolismo , NADPH Oxidases/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Formação Reticular/metabolismo , Caracteres Sexuais , Angiotensina II/metabolismo , Animais , Pressão Sanguínea/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Dendritos/ultraestrutura , Ciclo Estral/fisiologia , Feminino , Hormônios Esteroides Gonadais/metabolismo , Masculino , Bulbo/ultraestrutura , Microscopia Imunoeletrônica , NADPH Oxidases/química , Estresse Oxidativo/fisiologia , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Reprodução/fisiologia , Formação Reticular/ultraestrutura , Regulação para Cima/fisiologiaRESUMO
The alpha 7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the alpha7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the alpha7nAChR and VAChT. In both species, the alpha7nAChR-immunoreactivity ((-)ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable alpha7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of alpha7nAChR-labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n=240). Of the alpha7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic alpha7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed alpha7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the alpha7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.
Assuntos
Neurônios/metabolismo , Neurônios/ultraestrutura , Córtex Pré-Frontal/metabolismo , Receptores Nicotínicos/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The hippocampal formation (HF) is involved in modulating learning related to drug abuse. While HF-dependent learning is regulated by both endogenous opioids and estrogen, the interaction between these two systems is not well understood. The mossy fiber (MF) pathway formed by dentate gyrus (DG) granule cell axons is involved in some aspects of learning and contains abundant amounts of the endogenous opioid peptide dynorphin (DYN). To examine the influence of ovarian steroids on DYN expression, we used quantitative light microscopic immunocytochemistry to measure DYN levels in normal cycling rats as well as in two established models of hormone-treated ovariectomized (OVX) rats. Rats in estrus had increased levels of DYN-immunoreactivity (ir) in the DG and certain CA3 lamina compared with rats in proestrus or diestrus. OVX rats exposed to estradiol for 24 h showed increased DYN-ir in the DG and CA3, while those with 72 h estradiol exposure showed increases only in the DG. Six hours of estradiol exposure produced no change in DYN-ir. OVX rats chronically implanted with medroxyprogesterone also showed increased DYN-ir in the DG and CA3. Next, dual-labeling electron microscopy (EM) was used to evaluate the subcellular relationships of estrogen receptor (ER) alpha-, ERbeta and progestin receptor (PR) with DYN-labeled MFs. ERbeta-ir was in some DYN-labeled MF terminals and smaller terminals, and had a subcellular association with the plasmalemma and small synaptic vesicles. In contrast, ERalpha-ir was not in DYN-labeled terminals, although some DYN-labeled small terminals synapsed on ERalpha-labeled dendritic spines. PR labeling was mostly in CA3 axons, some of which were continuous with DYN-labeled terminals. These studies indicate that ovarian hormones can modulate DYN in the MF pathway in a time-dependent manner, and suggest that hormonal effects on the DYN-containing MF pathway may be directly mediated by ERbeta and/or PR activation.
Assuntos
Dinorfinas/efeitos dos fármacos , Dinorfinas/metabolismo , Estradiol/farmacologia , Hormônios Esteroides Gonadais/farmacologia , Hipocampo/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Animais , Ciclo Estral/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hormônios Esteroides Gonadais/classificação , Hipocampo/metabolismo , Microscopia Imunoeletrônica , Fibras Musgosas Hipocampais/efeitos dos fármacos , Fibras Musgosas Hipocampais/fisiologia , Ovariectomia , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/classificação , Receptores de Estrogênio/ultraestrutura , Fatores de TempoRESUMO
Estradiol modulates dendritic spine morphology and synaptic protein expression in the rodent hippocampus, as well as hippocampal-dependent learning and memory. In the rat, these effects may be mediated through nongenomic steroid signaling such as estradiol activation of the Akt and LIM kinase (LIMK) pathways, in addition to genomic signaling involving estradiol upregulation of brain-derived neurotrophic factor expression (BDNF). Due to the many species differences between mice and rats, including differences in the hippocampal response to estradiol, it is unclear whether estradiol modulates these pathways in the mouse hippocampus. Therefore, we investigated whether endogenous fluctuations of gonadal steroids modulate hippocampal activation of the Akt, LIMK, and the BDNF receptor TrkB in conjunction with spatial memory in female C57BL/6 mice. We found that Akt, LIMK, and TrkB were activated throughout the dorsal hippocampal formation during the high-estradiol phase, proestrus. Cycle phase also modulated expression of the pre- and post-synaptic markers synaptophysin and post-synaptic density 95. However, cycle phase did not influence performance on an object placement test of spatial memory, although this task is known to be sensitive to the complete absence of ovarian hormones. The findings suggest that endogenous estradiol and progesterone produced by the ovaries modulate specific signaling pathways governing actin remodeling, cell excitability, and synapse formation.
Assuntos
Ciclo Estral/fisiologia , Hipocampo/metabolismo , Quinases Lim/metabolismo , Aprendizagem em Labirinto/fisiologia , Proteína Oncogênica v-akt/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Análise de Variância , Animais , Comportamento Animal , Proteína 4 Homóloga a Disks-Large , Estradiol/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Guanilato Quinases , Hipocampo/anatomia & histologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Progesterona/metabolismo , Sinaptofisina/metabolismoRESUMO
17beta-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor alpha (ER-alpha) that potentially regulate spine formation, such as LIM-kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-alpha, phospho-LIM-kinase (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (P<0.02) in young animals, and decreased (43%) with age (P<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-alpha occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.
Assuntos
Envelhecimento/fisiologia , Estradiol/farmacologia , Estrogênios/farmacologia , Hipocampo/citologia , Quinases Lim/metabolismo , Sinapses/efeitos dos fármacos , Fatores Etários , Animais , Receptor alfa de Estrogênio/metabolismo , Feminino , Hipocampo/efeitos dos fármacos , Microscopia Imunoeletrônica/métodos , Ovariectomia , Fosforilação , Ratos , Ratos Sprague-Dawley , Sinapses/enzimologia , Sinapses/ultraestruturaRESUMO
Women are less susceptible to the cerebrovascular complications of hypertension, such as a stroke and vascular dementia. The mechanism of such protection may be related to a reduced vulnerability of women to the cerebrovascular actions of hypertension. To test this hypothesis, we used a model of hypertension based on infusion of angiotensin II (ANG II), an octapeptide that plays a key role in hypertension and produces cerebrovascular dysregulation. Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry in anesthetized (urethane-chloralose) C57BL/6J male and female mice equipped with a cranial window. ANG II administration (0.25 mug.kg(-1).min(-1) iv x 30-45 min) elevated arterial pressure equally in both sexes but attenuated the CBF increase induced by whisker stimulation or by the endothelium-dependent vasodilator acetylcholine (ACh) in male but not in female mice. The administration of ANG II for 7 days (2.74 mg.kg(-1).day(-1)), using osmotic minipumps, also attenuated these cerebrovascular responses in male, but not female, mice. The reduced susceptibility to the effect of ANG II in female mice was abolished by ovariectomy and reinstated by estrogen administration to ovariectomized mice. Administration of estrogen to male mice abolished the ANG II-induced attenuation of CBF responses. We conclude that female mice are less susceptible to the cerebrovascular dysregulation induced by ANG II, an effect related to estrogen. Such protection from the deleterious cerebrovascular effects of hypertension may play a role in the reduced vulnerability to the cerebrovascular complications of hypertension observed in women.
Assuntos
Angiotensina II/metabolismo , Circulação Cerebrovascular , Estradiol/metabolismo , Terapia de Reposição de Estrogênios , Hipertensão/complicações , Neocórtex/irrigação sanguínea , Acidente Vascular Cerebral/etiologia , Acetilcolina/farmacologia , Adenosina/farmacologia , Angiotensina II/administração & dosagem , Animais , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Estradiol/administração & dosagem , Feminino , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Bombas de Infusão Implantáveis , Fluxometria por Laser-Doppler , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Neocórtex/fisiopatologia , Ovariectomia , Reflexo , Fatores Sexuais , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo , Vasodilatadores/farmacologia , Vibrissas/inervaçãoRESUMO
Like estrogens in female rats, androgens can affect dendritic spine density in the CA1 subfield of the male rat hippocampus [J Neurosci 23:1588 (2003)]. Previous light microscopic studies have shown that androgen receptors (ARs) are present in the nuclei of CA1 pyramidal cells. However, androgens may also exert their effects through rapid non-genomic mechanisms, possibly by binding to membranes. Thus, to investigate whether ARs are at potential extranuclear sites of ARs, antibodies to ARs were localized by light and electron microscopy in the male rat hippocampal formation. By light microscopy, AR immunoreactivity (-ir) was found in CA1 pyramidal cell nuclei and in disperse, punctate processes that were most dense in the pyramidal cell layer. Additionally, diffuse AR-ir was found in the mossy fiber pathway. Ultrastructural analysis revealed AR-ir at several extranuclear sites in all hippocampal subregions. AR-ir was found in dendritic spines, many arising from pyramidal and granule cell dendrites. AR-ir was associated with clusters of small, synaptic vesicles within preterminal axons and axon terminals. Labeled preterminal axons were most prominent in stratum lucidum of the CA3 region. AR-containing terminals formed asymmetric synapses or did not form synaptic junctions in the plane of section analyzed. AR-ir also was detected in astrocytic profiles, many of which apposed terminals synapsing on unlabeled dendritic spines or formed gap junctions with other AR-labeled or unlabeled astrocytes. Collectively, these results suggest that ARs may serve as both a genomic and non-genomic transducer of androgen action in the hippocampal formation.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Hipocampo/citologia , Células Piramidais/ultraestrutura , Receptores Androgênicos/metabolismo , Animais , Western Blotting/métodos , Dendritos/metabolismo , Hipocampo/metabolismo , Masculino , Microscopia Imunoeletrônica/métodos , Modelos Neurológicos , Células Piramidais/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Neuropeptide Y-immunoreactive (NPY-I) interneurons in the dentate gyrus are vulnerable to various insults, including septohippocampal cholinergic deafferentation. The present study examined whether a loss of NPY-I neurons occurs during aging, when the functional integrity of the septohippocampal pathway is thought to be compromised. Sets of male Long Evans rats (consisting of young and aged rats, with and without spatial learning impairments assessed by the Morris water maze) were examined. Light microscopic analysis revealed that hilar NPY-I neuronal number in matched dorsal sections was significantly decreased in aged compared to young rats. Ultrastructural analysis disclosed that the microenvironment (the types of processes apposed to the plasmalemmal surface) of NPY-I neurons also differed significantly between young and aged rats. In particular, a subgroup of NPY-I neurons, distinguished by a higher percentage of unmyelinated axon coverage of the plasmalemmal surface, was present in young, but not aged, rats. Neither the number nor the microenvironment of NPY-I neurons significantly differed between aged animals that were impaired versus unimpaired in spatial learning performance. To our knowledge these findings represent the first report of an age-associated decline in the number of a specific, neurochemically identified neuronal subpopulation within the hippocampal formation. Additionally, they closely parallel observations in 192 IgG-saporin-lesioned animals, suggesting that a distinct subgroup of NPY-I interneurons is particularly dependent on the viability of septohippocampal cholinergic innervation for its survival. Since neuronal loss was not correlated with performance, this alteration by itself does not appear to be sufficient to produce learning impairment.
Assuntos
Envelhecimento/fisiologia , Hipocampo/metabolismo , Interneurônios/metabolismo , Neuropeptídeo Y/biossíntese , Fatores Etários , Animais , Comportamento Animal/fisiologia , Contagem de Células , Hipocampo/citologia , Interneurônios/citologia , Interneurônios/ultraestrutura , Masculino , Aprendizagem em Labirinto/fisiologia , Ratos , Ratos Long-EvansRESUMO
The hippocampal formation is a brain region sensitive to seizure development, a phenomenon thought to be mediated in part by mu-opioid receptor (MOR) activation. Previous studies have found a delayed increase in MOR immunoreactivity (IR) in the inner molecular layer (IML) of the dentate gyrus after experimentally induced seizures. However, whether these increases in MOR-IR are restricted to certain cell types or cellular compartments (i.e., presynaptic, postsynaptic, or glial profiles) has not been determined. Thus, the present study examined which subcellular profiles demonstrate changes in MOR-IR after kainic acid (KA)-induced seizures. Light microscopic (LM) analysis demonstrated seizure-induced increases in MOR-IR at three points of the IML (dorsal blade, ventral blade, and crest) at three levels of section (septal, mid-septotemporal, and temporal). Electron microscopic analysis of the IML revealed that MOR-IR was present in the same types of cellular profiles in both control and KA-treated rats. However, a significant increase in the number of MOR-labeled terminal profiles was revealed in KA-treated rats compared to controls. Additionally, some MOR-labeled terminals in KA-treated rats possessed excitatory-type morphology and contained enkephalin or dynorphin, peptides found in mossy fiber terminals. These data suggest that most of the seizure-induced increases in MOR expression in the IML are associated with terminals originating from several different neuronal populations, including granule cells, and possibly, surviving GABAergic interneurons, septal cholinergic, and/or supramamillary projection neurons.
Assuntos
Giro Denteado/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Opioides mu/metabolismo , Convulsões/metabolismo , Animais , Dendritos/ultraestrutura , Giro Denteado/patologia , Modelos Animais de Doenças , Encefalinas/biossíntese , Imuno-Histoquímica , Ácido Caínico , Masculino , Fibras Musgosas Hipocampais/metabolismo , Fibras Musgosas Hipocampais/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/patologiaRESUMO
In the dentate gyrus, mu opioid receptors (MORs) and their enkephalin agonists have overlapping distributions and influence excitability and plasticity. Released endogenous enkephalins can activate at least some of these MORs; however, whether these interactions involve synaptically associated profiles or more distant associations and whether some subcellular compartments (e.g., terminals or dendrites) are more likely to be targeted than others are not known. To elucidate the relationships between potential sites of enkephalin release and MORs, MOR1 and leucine-enkephalin (LE) immunoreactivities were localized in the hilus by electron microscopy, using immunoperoxidase and immunogold markers. Of the 573 MOR-immunoreactive (ir) profiles analyzed, most were axons and terminals (51 and 30%, respectively), and fewer were dendrites (12%), glia (3%), or unclassifiable (4%). Most MOR-ir profiles resembled interneuron processes, while most LE-ir terminals resembled mossy fibers. One third of MOR-ir profiles were within 3 microm and approximately half were within 4 microm of the nearest LE-ir profile. In contrast, few (3%) MOR-ir profiles contacted LE-ir profiles; only 16% of these contacts included observable synapses, and very few profiles (0.5%) colocalized MOR and LE immunoreactivity. MOR-ir axons, terminals, and dendrites were not distributed differently relative to LE-ir profiles. These results suggest that activation of hilar MORs by LE usually involves short-range volume transmission and that dendritic MORs are as likely as axonal and terminal MORs to be activated by released LE. However, the greater abundance of MOR-ir axons and terminals compared to dendrites indicates that presynaptic profiles are a more prominent target for enkephalins and exogenous MOR agonists such as morphine.
Assuntos
Comunicação Celular/fisiologia , Giro Denteado/metabolismo , Encefalina Leucina/metabolismo , Neurônios/metabolismo , Receptores Opioides mu/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Giro Denteado/citologia , Giro Denteado/ultraestrutura , Masculino , Neurônios/classificação , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Ligands of the delta-opioid receptor tonically influence sympathetic outflow. Some of the actions of delta-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain delta-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for delta-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few ( approximately 5% of 903) profiles dually labeled for PNMT and delta-opioid receptor were detected; most of these were dendrites with diameters < 1.5 microm. delta-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas delta-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority ( approximately 83% of 338) of delta-opioid receptor-immunoreactive profiles were axons and axon terminals. delta-Opioid receptor-immunoreactive terminals averaged 0.75 microm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of delta-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by delta-opioid receptor-immunoreactive profiles ( approximately 75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 microm. The prominent localization of delta-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that delta-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of delta-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that delta-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain delta-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle delta-opioid receptors.
Assuntos
Vias Eferentes/metabolismo , Epinefrina/metabolismo , Bulbo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Opioides delta/metabolismo , Formação Reticular/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Fenômenos Fisiológicos Cardiovasculares , Vias Eferentes/ultraestrutura , Imuno-Histoquímica , Masculino , Bulbo/ultraestrutura , Microscopia Eletrônica , Inibição Neural/fisiologia , Peptídeos Opioides/metabolismo , Feniletanolamina N-Metiltransferase/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/ultraestrutura , Formação Reticular/ultraestrutura , Sistema Nervoso Simpático/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
Neurotrophins are important modulators of structural synaptic plasticity. (Through trophic action (Jordan. J Neurobiol 40:434-445, 1999), astrocytes serve as permissive substrates to support axonal regrowth (Ridet et al. Trends Neurosci 20:570-571, 1997), and are involved in estrogen-induced synaptic structural plasticity (Garcia-Segura et al. Cell Mol Neurobiol 16:225-237, 1996). Previously, we reported that tyrosine kinase A receptor (TrkA) immunoreactivity was present both in presynaptic neuronal processes (axons and terminals) and in select astrocytes of the male rat hippocampal formation (Barker-Gibb et al. J Comp Neurol 430:182-199, 2001). We show that the number of TrkA-immunoreactive astrocytes in female rats fluctuates 16-fold across the estrous cycle in dendritic fields of the hippocampal formation, with the greatest number at estrus after the peak plasma estradiol concentration of proestrus. Few TrkA-labeled astrocytes were found in ovariectomized animals; after estrogen replacement, this number increased by 12-fold in the hippocampal formation, indicating estrogen-mediated induction. Dual-labeling studies showed that TrkA-labeled astrocytes were also immunoreactive for vimentin, a protein expressed by reactive astrocytes. Ultrastructural analysis of the dentate gyrus molecular layer demonstrated that TrkA immunoreactive astrocytes are positioned primarily next to dendrites and unmyelinated axons. Because nerve growth factor (NGF) has been reported to stimulate astrocytes to function as substrates for axon growth (Kawaja and Gage. Neuron 7:1019-1030, 1991), these findings are consistent with the theory that TrkA immunoreactive astrocytes serve a role in structural plasticity, axon guidance, and synaptic regeneration across the estrous cycle in the hippocampal formation.