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.

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.

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.

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.