Histochemical study of acute and chronic intraperitoneal nicotine effects on several glycolytic and Krebs cycle dehydrogenase activities in the frontoparietal cortex and subcortical nuclei of the rat brain.

The effects of nicotine on the activity of different dehydrogenases in frontoparietal regions and subcortical nuclei of the rat brain have been studied using histochemical methods. Nicotine sulphate was intraperitoneally administered in acute (4 mg/kg/day x 3 days) or chronic (ALZET osmotic pump providing 2 mg/kg/day x 15 days) doses. The enzymes analyzed were glyceraldehyde-3-phosphate, lactate, malate and succinate dehydrogenases (gly3PDH, LDH, MDH, and SDH, respectively). The results demonstrate that chronic as well as acute administration of nicotine produced strong increases in all these enzymatic activities in the superior layers (I, II and III) of the frontoparietal cortex (cingulate, motor and somatosensory regions); but high increases were not seen in the deeper layers of the cortex or in the subcortical nuclei (substantia nigra, caudate-putamen, nucleus accumbens or nucleus basalis magnocellularis). These hyperactivities were produced in brain regions with normally low enzymatic activity (cortex), but not in those with great intensity (subcortical nuclei). The results are in rough agreement with previous reports on nicotine-induced increases in glucose utilization, gly3PDH genic expression and neuronal hyperactivity in the brain cortex; but significant discrepancies between the cortical enzymatic maps and those obtained both in these studies and others on nicotine(N)-receptor localization have been appreciated. The results support the hypothesis that nicotinic cholinergic drugs can have metabolic, long-lasting stimulant effects on cortical neurons at specific points (probably layer III pyramidal cells and structures with alpha7-N-receptors) of the cortical circuits that could be of great interest in improving altered cognitive functions that are present in Alzheimer disease, as well as in other less severe mental disturbances. Mitochondrial hyperfunction should also be evaluated as a possible side-effect (as an oxidative stress inductor) of these kinds of drugs.

Localization of glyoxylate dehydrogenase and glyoxylate-complex molecules in the rat prefrontal cortex: enzymohistochemical and immunocytochemical study.

Glyoxylic acid is synthesized and catabolized in cells of vertebrates; several pathways have been described. In previous papers, we have demonstrated the localization in some areas of the rat cerebral cortex both of beta-NAD-dependent glyoxylate dehydrogenase (glyoDH), using an enzymohistochemical method, and of glyoxylate-complex molecules, using immunocytochemical procedures. In this study we have applied these two techniques in various areas of the prefrontal cortex with different histological cytoarchitecture. GlyoDH has been located in most neurons, in some glial cells, and in capillary wall structures in all cortical layers of all areas of the rat prefrontal cortex. Antibodies against glyoxylate-complex molecules showed positive immunoreactivity in scattered neurons, mostly of multipolar or stellate appearance, from layers III, IV, and V in the medial precentral area, but not in cortical areas 24, 25, or 32 of the prefrontal cortex. Immunoreaction was found in the periphery of neuronal perikarya and in some of their processes. These results demonstrate the existence of a particular area-dependent neuronal cortical system, of specific but uncertain function, related to glyoxylic acid and/or glyoxylate compounds. At the electron microscope level, positive reaction was associated with synaptic sites, axonal filaments, glial cells, and several components of the blood-brain barrier. These localizations suggest the involvement of glyoxylate derivatives in synaptic functioning and also in glial cell functions.

Glyoxylate oxidoreductase activity and glyoxylate-like molecules in the motor, somatosensory, cyngulate and olfactory areas of the rat frontoparietal cortex. Histoenzymological and immunohistochemical study.

In the present paper the presence of glyoxylate-oxidoreductase (GOR, GLYO-DH) activity and glyoxylate-like molecules has been investigated in several areas of the rat frontoparietal cortex-motor, somatosensory, cyngulate and olfactory areas-, using the histoenzymological and immunocytochemical methods. Antibodies against glyoxylate-like molecules have been obtained using a glyoxylate-BSA conjugate as immunogen. GOR activity as well as immunostaining for glyoxylate-like molecules were observed in the periphery of the perikaryon and neuronal processes of scattered neurons in the above mentioned cortical areas. At the electron microscopic level positive immunoreaction was found associated to synaptic vesicles in axon terminals, and also within glial cells. These findings reveal both the presence of glyoxylate-like molecules and glyoxylate metabolism in neurons of the motor, somatosensory, cyngulate and olfactory areas of the rat frontoparietal cortex. The presence of such molecules associated to synaptic vesicles within the axon terminals suggest the possible involvement of these molecules in some type of neurotransmission.

Distribution of glyoxylate dehydrogenase activity in cortical and subcortical regions of the rat brain. A light microscopic histoenzymological study.

The distribution of the glyoxylate dehydrogenase (GLYO-DH) (Glyoxylate oxidoreductase) activity has been investigated in rat brain using the corresponding histochemical method. The results have demonstrated a positive histochemical reaction in neurons of several cortical and subcortical areas. These neurons are located in the same regions in which operate monoaminergic neurotransmitters. These facts suggest a possible interaction between glyoxylic acid and monoaminergic neurotransmission.

Neuronal and non-neuronal localization of acid sulfated glycosaminoglycans (sGAG) and chondroitin proteoglycans in the rat medulla oblongata.

Acid sulfated glycosaminoglycans (sGAG) and chondroitin proteoglycans have been biochemically and immunohistochemically demonstrated in certain cortical areas of the CNS. Such molecules display an important role in cerebral function, modulating cell adhesion, migration and signal neuromediation. In the present study we have evidenced the presence of sGAG using the colloidal iron method and C0S, C4S- and C6S-proteoglycans by the immunohistochemical pre-embedding PAP method. Our results have demonstrated the presence of such molecules in several structures of nervous (axon terminals, neuronal bodies, dendrites) and non-nervous nature (glial processes, capillaries). This fact led us to suggest that the chondroitin localizations advocate for their different functions. In their various localization exist distinct proteoglycans constituted by chondroitins with diverse concentrations and other attached radicals.

Immunohistochemical demonstration of taurine in the rat cerebellar cortex. Evidence for its location within mossy fibers and Golgi axons.

Taurine, 2-aminoethanesulfonic acid, is one of the most abundant amino acid present in the Central Nervous System. Nevertheless, its functions are remain uncertain. Taking as a basis the immunocytochemical pre-embedding PAP-techniques for demonstrating Taurine-Like substances on cerebellar cortex of rats we have observed positive immunoreaction within Purkinje cell bodies and their dendrites. Likewise, taurine has been demonstrated within mossy fibers and Golgi axons, as well as in glial processes. Our results demonstrate the wide distribution of Taurine in the cerebellar cortex, justifying its possible involvement as an inhibitory neurotransmitter, neuromodulator or as a gliotransmitter.

L-aspartate aminotransferase and L-asparaginase in rat lymph node: a histoenzymological and immunohistochemical study.

L-asparaginase and L-aspartate aminotransferase are both involved in the synthesis of L-aspartic acid. It has been observed that L-asparaginase is involved in the immunosuppressor morphine-dependent syndrome in lymphoid cells whereas L-aspartic acid blocks the development of this syndrome. The aim of the present study was to clarify the localization of L-AATase activity and L-asparaginase in rat lymph nodes using histoenzymological and immunohistochemical methods, respectively. No positive reaction was demonstrated for L-AATase while L-asparaginase shown to be present in lymphocytes and lymphoblastic cells. These observations lead us to suggest that L-asparaginase is the enzyme mainly responsible for the synthesis of the L-aspartic acid necessary for satisfying the living requirements of lymphoid cells. Therapeutically administered L-asparaginase could exert its action intracellularly after crossing the cell membrane.

Light and electron microscopic immunolocalization of L-asparaginase in the rat central nervous system.

The investigation on the localization of L-asparaginase, the enzyme involved in the synthesis of L-aspartic acid, has been carried out using the immunohistochemical method. Antibodies against this enzyme were obtained immunizing BALB/c mice with purified Escherichia coli L-asparaginase. Light microscopic observation revealed positive immunoreactivity in the great majority of neurons and glial cells, and electron microscopic analysis demonstrated immunological localization of the enzyme in the cytosol. The ubiquitous distribution of L-asparaginase suggests its involvement in many important functions of the central nervous system.

Neuromediators in the cerebellar blood-brain barrier and its microenvironment. Immunocytochemical demonstration of taurine, glycine, serotonin, thiamin and AATase.

Cerebral capillaries represent the responsible structure for the establishment of the Blood-Brain Barrier (BBB). It is known that in the Central Nervous System a relationship exists between neuronal activity and microvascular blood flow and permeability. Our analysis demonstrates the presence of several putative neuromediators-taurine. glycine, serotonin, thiamine and aspartate-in endothelial cells of the rat cerebellar cortex capillaries, as well as in the perivascular glia, neuronal bodies, dendrites and axon terminals that come in close contact with the basement membrane surrounding capillary wall. Our results support the conclusion of other biochemical and pharmacological studies on the relationship between these neuromediators and microvascular function in the Central Nervous System.

Cytochemical and immunocytochemical comparative localization and characterization of acid sulfated glycolaminoglycans (sGAG) in several areas of the rat cerebral cortex during postnatal development.

There are some evidences demonstrating that Acid Sulfated Proteoglycans take part in several Central Nervous System (CNS) functions (Brittis et al., 1992; Carbonetto, 1989; Carey et al., 1990, 1992; Fichard et al., 1991; Kalb and Hockfield, 1990; Lafont et al., 1992; Schubert and Lacorbiere, 1985; Schubert et al., 1988, 1989; Snow et al., 1990, 1991, 1992). To date, the immunocytochemical methods have been developed to detect different proteglycans using specific monoclonal antibodies (Bertolotto et al., 1991; Watanabe et al., 1989; Zaremba et al., 1989). The aim of the present paper is to compare the localization of chondroitin-0-sulfate, -4-sulfate, -6-sulfate and keratan sulfate proteglycans in the rat cerebral cortex during the postnatal development, using both colloidal iron and immunocytochemical methods. Our observations, with the light microscope, revealed an intense immunocytochemical reaction closely associated to the neuronal membranes that, in most cases, were located in the III, IV, V and VI cortical layers of the 20 and 30 postnatal day rats, but not in the 7 and 15 postnatal ones. The colloidal iron reaction revealed similar distribution as that one observed with the immunocytochemical method for chondroitin-0-sulfate, -4-sulfate, -6-sulfate proteoglycans. At electron microscopic level it has been observed positive immunostaining for these sulfated proteoglycans on the plasma membrane of these scattered neurons. Positive immunoreaction for Keratan sulfate proteoglycan was demonstrated inside several astrocytes of 7, 15, 20 and 30 postnatal day rat cerebral cortexes, but it has not been observed in neurons. Taking into account the previous biochemical studies, our observation have led us to suggest that a unique membranous protein could be binded to several acid sulfated glycosaminoglycans (sGAG) types in a particular neuronal subset.

Electron microscopic immunolocalization of GABA and glutamic acid decarboxylase (GAD) in cerebellar capillaries and their microenvironment.

At present, it is known that a connection exists between neuronal activity and microvascular blood flow and permeability in the CNS. We have demonstrated the presence of immunoreactivities against both GABA and GAD as well in the endothelial capillary cells of rat cerebellar cortex, as in perivascular glia, neuronal bodies, dendrites and axon endings, in close association with the basal membrane of the pericyte or endothelial cells. These results support the suggestions from biochemical and pharmacological studies about the relationship between GABA and microvascular function in the CNS.

Molecular and ultrastructural basis of the blood-brain barrier function. Immunohistochemical demonstration of Na+/K+ ATPase, alpha-actin, phosphocreatine and clathrin in the capillary wall and its microenvironment.

Cerebral capillaries represent the responsible structure for the establishment of the BBB. We have demonstrated the presence within both pericytes and endothelial cells of abundant cytoplasmic vesicles. We have noticed the presence of Na+/K(+)-ATPase-, alpha-actin-, phosphocreatine- and clathrin-like molecules within cerebellar capillaries and their microenvironment. These facts suggest the importance of the contractile and transport mechanisms in the blood-brain barrier. We have also demonstrated the close contact between different nervous components of the cerebellar cortex with the basement membrane that surrounds capillary wall. We suggest that these observations represent the morphological evidence of neurogenic control of brain circulation.

Synaptic and non-synaptic immunolocalization of GABA and glutamate acid decarboxylase (GAD) in cerebellar cortex of rat.

The existence of a large number of GABA receptors in the cerebellar molecular layer, and the observation of numerous punctate immunoreactive deposits of GABA synthesizing enzyme (GAD) throughout this layer, could indicate the existence of numerous axon terminals that may be involved in neurotransmission modulated by GABA. These axon terminals may be different from those considered classically as cerebellar GABAergic axon terminals. Therefore, we have reinvestigated the localization of GABA- and GAD-immunoreactivities in the cerebellar cortex of the rat with the PAP method, using different antisera obtained from rabbits immunized with GABA, baclofen and GAD. The results observed in our investigation have demonstrated GABA- and GAD-immunoreactivities in the axon terminals considered classically as GABAergic, as well as in others which, until now, have not been considered GABAergic. This fact leads us to think that the distribution of GABA or molecules structurally similar to GABA is far more extended than previously thought in the cerebellum. We have also observed both GABA- and GAD-immunoreactivities within dendrites and glial cells. These facts suggest us a possible extrasynaptic release of GABA.

Axonal and non-axonal immunolocalization of cytosolic aspartate aminotransferase (cAATase), GABA and glutamic acid decarboxylase (GAD) in the rat cochlear nucleus.

The localization of both cAATase activity, by histoenzymological method, and the immunoreactivity against cAATase, were investigated in the cochlear nucleus of rats. The immunohistochemical determination of cAATase was carried out using the PAP method, with an antiserum obtained from rabbits immunized with porcine cAATase. It was also studied both the immunolocalization of GABA-like and GAD-like substances. Our observations, with light microscope, revealed weak cAATase activity in the small neurons, and a more intense one in the fibers surrounding neuronal bodies. The large neurons presented a very weakly activity within their neuronal bodies and dendrites, but it was strongly found in granulations that surround the perikaryon and dendrites. cAATase immunoreactivity presents the same distribution as the enzymological activity. In the same way, we have investigated the pattern of distribution of both GABA- and GAD-like substances. Immunolocalization of these substances was similar to that found for cAATase. In the control sections incubated with Gostatin (0.05 mM), cAATase activity was absent. The immunoreactivity was also negative in every immunohistochemical control sections. These facts suggest that aspartate could intervene as a co-neurotransmitter or neuromodulator in the rat cochlear nucleus, and that axonic endings could contain cAATase, GABA and GAD. It was also found immunoreactivity against cAATase, GABA and GAD, in neuronal bodies, dendrites and glial processes, in close association with capillary wall. These observations have led us to suggest the possible co-localization and co-release of both GABA and aspartate from synaptic and non-synaptic sites in the cochlear nucleus.

Immunocytochemical localization of GnRH in the hypothalamus of the bat Miniopterus schreibersii schreibersii.

The distribution of gonadotropin-releasing hormone (GnRH) has been examined in the hypothalamus of adult female bats, Miniopterus schreibersii schreibersii, along annual reproductive cycle by means of light-microscopic immunocytochemistry. GnRH-immunoreactive cells are localized throughout the medial basal hypothalamus, being specially numerous in the arcuate nucleus. They are generally bipolar or monopolar neurons with smooth contour, although spiny cells were also found exclusively in the arcuate nucleus from periovulatory bats. Depletion in both number and immunocytochemical labelling of GnRH perikarya is detected in pregnant and lactating bats. GnRH-immunoreactive fibers are distributed in the internal layer of the median eminence and the infundibular stalk. Pregnant and lactating animals show lower GnRH fibers content and a less intense labelling than in periovulatory and hibernating conditions. The results suggest that changes in the secretory activity of GnRH neurons may be associated with hibernation and delayed implantation that suffer these mammalian species.