RESUMO
We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.
Assuntos
Córtex Cerebral/química , Ácido gama-Aminobutírico/análise , Animais , Química Encefálica , Endopeptidase K , Resinas Epóxi , Microscopia Imunoeletrônica , Ratos , Sensibilidade e Especificidade , Serina Endopeptidases , Coloração e RotulagemRESUMO
Previous studies showed that various stressors can induce delayed (days) and long-lasting (weeks) increases of vasopressin (AVP) stores in terminals of CRH neurons in the external zone of the median eminence (ZEME) in adult rats. Here we tested whether this long-lasting neuroplastic change can be induced by mechanisms other than stressor provoked transsynaptic activation of CRH neurons. Single i.v. administration of a CRH antibody to adult rats causes a delayed (at least 1 day) and long-lasting (3 weeks) increase (2-3 fold) of AVP stores in the ZEME without affecting CRH stores. It suppresses ether-induced ACTH-responses for at least 8 days. In contrast, resting pm levels of ACTH and corticosterone (CORT) were suppressed only during the first 2 days. Suppletion of CORT levels on day 1 and 2, attenuates the antibody induced AVP-increase by 57%. CRH-immunoneutralization did not affect the AVP stores in CORT supplemented ADX rats. Thus, long-term increases of AVP stores induced by CRH-immunoneutralization largely depend on short-term suppression of pm CORT levels. Accordingly, single administration of metyrapone, which causes a transient suppression of pm CORT levels, increases AVP (1.5 fold) but not CRH stores one week later. We conclude that transient activation of hypothalamic CRH neurons results in long-lasting increases in AVP co-expression irrespective of the nature of the activating stimulus.
Assuntos
Arginina Vasopressina/metabolismo , Corticosterona/sangue , Hormônio Liberador da Corticotropina/fisiologia , Hipotálamo/citologia , Neurônios/metabolismo , Adrenalectomia , Hormônio Adrenocorticotrópico/sangue , Animais , Arginina Vasopressina/análise , Corticosterona/farmacologia , Hormônio Liberador da Corticotropina/análise , Hormônio Liberador da Corticotropina/imunologia , Éter , Imunização Passiva , Masculino , Eminência Mediana/química , Metirapona/farmacologia , Ratos , Ratos Wistar , Estresse Fisiológico/sangue , Estresse Fisiológico/induzido quimicamenteRESUMO
Recently, we demonstrated that single administration of interleukin-1 beta (IL-1) to adult rats induces a long-lasting (weeks) increase of vasopressin (AVP) stores in terminals of CRH neurons in the external zone of the median eminence (ZEME). This is accompanied by hypersecretion of AVP into the pituitary portal circulation and long-lasting hyperresponsiveness of the hypothalamo-pituitary-adrenal (HPA) axis to stressors. Here, we determine whether this form of plasticity of hypothalamic CRH neurons is specific for IL-1 or represents a general response to a stressor. Single exposure of rats to lipopolysaccharide (LPS), IL-1, brain surgery or electric footshocks increases the AVP stores in the ZEME 7 and 11 days later. Exposure to insulin or ether does not affect the AVP stores. The stressors have little or no effect on the CRH stores in the ZEME. The amplitude of the increase in AVP as measured 7-11 days after stimulation correlates with the overall ACTH response to the stressor (area under curve, r = 0.89, P < 0.0001), with the peak ACTH levels (r = 0.52, P < 0.05), but not with the duration of the ACTH response nor with any parameter of the corticosterone response. Administration of ACTH or corticosterone at doses that mimic stress-induced plasma levels does not increase AVP stores 7 days later. We conclude that long-lasting increases of AVP stores in CRH terminals in the ZEME can be induced by various stressors and postulate that the amplitude of such increases depends on the degree of activation of the CRH neurons by the stressor. (NWO grant: 900-543-101.)
Assuntos
Arginina Vasopressina/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Estresse Fisiológico/metabolismo , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/farmacologia , Animais , Corticosterona/sangue , Masculino , Ratos , Ratos Wistar , Estresse Fisiológico/sangue , Estresse Fisiológico/etiologiaRESUMO
The aim of this study was to investigate whether perinatal hypoxia-ischemia preferentially destroys GABAergic nerve endings in rat cerebral cortex tissue which, in its turn, could then account for the reported higher risk of developing epilepsy later in life. To that end rat pups, with an age of 12-13 days postnatally, were unilaterally exposed to hypoxic-ischemic conditions. After a survival period of 2 to 6 months, the animals were sacrificed by perfusion fixation and their brains were used for cutting transversal vibratome and frozen sections. These sections were double-stained with primary antibodies against one of the two GABA synthesizing enzymes, glutamic acid decarboxylase with a mol. wt. of 66,600 (GAD67) and one of the intrinsic membrane proteins of small synaptic vesicles, synaptophysin, followed by fluorophore-conjugated second antibodies. By using the confocal laser scanning microscope, we determined the ratio between the amount of GAD67/synaptophysin immunofluorescence in nerve endings per unit volume of tissue in the hypoxia-damaged neocortex. It turned out that this ratio, contrary to expectations, was significantly higher in the hypoxia-damaged cortical areas than in matched areas on the contralateral side. It appeared, moreover, that this effect was directly proportional to the severity of the incurred damage. The conclusion was drawn that these observations do not support the hypothesis that perinatal hypoxia-ischemia ultimately leads to a preferential loss of GABAergic nerve endings in the damaged neocortex and, as such, to a shortage of inhibition.
Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Glutamato Descarboxilase/metabolismo , Hipóxia Encefálica/metabolismo , Terminações Nervosas/metabolismo , Sinaptofisina/metabolismo , Animais , Animais Recém-Nascidos , Dano Encefálico Crônico/etiologia , Dano Encefálico Crônico/metabolismo , Isquemia Encefálica/complicações , Hipóxia Encefálica/complicações , Lasers , Microscopia/métodos , Peso Molecular , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The aim of the present study was 2-fold: (1) to determine the ratio between the amount of GAD67 and GAD65 (two isoforms of the GABA synthetizing enzyme glutamic acid decarboxylase) in nerve endings in the mature rat cerebral cortex damaged by hypoxia-ischemia during early postnatal life; and (2) to compare two different computer-assisted procedures developed for quantitative analysis of immunofluorescence images obtained with a confocal laser scanning microscope (CLSM). One procedure was based on a program present in the standard Leica CLSM software packet for full-field analysis, the other on a specially written program for object-oriented analysis run on a Kontron IBAS-KAT image analysis system. To this end, rat pups were unilaterally exposed to hypoxic-ischemic conditions and, after a survival period of 6.5 months, sacrificed by perfusion fixation. After dissection of the brain and vibratome sectioning, three animals with substantial damage on one cortical side were selected. Sections of these animals were double-stained with primary antibodies against GAD67 and GAD65 and fluorophore-conjugated secondary antibodies and subsequently sampled with a CLSM. Analysis of the CLSM images with both computer-assisted procedures showed for all three animals a clear tendency to higher GAD67/GAD65 ratios in cortical GABAergic nerve endings on the hypoxia-damaged side than in matched areas on the contralateral side. This outcome led to the following conclusions. (1) The correspondence between the outcome of both analysis procedures indicates that both procedures are valid for quantification of immunofluorescence images of nerve endings obtained with a CLSM. (2) The outcome lends further support to our view that hypoxic-ischemic encephalopathy, sustained during early postnatal life, may result in an unstable cortical network generating abnormal synchronizations and oscillations which can be amplified and propagated as true epileptic discharges. In such a network both excitatory and inhibitory processes are tonically enhanced, the latter probably as a homeostatic reaction tending to keep abnormal excitation within physiological limits.
Assuntos
Isquemia Encefálica/enzimologia , Córtex Cerebral/enzimologia , Glutamato Descarboxilase/metabolismo , Hipóxia Encefálica/enzimologia , Isoenzimas/metabolismo , Terminações Nervosas/enzimologia , Animais , Imunofluorescência , Processamento de Imagem Assistida por Computador , Microscopia , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The hypothesis was tested whether perinatal hypoxic ischemia leads to a preferential degeneration of the GABAergic inhibitory neurons in the cerebral cortex which, in turn, could account for the reported higher risk of developing epilepsy later in life. To that end rat pups, aged 12-13 days, were made hypoxic by employing a combination of unilateral ligation of one of the carotid arteries and a 90-min exposure to 8% O2. After recovery periods of 3, 7, 35 and 150 days, the animals were sacrificed by perfusion fixation and the brains embedded in Epon. Transverse semi-thin sections were alternately stained with an antibody against GABA and with Toluidine blue. By using an unbiased morphometric method (the disector) the number of GABA-immunoreactive (GABA-IR) neurons and the total number of nerve cells per unit volume of tissue were estimated in corresponding neocortical areas in the ipsilateral (damaged) and contralateral ('control') hemisphere. For all animals with post-ischemic survival times of 3 and 7 days GABA-IR cells constituted a lower proportion of the total number of nerve cells in the damaged than in the 'control' cortical areas. This finding was consistent with the outcome of an earlier in vitro study. By contrast, in all animals with a survival time of 35 and 150 days, the proportion of GABA-IR neurons was higher on the damaged than on the 'control' side. This switch in the direction of the left/right differences, apparently depending on the length of the post-ischemic survival time, was statistically significant. No lateralization in the proportion of GABA-IR cells was detected in the cerebral cortex of the control rats. These observations, therefore, do not support the hypothesis that perinatal hypoxic ischemia ultimately leads to a preferential loss of GABAergic neurons in the cerebral cortex.
Assuntos
Isquemia Encefálica/patologia , Córtex Cerebral/patologia , Epilepsia/etiologia , Hipóxia Encefálica/patologia , Ácido gama-Aminobutírico/análise , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Isquemia Encefálica/complicações , Isquemia Encefálica/metabolismo , Contagem de Células , Sobrevivência Celular , Córtex Cerebral/química , Epilepsia/metabolismo , Hipóxia Encefálica/complicações , Hipóxia Encefálica/metabolismo , Imuno-Histoquímica , Neurônios/química , Neurônios/patologia , Ratos , Ratos WistarRESUMO
In view of the possible role of the hypothalamus-pituitary-adrenal axis in the long-term effects of drugs of abuse, we studied the response of the hypothalamus-pituitary-adrenal axis to cocaine challenges 3 and 14 days after cocaine withdrawal. Three days after intermittent cocaine exposure, the cocaine-induced increase of plasma adrenocorticotropic hormone (ACTH) is unchanged, whereas after 14 days the ACTH response is enhanced 2-fold. The cocaine-induced increase of plasma corticosterone is enhanced approximately 1.5-fold both 3 and 14 days after cocaine withdrawal. Apparently, prior cocaine treatment causes a delayed sensitization of cocaine-induced ACTH secretion and long-lasting corticosterone hyper-responsiveness. We propose that the long-lasting changes in the hypothalamus-pituitary-adrenal axis may facilitate drug-induced long-term behavioral sensitization.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Cocaína/farmacologia , Animais , Corticosterona/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Prolactina/farmacologia , Ratos , Ratos WistarRESUMO
A former study indicated that hypoxic-ischemic encephalopathy in rat sustained during early postnatal life may result in permanent epileptic activity in the baseline electroencephalogram. We, therefore, investigated whether the presumed higher firing frequency and metabolic activity of neurons in such hypoxia-damaged cortical areas would be reflected by an enhanced light microscopic immunoreactivity of gamma-aminobutyric acid (GABA), the two isoforms of glutamic acid decarboxylase (GAD67 and GAD65), the mitochondrial enzymes cytochrome c oxidase and ATP synthase, and/or glial fibrillary acidic, protein (GFAP). To that end rat pups, 12-13 days of age, were unilaterally exposed to hypoxic-ischemic conditions and, after a survival period of 2 and 6 1/2 months, respectively, killed by perfusion fixation. After dissection of the brain, coronal vibratome sections of animals showing cortical damage were immunostained for the presence of the above-mentioned antigens. Subsequent qualitative analysis revealed that the surroundings of cortical infarctions were unambiguously characterized by a disordered neural network containing numerous nerve cells, fibers and/or endings showing an enhanced immunoreactivity for GABA, both isoforms of glutamic acid decarboxylase, and cytochrome c oxidase and ATP synthase, while the astrocytes showed an enhanced immunoreactivity for GFAP. The diverse patterns of enhanced immunoreactivity suggested, furthermore, a wider low-to-high range of metabolic activities in both excitatory and inhibitory neurons.
Assuntos
Animais Recém-Nascidos/fisiologia , Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutamato Descarboxilase/metabolismo , Hipóxia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos Wistar , Coloração e RotulagemRESUMO
Hypothalamic CRH neurons that control ACTH secretion from the pituitary gland have secretory terminals in the external zone of the median eminence (ZEME). These neurons can coproduce vasopressin (AVP), a neuropeptide that potentiates the ACTH releasing effects of CRH. Recently, we found increased AVP production in adult rats weeks after single exposure to a stressor, which may play a role in event-induced stress disorders. Here, we describe the long-term changes in the HPA axis of adult male rats following a single exposure to a stressor, the cytokine interleukin-1 beta (IL-1 beta). The effects on storage and release of AVP and CRH were established by quantitative immunocytochemistry, the effects on ACTH and corticosterone responses by radioimmunoassay. Single administration of IL-1 beta (5 micrograms/kg i.p.) induces a delayed (at least 4 d) and a long-lasting (at least 3 weeks) increase of vasopressin (AVP) stores in CRH terminals of the ZEME without affecting the CRH stores, and a marked increase of the fraction of CRH terminals that costore AVP. Eleven days after IL-1 beta administration, a second IL-1 beta challenge causes a marked depletion of the AVP stores in the ZEME within 2 hr, which is not seen in rats treated with vehicle 11 d earlier. This is accompanied by twofold higher ACTH and corticosterone responses, as compared to those in vehicle pretreated rats. IL-1 beta-pretreated rats also showed increased ACTH and corticosterone responses to electric footshocks. We conclude that transient activation of the HPA axis by a single administration of IL-1 beta induces a delayed and long-lasting hyperproduction, hyperstorage, and hypersecretion of AVP from hypothalamic CRH neurons that results in hyperresponsiveness of the HPA axis to subsequent stimuli.