RESUMO
Novel antidepressants are predominantly evaluated preclinically in rodent models of chronic stress in which animals experience a single prolonged exposure to chronic stress prior to treatment. Rodent models of a single episode of chronic stress translate poorly to human depressive disorders, which are commonly marked by recurring depressive episodes. Intravenous administration of Reelin has previously been shown to resolve immobility in the forced swim test of rats exposed to a single prolonged exposure to chronic stress. To determine whether Reelin has antidepressant-like properties in a model of recurring depressive episodes, Long-Evans rats (N = 57) were exposed to multiple cycles of chronic stress and stress-free periods before the administration of a single injection of Reelin during the final cycle of chronic stress. The animals then performed in the forced swim test and open field test before the post-mortem evaluation of Reelin cell counts in the sub-granular zone of the dentate gyrus to determine the impact of treatment on hippocampal Reelin levels and spleen white pulp to evaluate the role of Reelin treatment in peripheral inflammation. The results show a single Reelin injection reversed elevated levels of immobility in the forced swim test in both male and female subjects exposed to the cyclic chronic stress model of recurring depressive episodes. Treatment with Reelin also restored Reelin-positive cell counts in the dentate gyrus sub-granular zone and reversed atrophy of spleen white pulp. The results shown here indicate that treatment with Reelin could effectively resolve alterations in forced swim test behavior caused by the cyclic corticosterone model of recurring depressive episodes and that Reelin homeostasis is important for regulating stress-related inflammation. Future preclinical antidepressant research should incorporate models of multiple depressive episodes to improve the translation of preclinical rodent research to human depressive disorders.
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Dopamine and its 5 receptors, which are grouped into two families (D1-like and D2-like), modulate functions at a systemic level in both the central nervous system and periphery. The central nervous system and the immune system are the main adaptive systems, which participate in a continuous and functional crosstalk to guarantee homeostasis. On binding to its 5 dopamine receptors, dopamine acts as a co-regulator of the immune system, contributing to the interaction of the central nervous system and inflammatory events and as a source of communication between the different immune cells. Dopaminergic perturbations in the central nervous system are observed in several neurological and psychiatric disorders. Schizophrenia is one of the most common mental disorders with a poorly understood pathoaetiology that includes genetic and environmental components that promote alterations in the dopaminergic system. Interestingly, abnormalities in dopamine receptors expression in lymphocytes of schizophrenia patients have been reported, often significantly correlating with the severity of the psychotic illness. Here, we review the current literature regarding the dopaminergic system in human lymphocytes and its alterations in schizophrenia.
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The vulnerability and plasticity of hippocampal GABAergic interneurons is a topic of broad interest and debate in the field of epilepsy. In this experiment, we used the electrical kindling model of epilepsy to determine whether seizures that originate in different brain regions have differential effects on hippocampal interneuron subpopulations. Long-Evans rats received 99 electrical stimulations of the hippocampus, amygdala, or caudate nucleus, followed by sacrifice and immunohistochemical or western blot analyses. We analyzed markers of dendritic (somatostatin), perisomatic (parvalbumin), and interneuron-selective (calretinin) inhibition, as well as an overall indicator (GAD67) of interneuron distribution across all major hippocampal subfields. Our results indicate that kindling produces selective effects on the number and morphology of different functional classes of GABAergic interneurons. In particular, limbic kindling appears to enhance dendritic inhibition, indicated by a greater number of somatostatin-immunoreactive (-ir) cells in the CA1 pyramidal layer and robust morphological sprouting in the dentate gyrus. We also found a reduction in the number of interneuron-selective calretinin-ir cells in the dentate gyrus of hippocampal-kindled rats, which suggests a possible reduction of synchronized dendritic inhibition. In contrast, perisomatic inhibition indicated by parvalbumin immunoreactivity appears to be largely resilient to the effects of kindling. Finally, we found a significant induction in the number of GAD67-cells in caudate-kindled rats in the dentate gyrus and CA3 hippocampal subfields. Taken together, our results demonstrate that kindling has subfield-selective effects on the different functional classes of hippocampal GABAergic interneurons. J. Comp. Neurol. 525:389-406, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Neurônios GABAérgicos/fisiologia , Hipocampo/fisiopatologia , Interneurônios/fisiologia , Excitação Neurológica/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Western Blotting , Modelos Animais de Doenças , Epilepsia/fisiopatologia , Neurônios GABAérgicos/patologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Imuno-Histoquímica , Interneurônios/patologia , Masculino , Ratos , Ratos Long-EvansRESUMO
Seizures dramatically increase the number of adult generated neurons in the hippocampus. However, it is not known whether this effect depends on seizures that originate in specific brain regions or whether it is nonspecific to seizure activity regardless of origin. We used kindling of different brain sites to address this question. Rats received 99 kindling stimulations of the basolateral amygdala, dorsal hippocampus, or caudate nucleus over a 6-week period. After kindling, we counted the number of adult generated hippocampal neurons that were birth-dated with the proliferative marker bromodeoxyuridine (BrdU) to evaluate cell proliferation and survival under conditions of repeated seizures. Next, we counted the number of doublecortin immunoreactive (DCX-ir) cells and evaluated their dendritic complexity to determine if limbic and nonlimbic seizures have differential effects on neuronal maturation. We also quantified hippocampal brain-derived neurotrophin factor (BDNF) protein levels using an ELISA kit and assessed memory performance using a hippocampal-dependent fear conditioning paradigm. We found that limbic, but not nonlimbic, seizures dramatically increased hippocampal cell proliferation and the number of hilar-CA3 ectopic granule cells. Further, limbic kindling promoted dendritic outgrowth of DCX-ir cells and the number of DCX-ir cells containing basal dendrites. Limbic kindling also enhanced BDNF protein levels throughout the entire hippocampus and impaired the retrieval of fear memories. Collectively, our results suggest a relationship between limbic seizures, neurogenesis, BDNF protein, and cognition.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Excitação Neurológica/fisiologia , Neurogênese/fisiologia , Neurônios/efeitos da radiação , Convulsões/fisiopatologia , Animais , Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Proteína Duplacortina , Medo , Hipocampo/fisiopatologia , Hipocampo/efeitos da radiação , Excitação Neurológica/efeitos da radiação , Masculino , Memória/efeitos da radiação , Neurônios/metabolismo , Neurônios/fisiologia , Radiossensibilizantes , Ratos , Convulsões/metabolismoRESUMO
Reelin is an extracellular matrix protein that plays a critical role in neuronal guidance during brain neurodevelopment and in synaptic plasticity in adults and has been associated with schizophrenia. Reelin mRNA and protein levels are reduced in various structures of post-mortem schizophrenic brains, in a similar way to those found in heterozygous reeler mice (HRM). Reelin is involved in protein expression in dendritic spines that are the major location where synaptic connections are established. Thus, we hypothesized that a genetic deficit in reelin would affect the expression and function of dopamine D2 and serotonin 5-HT2A receptors that are associated with the action of current antipsychotic drugs. In this study, D2 and 5-HT2A receptor expression and function were quantitated by using radioligand binding studies in the frontal cortex and striatum of HRM and wild-type mice (WTM). We observed increased expression (p<0.05) in striatum membranes and decreased expression (p<0.05) in frontal cortex membranes for both dopamine D2 and serotonin 5-HT2A receptors from HRM compared to WTM. Our results show parallel alterations of D2 and 5-HT2A receptors that are compatible with a possible hetero-oligomeric nature of these receptors. These changes are similar to changes described in schizophrenic patients and provide further support for the suitability of using HRM as a model for studying this disease and the effects of antipsychotic drugs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Corpo Estriado/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Lobo Frontal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Dopamina D2/metabolismo , Serina Endopeptidases/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Feminino , Guanosina Trifosfato/análogos & derivados , Dietilamida do Ácido Lisérgico , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/genética , Ensaio Radioligante , Proteína Reelina , Serina Endopeptidases/genética , Radioisótopos de Enxofre , TrítioRESUMO
Epileptic seizures negatively affect cognition. However, the mechanisms that contribute to cognitive impairments after seizures are largely unknown. Here, we examined the effects of long-term kindling (i.e., 99 stimulations) of limbic (basolateral amygdala, dorsal hippocampus) and non-limbic (caudate nucleus) brain sites on conditioned fear and hippocampal plasticity. We first showed that kindling had no effect on acquisition of a hippocampal-dependent trace fear-conditioning task but limbic kindling impaired the retrieval of these fear memories. To determine the relationship between memory and hippocampal neuronal activity, we examined the expression of Fos protein 90 min after memory retrieval (i.e., 4 days after the last kindling stimulation). We found that limbic kindling, but not non-limbic kindling, decreased Fos expression in the granule cell layer, hilus, CA3 pyramidal cell layer, and CA1 pyramidal cell layer. Next, to investigate a mechanism that could contribute to dampen hippocampal neuronal activity in limbic-kindled rats, we focused on the endogenous anticonvulsant neuropeptide Y (NPY), which is expressed in a subset of GABAergic interneurons and can prevent glutamate release through interactions with its Y2 receptor. We found that limbic kindling significantly decreased the number of NPY-immunoreactive cells in several hippocampal subfields despite minimal staining of the neurodegenerative marker Fluoro-Jade B. However, we also noted that limbic kindling enhanced NPY immunoreactivity throughout the mossy fiber pathway. In these same regions, we observed limbic kindling-induced de novo expression of the NPY Y2 receptor. These novel findings demonstrate the site-specific effects of kindling on cognition and NPY plasticity, and they provide evidence that altered hippocampal NPY after limbic seizures coincides with dampened neural activity and cognitive impairments.
Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiopatologia , Núcleo Caudado/fisiopatologia , Medo/fisiologia , Hipocampo/fisiopatologia , Excitação Neurológica , Plasticidade Neuronal , Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Núcleo Caudado/metabolismo , Hipocampo/metabolismo , Masculino , Rememoração Mental/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Long-EvansRESUMO
We investigated the effect of two well characterized preclinical animal models of depression - repeated injections of corticosterone (CORT) and repeated restraint stress - on markers of GABAergic and glutamatergic activity in the hippocampus and amygdala. Stress is an identified risk factor for the onset of major depression, but the neurobiological mechanisms by which stress may produce depressogenic effects are not clear. Rats received one of the following four treatments for 21 consecutive days: daily single CORT injections (40mg/kg), daily single vehicle injections, daily 6h of restraint stress, or daily handling. After the 21-day stress period, all rats were sacrificed and hippocampal and amygdalar tissue was collected and prepared for Western blot analyses. We examined the effect of CORT and restraint stress on glutamate decarboxylase (GAD)-65 and GAD67, as well as the α1, α2, α3, and ß2-3 GABA(A) receptor subunits, and the vesicular glutamate transporter (VGLUT)-2. We found that CORT significantly decreased GAD65 and the α2 receptor subunit and increased VGLUT2 within the hippocampus. We also found that CORT decreased GAD67 and the α2 receptor subunit in the amygdala. However, restraint stress had no significant effect on protein expression in either the hippocampus or the amygdala. These findings parallel our previous results showing that repeated CORT injections, but not restraint stress, increase depression-like behavior in rats, and suggest that the depressogenic effects of CORT may be related to alterations in GABAergic and glutamatergic neurotransmission in stress-sensitive regions of the brain.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Corticosterona/farmacologia , Hipocampo/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Estresse Fisiológico/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Tonsila do Cerebelo/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Corticosterona/administração & dosagem , Depressão/metabolismo , Transtorno Depressivo/metabolismo , Modelos Animais de Doenças , Glutamato Descarboxilase/metabolismo , Hipocampo/metabolismo , Masculino , Ratos , Ratos Long-Evans , Restrição FísicaRESUMO
Temporal lobe seizures can induce the proliferation and abnormal migration of newly generated dentate granule cells, but little is known about the molecular mechanisms that govern these pathological events. Reelin and DISC1 (disrupted-in-schizophrenia 1) are proteins that play a regulatory role in the maturation and integration of new neurons in the developing and adult brain. In this study, we examined whether amygdala kindling results in aberrant neurogenesis and altered expression of reelin and DISC1 in the adult dentate gyrus. Using doublecortin immunohistochemistry, we found that short-term kindling (i.e., 30 electrical stimulations) significantly increased the number of immature neurons in the dentate subgranular zone (SGZ), whereas long-term kindling (i.e., 99 electrical stimulations) did not. However, doublecortin-labeled neurons in long-term kindled rats showed greater dendritic complexity than they did in short-term kindled or control rats. We also found that long-term kindling decreased the number of reelin-positive cells and decreased DISC1 expression in the dentate granule cell layer and subgranular zone. Interestingly, kindling-induced changes in reelin and DISC1 expression coincided with the appearance of ectopically located Prox1-labeled granule cells in the hilus. These effects occurred independently of alterations in granule cell layer length, dentate volume, or the number of hilar neurons. Taken together, these findings suggest a novel role for DISC1 in the pathophysiology of temporal lobe epilepsy and further suggest that changes in reelin and DISC1 expression may contribute to aberrant neurogenesis in the kindling model.
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Moléculas de Adesão Celular Neuronais/metabolismo , Giro Denteado/metabolismo , Giro Denteado/fisiopatologia , Regulação para Baixo/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Excitação Neurológica/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Serina Endopeptidases/metabolismo , Animais , Modelos Animais de Doenças , Proteína Duplacortina , Estimulação Elétrica/efeitos adversos , Epilepsia/patologia , Masculino , Naftalenos , Neurônios/metabolismo , Oxepinas , Ratos , Ratos Long-Evans , Proteína Reelina , Fatores de TempoRESUMO
A down-regulation of reelin and glutamic acid decarboxylase (GAD) 67 mRNAs was detected in gamma-aminobutyric acid (GABA)ergic cortical interneurons of schizophrenia (SZ) postmortem brains (10), suggesting that the availability of GABA and reelin may be decreased in SZ cortex. In situ hybridization of the mRNA encoding for DNA-methyltransferase 1, which catalyzes the methylation of promoter CpG islands, shows that the expression of this mRNA is increased in cortical GABAergic interneurons but not in pyramidal neurons of SZ brains. Counts of reelin mRNA-positive neurons in Brodmann's area 10 of either nonpsychiatric subjects or SZ patients show that the expression of reelin mRNA is decreased in layer-I, -II, and -IV GABAergic interneurons of SZ patients. These findings are consistent with the hypothesis that the increase of DNA-methyltransferase 1 expression in telencephalic GABAergic interneurons of SZ patients causes a promoter hypermethylation of reelin and GAD(67) and perhaps of other genes expressed in these interneurons. It is difficult to decide whether this dysfunction of GABAergic neurons detected in SZ is responsible for this disease or is a consequence of this disorder. Although at present we cannot differentiate between these two alternatives, it is important to consider that so far a molecular pathology of cortical GABAergic neurons appears to be the most consistent finding associated with SZ morbidity.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esquizofrenia/enzimologia , Esquizofrenia/genética , Telencéfalo/enzimologia , Adulto , Idoso , Estudos de Casos e Controles , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Humanos , Interneurônios/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso , Regiões Promotoras Genéticas , Proteína Reelina , Serina Endopeptidases , Ácido gama-Aminobutírico/metabolismoRESUMO
Most studies of 5-HT(2) receptor regulation have been carried out on the central nervous system (CNS) (which expresses 5-HT(2A) and 5-HT(2C) receptors); very few in vitro studies have addressed the peripheral receptors 5-HT(2A) and 5-HT(2B). The aim of this investigation was to compare the possible short- and long-term processes regulating these peripheral receptors in the rat. The in vitro contractile response elicited by serotonin (5-HT, 10 micro M) in the rat gastric fundus (5-HT(2B) receptor system) was rapid and followed by a partial fade to a steady state, in contrast with the rat thoracic aorta response (5-HT(2A) receptor system), which was more stable, slower and sustained. To characterize drug-receptor interactions, cumulative concentration/response curves (CCRCs) for 5-HT were constructed ex vivo for rat tissues treated with drugs acting at these receptors. Rats were examined 4 or 24 h after a single, i.p. administration of (+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(+/-)DOI, 1 or 2.5 mg/kg], clozapine, cyproheptadine or rauwolscine (10 mg/kg), 48 h after a single i.p. administration of (+/-)DOI (2.5 mg/kg), clozapine or cyproheptadine (10 mg/kg) or 24 h after the last of with 15 daily i.p. administrations of (+/-)DOI (1 or 2.5 mg/kg), clozapine, cyproheptadine or rauwolscine (10 mg/kg). In the aorta, E(max) (the maximum response elicited by 5-HT) was unchanged 4 h after a single dose of any of the drugs tested. However, 24 h after a single dose, E(max) was lower in animals treated with (+/-)DOI (2.5 mg/kg), clozapine or cyproheptadine than in controls, whilst 48 h after a single dose of (+/-)DOI (2.5 mg/kg), clozapine or cyproheptadine there was no difference in E(max) between experimental and control animals. After chronic treatment with (+/-)DOI (2.5 mg/kg), clozapine and cyproheptadine, E(max) was lower than in controls. In the gastric fundus, E(max) 4 h after a single dose of each drug was lower than in controls, and the response recovered by 24 or 48 h. Following chronic treatment, E(max) was significantly lower than in controls for each drug used. These findings suggest first, that regulation of peripheral 5-HT(2) receptors (5-HT(2A) and 5-HT(2B)) is a functionally significant phenomenon in vivo, and occurs after administration of both agonists and antagonists. Second, the kinetics of peripheral 5-HT(2) receptor regulation were similar in both in vivo and ex vivo experiments. The 5-HT(2B) receptors in rat gastric fundus are more sensitive to drug-induced regulation than the 5-HT(2A) rat aortic receptors. Finally, long-term regulation of both receptors stabilizes short-term desensitization for longer.
Assuntos
Músculo Liso/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Anfetaminas/farmacologia , Animais , Antipsicóticos/farmacologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Clozapina/farmacologia , Ciproeptadina/farmacologia , Fundo Gástrico/efeitos dos fármacos , Fundo Gástrico/metabolismo , Fundo Gástrico/fisiologia , Técnicas In Vitro , Ligantes , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Ioimbina/farmacologiaRESUMO
It is not currently clear whether the cortical atrophy observed in Huntington disease (HD) is entirely a direct consequence of the disease or at least partially a secondary consequence of striatal atrophy. This is of major importance for evaluating the possible therapeutic value of intrastriatal fetal-striatum grafts in HD. Cresyl violet-stained sections from rats that had received striatal excitotoxic lesions 1 wk or 4 wk previously showed small and statistically nonsignificant decreases in the thickness of cortical layers V and VI, while series from rats lesioned 12 months previously showed marked decreases in the thickness of the whole cortex (approximately 35% decrease), layer V (approximately 45%-50%) and layer VI (approximately 45%-50%), together with marked neuron loss in these layers. In deep layer V and layer VI, Fluoro-Jade staining showed labeled neurons in animals lesioned 1 wk previously, labeled neurons and astrocytes in animals lesioned 4 wk previously, and practically no labeling in animals lesioned 12 months previously. Intracortical injection of Phaseolus vulgaris leucoagglutinin revealed that corticostriatal fibers were practically absent from the lesioned area of striata lesioned 12 months previously. However, rats that received intrastriatal fetal-striatum grafts shortly after the lesion and were killed 12 months later showed a significant reduction in cortical atrophy, and a large number of labeled corticostriatal fibers surrounding and innervating the graft. In addition, a reduction in the number of Fluoro-Jade-labeled cells in the cortex was already apparent at 3 wk post-grafting. Regardless of whether HD has a primary effect on the cortex, the present results suggest that the striatal degeneration caused by HD contributes markedly to the cortical atrophy, and that intrastriatal grafts may ameliorate this secondary component of the cortical degeneration.
Assuntos
Encefalopatias/patologia , Córtex Cerebral/patologia , Corpo Estriado/patologia , Corpo Estriado/cirurgia , Transplante de Tecido Fetal , Doença de Huntington/cirurgia , Animais , Atrofia , Encefalopatias/induzido quimicamente , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/embriologia , Feminino , Ácido Ibotênico/farmacologia , Degeneração Neural/patologia , Degeneração Neural/cirurgia , Neurotoxinas/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
In order to study the most abundant GABAA receptor subtypes expressed in cholinergic, dopaminergic, noradrenergic and serotonergic neurons (i.e., in neurons of the so-called "global" projection systems), we employed double-immunocytochemical techniques combining the labeling of GABAA receptor alpha1, alpha2 and alpha3 subunit with markers for these cells. Cholinergic neurons in the striatum, habenula, and pedunculo-pontine nucleus were immunonegative for the alpha1 subunit, and most were also alpha2-immunonegative. However, cholinergic neurons in the striatum, septum and pedunculo-pontine nucleus were alpha3 immunopositive. Dopaminergic neurons in the substantia nigra pars compacta were highly immunopositive for the alpha3, and noradrenergic neurons in the locus coeruleus were immunoreactive for the alpha3 and the alpha2-subunit; although neurons of these areas were negative for alpha1. Similarly, serotonergic neurons in raphe also showed a high level of labeling of alpha3, while there was a lack of immunoreactivity for the alpha1-subunit, and only some individual neurons were positive for the alpha2 subunit. As the presence of different alpha-subunits confers specific physiological and pharmacological properties to GABAA receptors, the abundance of receptors containing the alpha3 subunit (and the scarcity of receptor subtypes including the other alpha-subunits studied) may have important implications for the GABAergic regulation of brain "global" or "diffuse" projection systems.
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Encéfalo/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Serotonina/metabolismo , Adrenérgicos , Animais , Biomarcadores , Colina O-Acetiltransferase/metabolismo , Colinérgicos , Dopaminérgicos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Neurônios/metabolismo , Neurotransmissores , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Serotoninérgicos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The morphological characteristics, distribution and neurochemical phenotype of striatal neuronal subtypes expressing the GABA(A) receptor alpha3-subunit were investigated using immunocytochemical and immunofluorescent techniques with an antibody specific for this subunit. alpha3-immunopositive neurons were infrequent in the rat striatum, but two morphologically different subtypes were observed: Cholinergic neurons, and a second cellular type that may correspond to neurogliaform neurons, although it may also be a novel subtype of striatal interneuron. To identify the second cellular subtype, co-localization of the GABA(A) receptor alpha3-subunit with markers of different classes of striatal interneurons was studied using specific antibodies. It was found that there was a lack of co-localization between all interneuronal markers used in this study and the alpha3-subunit. Although the alpha3-subunit immunopositive neurons represent a small percentage of the total of striatal neuronal populations, they may play an important role in the regulation of the microcircuitry of the striatum.
Assuntos
Corpo Estriado/citologia , Corpo Estriado/metabolismo , Proteínas do Tecido Nervoso , Neurônios/citologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Animais , Biomarcadores , Colina O-Acetiltransferase/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina , Feminino , Imuno-Histoquímica , Interneurônios/metabolismo , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
To compare the expression of GABAA receptor subunits in the normal substantia nigra and in fetal mesencephalic neurons ectopically transplanted into the dopamine-depleted striatum, we have employed single and double immunocytochemical approaches using tyrosine hydroxylase (TH) and alpha 1, alpha 2, alpha 3, and beta 2/3 GABAA receptor subunit specific antibodies. In the substantia nigra, alpha 1 and beta 2/3 GABAA receptor subunits were labeled in processes in the pars compacta (SNc) and, more intensely, in both somata and processes in the pars reticulata (SNr). There was no clear TH and alpha 1 or beta 2/3 colocalization, with the exception of some TH-immunoreactive (-ir) neurons that showed a weak immunoreactivity for beta 2/3. Sections immunolabeled for alpha 2 showed a faint diffuse labeling for this subunit both in the SNr and in the SNc. Scattered somata were immunopositive for alpha 2, and some of them were also TH-ir. The labeling for alpha 3 and TH showed that TH-positive neurons expressed intense alpha 3 immunoreactivity, although some TH-negative somata in the SNr expressed weak alpha 3 immunoreactivity. In the transplants, double immunostaining procedures showed that the labeling for alpha 1 or beta 2/3 appeared particularly concentrated in patches of intensely immunoreactive neuronal processes that surrounded TH-ir cells, but these processes were not TH-ir. In the case of alpha 2, diffuse immunostaining was observed all over the graft, with some scattered positive somata. Only a few of them were also TH positive. Sections immunoreacted for alpha 3 and TH revealed that TH-ir neurons expressed intense alpha 3 immunoreactivity, and that only a few TH-negative neurons were weakly positive for alpha 3. These results show that mesencephalic tissue ectopically grafted into the striatum develops a pattern of GABAA receptor expression similar to that normally expressed in situ, and particularly that the grafted dopaminergic neurons express similar GABAA receptors, including the alpha 3 subunit. This might be due to the similarity of GABAergic afferents to these neurons in the SNc and the graft, or that at the time of transplantation this expression had already been determined.
Assuntos
Transplante de Tecido Encefálico/fisiologia , Mesencéfalo/metabolismo , Neostriado/metabolismo , Receptores de GABA-A/biossíntese , Animais , Transplante de Tecido Fetal/fisiologia , Hidroxidopaminas/farmacologia , Imuno-Histoquímica , Mesencéfalo/transplante , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The mechanisms by which dopaminergic and glutamatergic inputs interact to regulate striatal neuropeptide expression during physiological motor activity are poorly understood. In this work, striatal expression of preprotachykinin (PPT) and preproenkephalin (PPE) mRNA was studied by in situ hybridization in rats killed 2 h after treadmill running (36 m/min for 20 min). Treadmill running induced a significant increase in the levels of both PPT (60% increase) and PPE (90% increase) mRNA in the striatum of normal rats. The increase in the level of PPT mRNA was blocked in rats previously subjected to nigrostriatal deafferentation (i.e., 6-hydroxydopamine lesion) or pretreated with D1-receptor antagonist SCH-23390 (0.1 mg/kg), the D2-receptor antagonist eticlopride (0.5 mg/kg), or the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 (0.1 mg/kg). The running-induced increase in the level of PPE mRNA was blocked in rats pretreated with SCH-23390 or MK-801. Rats subjected to nigrostriatal deafferentation or pretreated with eticlopride showed an increase in PPE mRNA levels (around 150% and 40% increase, respectively), that was enhanced by running (around 230% and 160% increase, respectively). These results suggest that locomotor activity increases, in a NMDA receptor dependent fashion, the excitatory influence of the corticostriatal glutamatergic system on the two populations of striatal projection neurons, as reflected by increases in the levels of PPT and PPE mRNA. The results obtained after dopamine depletion or injection of dopamine receptor antagonists suggest that a concomitant increase in dopamine release may enhance PPT mRNA level in striatonigral neurons via D1 receptors, and reduce PPE mRNA level in striatopallidal neurons via D2 receptors. Additionally, levels of dopamine and glutamate may be regulated by other complex indirect mechanisms.
Assuntos
Corpo Estriado/metabolismo , Dopamina/fisiologia , Encefalinas/biossíntese , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/fisiologia , Atividade Motora/genética , Proteínas do Tecido Nervoso/biossíntese , Precursores de Proteínas/biossíntese , RNA Mensageiro/biossíntese , Taquicininas/biossíntese , Animais , Apomorfina/farmacologia , Benzazepinas/farmacologia , Denervação , Maleato de Dizocilpina/farmacologia , Antagonistas de Dopamina/farmacologia , Encefalinas/genética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Proteínas do Tecido Nervoso/genética , Oxidopamina/toxicidade , Precursores de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Corrida/fisiologia , Taquicininas/genéticaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP-32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP-32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP-32-positive neurons and TH-immunoreactive (TH-ir) fibers. In 1-week-old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH-positive fibers, the ones that were seen were located in GDNF-positive areas. This was clearly evident in 2-week-old transplants, where TH-ir fibers appeared selectively concentrated in GDNF-positive patches. This pattern was repeated in 3-week-old grafts. In co-transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH-ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH-ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF. These results demonstrate that GDNF-ir is coincident with that for TH and DARPP-32, and suggest that GDNF release by fetal striatal neurons both in normal development and in developing striatal grafts may have not only a trophic but also a tropic influence on TH-ir fibers and may be one of the factors that regulate dopaminergic innervation of the striatum.
Assuntos
Animais Recém-Nascidos/crescimento & desenvolvimento , Corpo Estriado/fisiologia , Dopamina/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/fisiologia , Neurônios Aferentes/transplante , Animais , Animais Recém-Nascidos/metabolismo , Animais Recém-Nascidos/fisiologia , Transplante de Células , Corpo Estriado/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Mesencéfalo/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Reelin (Reln) is a protein with some structural analogies with other extracellular matrix proteins that functions in the regulation of neuronal migration during the development of cortical laminated structures. In the cortex of adult animals, Reln is expressed primarily in gamma-aminobutyric acid (GABA)ergic neurons and is secreted into perineuronal nets. However, only 50-60% of GABAergic interneurons express Reln. We have characterized this subpopulation of cortical GABAergic neurons that expresses Reln by using two strategies: (i) a double immunolabeling procedure to determine the colocalization of Reln with neuropeptides and Ca2+-binding proteins and (ii) a combination of Golgi staining and Reln immunolabeling to determine the morphology of the rat cortical cells that store Reln. Many interneurons that express Neuropeptide Y (NPY) or somatostatin (but none of those that express parvalbumin) are Reln-immunopositive. A small population of calbindin-positive interneurons and very few calretinin-positive cells express Reln immunopositivity. Golgi staining revealed that layer I horizontal cells, layer II-V bitufted neurons, and some deep cortical layer Martinotti cells express Reln. Basket and chandelier cells are often immunopositive to parvalbumin, but never to Reln. Although Reln is secreted by GABAergic neurons, its target are not the GABA receptors, but rather may be extrasynaptically located in perineuronal nets and concerned with the modulation of neuronal plasticity. Dab1, the target adapter protein that presumably mediates transcription regulation via the extrasynaptic actions of Reln, is expressed predominantly in pyramidal neurons, but it can also be detected in a small population of GABAergic neurons that are neither horizontal nor bitufted neurons.
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Córtex Cerebral/fisiologia , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/fisiologia , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Endogâmicos F344 , Proteína Reelina , Serina Endopeptidases , Sinapses/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
The combination of in situ hybridization and immunocytochemical technique is an important tool to detail the biochemical phenotype of individual neurons. In this work, we have developed a double fluorescence method to show the presence of reelin mRNA in GABAergic cells. This was achieved by demonstrating the colocalization of glutamic acid decarboxylase67, the synthesizing enzyme for GABA, with the mRNA for reelin, a novel factor involved in brain development and possibly the maintenance of the synaptic organization of layered structures in adult brain. The results demonstrated that reelin is expressed primarily in GABAergic cells in the adult rat cerebrum, but not in the cerebellum.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Animais , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/química , Neurônios/enzimologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Proteína Reelina , Serina EndopeptidasesRESUMO
A between-side comparison of GABAA receptor subunit expression levels in the globus pallidus and anterior-pole motor thalamic nuclei of rats with an ibotenate lesion of the striatum, and rats receiving a fetal striatal graft in the lesioned area was made by using immunocytochemistry with subunit-specific antibodies, at different times post-lesion or different times post-grafting. At 10 days post-lesion, there was already an increase in the labeling of the alpha 1- and beta 2/3-subunits in the globus pallidus, entopeduncular nucleus and ventrolateral nucleus ipsilateral to the lesion when compared with the contralateral side, while there were no significant changes at the level of the ventromedial nucleus. Labeling of the alpha 2-subunit showed a clear increase in the entopeduncular nucleus compared with the contralateral side at 10 days post-lesion. Similar changes were also observed for the different subunits studied at 30 and 120 days after lesioning. Rats with 20-day old transplants of fetal striatal neurons that were implanted in the ibotenate lesioned striatum at 10 days post-lesioning, continued to show changes in the expression of GABAA receptor subunits, albeit at a lower level than those of ibotenate lesioned rats at similar age post-lesion. However, when examining rats with 70-day old transplants, the ibotenate-lesion induced between-side changes were almost completely compensated. These findings suggest a correlation between the maturation of the grafts and their capability to function in reestablishing neuronal circuits as shown by the reduction of changes in GABAergic transmission induced by ibotenate lesions, as indicated by the reversal of changes in GABAA receptor subunit in several areas of the basal ganglia circuit.
Assuntos
Corpo Estriado/transplante , Transplante de Tecido Fetal , Globo Pálido/química , Fragmentos de Peptídeos/análise , Receptores de GABA-A/química , Tálamo/química , Animais , Gânglios da Base/química , Corpo Estriado/embriologia , Corpo Estriado/fisiologia , Ácido Ibotênico/toxicidade , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Neurônios/transplante , RatosRESUMO
During embryonic development of brain laminated structures, the protein Reelin, secreted into the extracellular matrix of the cortex and hippocampus by Cajal-Retzius (CR) cells located in the marginal zone, contributes to the regulation of migration and positioning of cortical and hippocampal neurons that do not synthesize Reelin. Soon after birth, the CR cells decrease, and they virtually disappear during the following 3 weeks. Despite their disappearance, we can quantify Reelin mRNA (approximately 200 amol/ g of total RNA) and visualize it by in situ hybridization, and we detect the translated product of this mRNA by immunocytochemistry preferentially in gamma-aminobutyric acid (GABA)ergic neurons of adult rat cortex and hippocampus. In adult rat cerebellum, Reelin is expressed in glutamatergic neurons (granule cells). The translated product of this mRNA is readily exported from the granule cell somata to the parallel fibers, where it has been detected by electron microscopy in axon terminals located presynaptically to Purkinje cell dendrites.