RESUMO
The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Etanol/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sinapses/efeitos dos fármacos , Sinapses/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Forma do Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/fisiologia , Ratos Wistar , Sinapses/fisiologiaRESUMO
Recent research involving human and animals has shown that aerobic exercise of moderate intensity produces the greatest benefit on brain health and behavior. In this study we investigated the effects on cognitive function and anxiety-related behavior in rats at different ages of aerobic exercise, performed regularly throughout life. We designed an aerobic training program with the treadmill running following the basic principles of human training, and assuming that rats have the same physiological adaptations. The intensity was gradually adjusted to the fitness level and age, and maintained at 60-70% of maximum oxygen consumption (max.VO(2)). In middle age (8 months) and old age (18 months), we studied the cognitive response with the radial maze (RM), and anxiety-related behaviors with the open field (OF) and the elevated plus maze (EPM). Aerobically trained (AT) rats had a higher cognitive performance measured in the RM, showing that exercise had a cumulative and amplifier effect on memory and learning. The analysis of age and exercise revealed that the effects of aerobic exercise were modulated by age. Middle-aged AT rats were the most successful animals; however, the old AT rats met the criteria more often than the middle-aged sedentary controls (SC), indicating that exercise could reverse the negative effects of sedentary life, partially restore the cognitive function, and protect against the deleterious effects of aging. The results in the OF and EPM showed a significant decrease in key indicators of anxiety, revealing that age affected most of the analyzed variables, and that exercise had a prominent anxiolytic effect, particularly strong in old age. In conclusion, our results indicated that regular and chronic aerobic exercise has time and dose-dependent, neuroprotective and restorative effects on physiological brain aging, and reduces anxiety-related behaviors.
Assuntos
Envelhecimento/psicologia , Ansiedade/psicologia , Cognição/fisiologia , Condicionamento Físico Animal/fisiologia , Limiar Anaeróbio/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Defecação/fisiologia , Asseio Animal/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Memória de Curto Prazo/fisiologia , Atividade Motora/fisiologia , Destreza Motora/fisiologia , Plasticidade Neuronal/fisiologia , Equilíbrio Postural/fisiologia , Ratos , Ratos WistarRESUMO
2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.
Assuntos
Ácido 2,4-Diclorofenoxiacético/toxicidade , Anfetamina/farmacologia , Cerebelo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Ácido 2,4-Diclorofenoxiacético/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fármacos Neuroprotetores/farmacologia , Gravidez , Ligação Proteica/fisiologia , Ratos , Ratos WistarRESUMO
The functional expression of neuronal CB2 cannabinoid receptors (CB2-Rs) in the brain has been controversial. We and others have now demonstrated that CB2-Rs are expressed in neurons and glial cells in the brain. However, the subcellular localization of these receptors has not been characterized. In this study we used immunohistochemical electron microscopy to determine the subcellular distribution of CB2-Rs in two brain regions. Brain sections from the CA1 hippocampal area and substantia nigra were immunostained for CB2-Rs and analyzed by electron microscopy. In each region immunoperoxidase labeling for CB2-Rs was detected in neurons as well as in glial and endothelial cells. In neuronal cells, CB2-R immunoreactivity was observed in somata and large and medium-sized dendrites. In the soma, the CB2-R labeling was mainly associated with the rough endoplasmic reticulum and Golgi apparatus, suggesting its endogenous synthesis. In the dendrites, the CB2-R labeling was observed in the cytoplasm and was associated with the plasma membrane near the area of synaptic contact with axon terminals, indicating a postsynaptic distribution of these receptors. In CB2-Rs in immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. In the substantia nigra, some unmyelinated axons were immunoreactive for CB2-Rs, but we rarely found CB2-R-labeled axon terminals. These results extend our previous detection of postsynaptic cortical CB2-Rs and provide additional ultrastructural evidence that CB2-Rs are mainly postsynaptic in the CA1 area of the hippocampus and substantia nigra. The functional implication of pre- and/or postsynaptic localization of CB2-Rs remains to be determined.
Assuntos
Química Encefálica , Encéfalo/ultraestrutura , Neurônios/química , Receptor CB2 de Canabinoide/análise , Animais , Encéfalo/citologia , Dendritos/química , Células Endoteliais/química , Células Endoteliais/ultraestrutura , Hipocampo/citologia , Masculino , Neuroglia/química , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/química , Ratos , Ratos Sprague-Dawley , Substância Negra/citologiaRESUMO
Dopaminergic neurons from the midbrain nuclei substantia nigra (SN; A9) and ventral tegmental area (VTA; A10) were investigated by tyrosine hydroxylase (TH) immunostaining in neonate rat brains exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through lactation. Dorsal raphe serotonin (5-HT) projections to SN and VTA were also studied by 5-HT transporter (5-HTT) immunostaining and results were quantified by image analysis. Twenty-five-day-old pups exposed to 2,4-D through mothers milk were used. Dams were intraperitoneally administered 70 or 100mg/kg/day of 2,4-D from the 9th to the 25th postpartum day. After 100mg/kg of 2,4-D exposure, a 25% diminution in the SN and a 33% diminution in the VTA neurons' TH immunostaining along with a significantly 5-HT fiber density diminution were observed. The present work supports previous reports which suggest that exposure to 2,4-D during development has multiple effects on CNS.
Assuntos
Ácido 2,4-Diclorofenoxiacético/toxicidade , Lactação/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/análise , Animais , Animais Recém-Nascidos , Feminino , Imunoquímica , Neurônios/enzimologia , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.
Assuntos
Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Animais , Fenclonina/farmacologia , Masculino , NADPH Desidrogenase , Neurônios/química , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/efeitos dos fármacos , Núcleos da Rafe/química , Núcleos da Rafe/efeitos dos fármacos , Ratos , Ratos Wistar , Serotonina/análise , Antagonistas da Serotonina/farmacologiaRESUMO
2,4-D is a chlorophenoxyherbicide used worldwide. We have studied the morphological alterations of 5-HT neurons and glial cells in the mesencephalic nuclei of adult rats exposed to 2,4-D both perinatally (during pregnancy and lactation) and chronically (during pregnancy, lactation and after weaning) with quantitative methods. Pregnant rats were daily exposed to 70 mg/kg of 2,4-D from gestation day (GD) 16 to post-natal day (PND) 23 through diet. After weaning, pups were assigned to one of two sub-groups: T1 (fed with untreated diet until PND 90) and T2 (maintained with 2,4-D diet until PND 90). Brain sections were immunocytochemically stained using polyclonal anti-5-HT, anti-GFAP and anti-S-100 protein antibodies as cells markers. 2,4-D exposure during pregnancy and lactancy (T1 group) produced an increase in 5-HT neuronal area and immunoreactivity (IR) in the mesencephalic nuclei studied. However, with the chronical 2,4-D exposure (T2 group) only the 5-HT neuronal area from the dorsal raphe nucleus (DRN) was increased, suggesting an adaptable response of 5-HT neurons in median raphe nucleus (MRN). The presence of reactive astrocytes in mesencephalic nuclei and in hippocampus were also different for the two 2,4-D exposure designs, showing the existence of a correspondence between neuronal changes and astrogliosis. Results support evidences that 2,4-D alters the serotoninergic system and that 5-HT neurons of each mesencephalic nuclei show different responses to the 2,4-D exposure designs which are parallel to astrogliosis.
Assuntos
Ácido 2,4-Diclorofenoxiacético/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Encéfalo/citologia , Herbicidas/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Serotonina/fisiologia , Animais , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/ultraestrutura , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Indicadores e Reagentes , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/patologia , Núcleos da Rafe/ultraestrutura , Ratos , Proteínas S100/metabolismoRESUMO
Serotonin (5HT) is involved in the development and plasticity of the CNS through the release of S-100beta, a glial trophic factor which stabilizes synapses and neuronal cytoskeleton and promotes neuronal development. S-100beta is released from glial cells after activation of glial 5HT(1A) receptors. We present in this paper the effects upon neurons and glia of a 5HT depletion induced by 14 days of treatment with para-chlorophenylalanine (PCPA) in adult rats. S-100beta, 5HT, 5HT-transporter (5HT-T) and neurofilaments (Nf-200 and Nf-68) expressions were studied by immunohistochemistry and image analysis in striatum, hippocampus, parietal and frontal cortex. Immediately after ending PCPA treatment we found increased intracellular S-100beta immunoreactivity in glial cells, reduced 5HT immunolabelling, reduced density of 5HT-T, Nf-200 and Nf-68 fibers and morphological alterations in neuronal cytoskeleton. One week after PCPA treatment S-100beta immunoreactivity decreased towards control levels, 5HT was normalized in dorsal raphe nucleus, but not in innervation areas; 5HT-T, Nf-200 and Nf-68 fiber densities increased but some neuronal cytoskeletal alterations were still present in striatum. Two weeks after PCPA treatment S-100beta had returned to control levels in most studied regions; 5HT immunoreactivity was normalized, meanwhile 5HT-T, Nf-200 and Nf-68 fiber densities increased reaching values over the control level. We propose that S-100beta could be accumulated in glial cells during the 5HT depletion period, to be released once 5HT levels have recovered. Neuronal cytoskeletal alterations and reduced fiber density may be the expression of decreased extracellular availability of S-100beta. Conversely, increased 5HT-T, Nf-200 and Nf-68 expressions, once S-100beta is normalized, may be the biological response to the growth factor release.
Assuntos
Comunicação Celular/fisiologia , Fenclonina/farmacologia , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neuroglia/fisiologia , Proteínas S100 , Antagonistas da Serotonina/farmacologia , Serotonina/deficiência , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100 , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
Nitric oxide (NO) is a gas involved in neurotransmission in the central nervous system (CNS) and in vertebrate retinas. This paper describes five types of nitrergic neurons in developing and adult chick retina using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reaction. Three of them, nitrergic types 1, 2 and 3, were observed in the inner nuclear layer, while nitrergic type 4 was observed in the ganglion cell layer; nitrergic type 5 were the retinal photoreceptors. Cell processes formed four nitrergic networks, which could be observed in the inner plexiform layer (IPL), at sublayers 1, 3a, 3b and 4. Another nitrergic network was observed in the outer plexiform layer (OPL). From hatching, the dendritic branches were completely developed in the IPL and in the OPL, forming the mentioned networks. Current evidence suggests that NO is coexpressed with other neurotransmitters in neurons of the CNS. Double-staining procedures, using NADPHd and 5HT immunohistochemistry in chicken retina, in a sequential or in an alternative manner, did not reveal the coexistence of these two neurotransmitters in the same neurons, but their networks matched in sublayers 1 and 4 of the IPL.
Assuntos
Neurônios Aferentes/enzimologia , Óxido Nítrico/análise , Células Fotorreceptoras de Vertebrados/enzimologia , Retina/embriologia , Animais , Tamanho Celular , Embrião de Galinha , Galinhas , NADPH Desidrogenase/análise , Neurônios Aferentes/química , Neurônios Aferentes/citologia , Células Fotorreceptoras de Vertebrados/química , Células Fotorreceptoras de Vertebrados/citologia , Retina/citologia , Retina/enzimologia , Serotonina/análiseRESUMO
Tryptamine, a serotonin-related indolamine, could be involved in the modulation of catecholaminergic and serotoninergic systems interaction. Despite previous reports on this topic, the morphological relationship among these systems is not well described. We studied the interaction among serotoninergic and catecholaminergic with tryptaminergic systems by double immunostaining at the level of light microscopy. Mesencephalic rat brain sections treated according to the Schiff quenching method were double immunostained using peroxidase and fluorescein labeled antibodies. Primary antibodies to anti-tryptophan hydroxylase (TrpOH), anit-tyrosine hydroxylase (TH) and anti-tryptamine (T) were used to demonstrate serotoninergic, catecholaminergic and tryptaminergic neurons respectively. A morphometric study was performed in order to analyze the different morphological characteristics of each system. The results showed that (i) T+ and TrpOH+ neurons are localized in the same areas but their morphology is significantly different. Moreover morphometric parameters of T+ neurons were significantly different from those TrpOH+ or TH+ neurons; (ii) The number of TrpOH+ neurons was larger than T+ neurons; (iii) T+ neurons were dominant in the lateral dorsal raphe nucleus. TrpOH+ neurons were more numerous in the central area of the dorsal raphe nucleus; (iv) Coexpression of TrpOH and T was demonstrated in the somata of dorsal raphe nucleus neurons; (v) TrpOH+ neurons from raphe nuclei and TH+ neurons from substantia nigra are contacted by T+ fibres. The present morphological evidence supports a functional relationship among these three aminergic systems.
Assuntos
Química Encefálica , Dopamina/análise , Serotonina/análise , Triptaminas/análise , Animais , Anticorpos , Especificidade de Anticorpos , Reações Cruzadas , Dopamina/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas Imunoenzimáticas , Masculino , Núcleos da Rafe/química , Ratos , Ratos Wistar , Serotonina/imunologia , Substância Negra/química , Triptaminas/imunologiaRESUMO
The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc
Assuntos
Animais , Masculino , Coelhos , Ratos , Epêndima/citologia , Epêndima/química , Serotonina , Imuno-Histoquímica , Microscopia Imunoeletrônica , Fibras Nervosas , Ratos WistarRESUMO
The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc
Assuntos
Animais , Masculino , Coelhos , Ratos , Epêndima/química , Epêndima/citologia , Serotonina/análise , Imuno-Histoquímica , Microscopia Imunoeletrônica , Fibras Nervosas/química , Fibras Nervosas/ultraestrutura , Ratos WistarRESUMO
The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc.
Assuntos
Epêndima/química , Epêndima/citologia , Serotonina/análise , Animais , Cebus , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Fibras Nervosas/química , Fibras Nervosas/ultraestrutura , Coelhos , Ratos , Ratos WistarRESUMO
Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde-induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic-bipolar neurons are also described. SHT-amacrine neurons have round or pear-shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1-the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL-the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not "true" 5HT neurons.
Assuntos
Neurônios/química , Retina/química , Serotonina/análise , Animais , Diferenciação Celular/fisiologia , Embrião de Galinha , Fenótipo , Retina/embriologiaRESUMO
Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mother's milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.
Assuntos
Ácido 2,4-Diclorofenoxiacético/efeitos adversos , Encéfalo/patologia , Gliose/induzido quimicamente , Herbicidas/efeitos adversos , Lactação/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Química Encefálica , Feminino , Proteína Glial Fibrilar Ácida/imunologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Coloração e RotulagemRESUMO
Serotonin (5HT) is one of the classical neurotransmitters expressed earlier in the embryonic rat brain, and it was proposed as a developmental signal in the central nervous system. In the adult brain, 5HT seems to be involved in neuronal plasticity. It was postulated that S-100 protein, a glial neurotrophic factor, could be modulated by 5HT probably through the glial 5HT1A receptors. In a model of chronic inhibition of endogenous 5HT synthesis produced by the daily administration of parachlorophenylalanine (PCPA) for 2 wk, we have studied by immunohistochemical methods and digital morphometric analysis the expression of two proteins present in rat brain astrocytes: glial fibrillary acidic protein (GFAP) and S-100 protein. The effectiveness of the PCPA treatment was tested by the use of specific anti-5HT antibodies that showed absence of 5HT fibers in 5HT innervation areas like frontal cortex and hippocampus. Different effects of PCPA treatment on serotoninergic raphe nuclei were observed: dorsal raphe nucleus (DRN) seemed to be more sensitive to the PCPA's action than ventral raphe nucleus (VRN). In DRN and in the two 5HT innervation areas studied, glial cells responded to the 5HT depletion induced by PCPA showing astrocytes with large and tortuous processes. Astrocytes from 5HT-depleted regions showed higher immunostaining for S-100 protein than controls. There was not any modification in optical density of S-100 protein immunostaining in VRN, the area less sensitive to PCPA treatment. These observations indicated that astrocytes are sensitive to the 5HT level, and in presence of low 5HT concentration in the intercellular space, astrocytes could react by synthesizing glial proteins like GFAP and S-100 protein.
Assuntos
Astrócitos/metabolismo , Fenclonina/farmacologia , Neurônios/metabolismo , Antagonistas da Serotonina/farmacologia , Serotonina/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/metabolismo , Ratos , Ratos Wistar , Proteínas S100/metabolismoRESUMO
The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.
Assuntos
Técnica Direta de Fluorescência para Anticorpo , Glutaral , Compostos de Sulfidrila , Fixação de Tecidos/métodos , Animais , Anticorpos , Boroidretos , Corantes , Reagentes de Ligações Cruzadas , Fluoresceína-5-Isotiocianato , Técnica Direta de Fluorescência para Anticorpo/normas , Proteína Glial Fibrilar Ácida/imunologia , Glutaral/química , Indicadores e Reagentes , Masculino , Mesencéfalo/química , Mesencéfalo/citologia , Oxirredução , Proteínas/análise , Ratos , Ratos Wistar , Coloração e RotulagemRESUMO
This paper describes the development of the serotonergic innervation of the chick tectum opticum as revealed by an immunohistochemical methodology. The development of this innervation was previously described simply as the formation of an irregular network of serotonergic fibers that gradually invades the organ and increases in density. Our results show that the developmental pattern of serotonergic innervation differs significantly through the distinct tectal layers and that it progresses through a characteristic temporospatial pattern related to the lamination process. These findings support the idea that the concept of laminar segregation can be applied to describe the development of the serotonergic innervation. On the other hand, it is clear that the existence of a typical ordered developmental pattern of innervation makes it possible to detect embryonic or post-hatching alterations. Thus, the tectal serotonergic innervation could be used as a suitable model to investigate possible plastic changes in experimental conditions.
Assuntos
Serotonina/fisiologia , Colículos Superiores/embriologia , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Embrião de Galinha , Imuno-Histoquímica , Neuroglia/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Colículos Superiores/citologia , Sinapses/fisiologiaRESUMO
Several ontogenetic studies have been devoted to the structural organization of the developing tectum opticum. They disagree in many respects because they are based on histological preparations performed with differently oriented planes of section. According to our results the differences found in the literature mainly result from the fact that the developmental gradient axis undergoes remarkable positional changes with respect to both optic lobe and neural tube longitudinal anatomical axes during the early stages of development. The present work is a dynamic description of the tectum opticum lamination based on sections coinciding with the developmental gradient. Since this latter displays a curved disposition, several slightly modified planes of section had to be used to obtain a complete picture along the developmental gradient. The development of the tectal architecture proceeds from a relatively simple organization through increasingly complex multilaminated patterns. A dynamic interpretation of successive images of a particular region observed at increasing developmental stages or of images observed at a particular stage along the entire length of the developmental gradient axis, allows us to propose that embryonic laminae are only transient spatial arrangements of cells actively migrating from the sites where they were generated to those where they will definitively reside. These considerations led us to define a nomenclature that establishes clear correlations between the early transient organizations and the definitive one of the fully developed optic tectum. This type of nomenclature could be usefully applied to describe dynamically the development of structures displaying multilaminated patterns such as other cortical zones of the central nervous system.
Assuntos
Galinhas/fisiologia , Colículos Superiores/anatomia & histologia , Colículos Superiores/embriologia , Animais , Embrião de Galinha , Terminologia como AssuntoRESUMO
Embryo and adult rat spinal cord sections immunostained for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), or phosphorylated 160 kDa neurofilament protein (NF), were used to study glial participation in nerve tissue assembly. Radial glial fibers (RGF) expressed vimentin since E12. In the peripheral ventral zone, RGF formed cephalocaudal plates (CP) within the white matter, ensheathing developing axonal tracts first expressing NF at E13. As axonal tracts increased, CP became denser and extended ventrolaterally. S-100 protein appeared at E17 in the midline and, at E18, it overlapped vimentin expression. Astrocytes expressed GFAP since E20. We conclude that the pattern found in the spinal cord results from interactions between developing axonal tracts and radial glia, and that it could set the structural basis for the organized assembly of the spinal cord found in adults.