Enhancement of glutaminase-like immunoreactivity in rat brain by an irreversible inhibitor of the enzyme.

Changes of glutaminase immunoreactivity in rat brain were examined after intracranial injection of 6-diazo-5-oxo-L-norleucine (DON), an irreversible inhibitor of glutaminase. When 1 M DON was injected into the lateral ventricle, a half-lethal dose was 7.5-10 mumol. After intraventricular injection of 2-7.5 mumol DON, glutaminase immunoreactivity was dose dependently enhanced with the maximum enhancement 3-5 days after the injection. The enhanced glutaminase immunoreactivity was recognized by enlarged granular immunodeposits in both perikarya and neuropil in many regions, such as the hippocampus, thalamus, hypothalamus, periaqueductal gray, and some brain stem, cerebellar, and spinal cord regions. Intrathalamic injection of 0.2 mumol DON enhanced glutaminase immunoreactivity in many neuronal perikarya in the thalamus and in some perikarya in layer VI of the cerebral cortex. Intrastriatal injection of the same dose of DON enhanced glutaminase immunoreactivity in neuropil of the caudoputamen and in many neuronal perikarya of the intralaminar thalamic nuclei. These results suggested that DON induced a new massive synthesis of glutaminase in the affected neurons.     

Distribution and morphological characterization of phosphate-activated glutaminase-immunoreactive neurons in cat visual cortex

Phosphate-activated glutaminase (PAG) is the major enzyme involved in the synthesis of the excitatory neurotransmitter glutamate in cortical neurons of the mammalian cerebral cortex. In this study, the distribution and morphology of glutamatergic neurons in cat visual cortex was monitored through immunocytochemistry for PAG. We first determined the specificity of the anti-rat brain PAG polyclonal antibody for cat brain PAG. We then examined the laminar expression profile and the phenotype of PAG-immunopositive neurons in area 17 and 18 of cat visual cortex. Neuronal cell bodies with moderate to intense PAG immunoreactivity were distributed throughout cortical layers II-VI and near the border with the white matter of both visual areas. The largest and most intensely labeled cells were mainly restricted to cortical layers III and V. Careful examination of the typology of PAG-immunoreactive cells based on the size and shape of the cell body together with the dendritic pattern indicated that the vast majority of these cells were pyramidal neurons. However, PAG immunoreactivity was also observed in a paucity of non-pyramidal neurons in cortical layers IV and VI of both visual areas. To further characterize the PAG-immunopositive neuronal population we performed double-stainings between PAG and three calcium-binding proteins, parvalbumin, calbindin and calretinin, to determine whether GABAergic non-pyramidal cells can express PAG, and neurofilament protein, a marker for a subset of pyramidal neurons in mammalian neocortex. We here present PAG as a neurochemical marker to map excitatory cortical neurons that use the amino acid glutamate as their neurotransmitter in cat visual cortex.     

Distribution of phosphate-activated glutaminase in the human cerebral cortex

Phosphate-activated glutaminase (PAG), which catalyses conversion of glutamine to glutamate, is a potential marker for glutamatergic, and possibly GABA, neurons in the central nervous system. A polyclonal antibody, raised in rabbits against rat brain PAG, was applied to postmortem human brain tissue to reveal the distribution of PAG in the cerebral cortex. PAG immunoreactivity was observed in pyramidal and non-pyramidal neurons but not in glial cells. In the neocortex, large to medium-sized pyramidal neurons in layers III and V were stained most intensely, while the majority of smaller pyramidal cells were labeled either lightly or moderately. Such modified pyramids as the giant Betz cells, the large pyramidal cells of Meynert, and the solitary cells of Ramón y Cajal were also stained intensely. Fusiform cells in layer VI showed moderate to intense labeling. A number of cortical non-pyramidal neurons of various sizes stained moderately to intensely. These included large basket cells which were identified by their characteristic morphology and size in primary cortical areas. Pyramidal cells in the hippocampal formation as well as basket cells of the stratum oriens stained moderately to intensely. Since pyramidal cells are believed to be glutamatergic and large basket cells GABAergic, these results suggest that PAG plays a role in generating not only transmitter glutamate, but also GABA precursor glutamate.     

Periaqueductal gray and cerebral cortex modulate responses of medial thalamic neurons to noxious stimulation

The response of medial thalamic neurons to noxious peripheral stimulation were studied with intracellular recording methods in the cat. Electrical stimulation of the contralateral forepaw produced an EPSP-IPSP sequence followed by rebound excitation in these medial thalamic neurons. Action potentials appeared with the initial EPSP or with the rebound excitation. The mean latency to onset was 15 ms for the EPSP and 33 ms for IPSP. In contrast, electrical stimulation of the PAG or of the pericruciate cerebral cortex produced large IPSPs in the medial thalamic neurons. When PAG or cortex stimulation were paired with noxious stimulation, both the PAG and cortex responses predominated over the noxious response. This shows that the PAG and the cerebral cortex have the capabilities of influencing the responses of the medial thalamus to noxious stimulation. The medial thalamus is part of the relay system which sends information about noxious stimulation to the cerebral cortex where the noxious information reaches conscious awareness, so influencing the message at the level of the medial thalamus would probably alter the conscious perception of pain. The data suggest the existence of an ascending pain modulation system from the midbrain to the thalamus and also suggests a mechanism of cortical control over pain perception.     

Parallel but separate inputs from limbic cortices to the mammillary bodies and anterior thalamic nuclei in the rat

The proposal that separate populations of subicular cells provide the direct hippocampal projections to the mammillary bodies and anterior thalamic nuclei was tested by placing two different fluorescent tracers in these two sites. In spite of varying the injection locations within the mammillary bodies and within the three principal anterior thalamic nuclei and the lateral dorsal thalamic nucleus, the overall pattern of results remained consistent. Neurons projecting to the thalamus were localized to the deepest cell populations within the subiculum while neurons projecting to the mammillary bodies consisted of more superficially placed pyramidal cells within the subiculum. Even when these two cell populations become more intermingled, e.g., in parts of the intermediate subiculum, almost no individual cells were found to project to both diencephalic targets. In adjacent limbic areas, i.e., the retrosplenial cortex, postsubiculum, and entorhinal cortex, populations of cells that project to the anterior thalamic nuclei and mammillary bodies were completely segregated. This segregated pattern included afferents to those nuclei comprising the head‐direction system. The sole exception was a handful of double‐labeled cells, mainly confined to the ventral subiculum, that were only found after pairs of injections in the anteromedial thalamic nucleus and mammillary bodies. The projections to the anterior thalamic nuclei also had a septal‐temporal gradient with relatively fewer cells projecting from the ventral (temporal) subiculum. These limbic projections to the mammillary bodies and anterior thalamus comprise a circuit that is vital for memory, within which the two major components could convey parallel, independent information. J. Comp. Neurol. 518:2334–2354, 2010. © 2010 Wiley‐Liss, Inc.     

Somato-, branchio- and viscero-motor neurons contain glutaminase-like immunoreactivity

Immunocytochemistry combined with a fluorescent dye tracer method revealed that somatic, branchial and visceral motoneurons in the brainstem and spinal cord of the rat contain phosphate-activated glutaminase (PAG). An excitatory neurotransmitter glutamate is synthesized mainly through this enzyme. Among these motoneurons, neurons in the dorsal motor nucleus of the vagus nerve (dmnX), autonomic preganglionic neurons in the spinal cord and urethral sphincter motoneurons (DL) were most intensely immunostained. PAG is co-expressed with choline acetyltransferase, calcitonin gene-related peptide or galanin in these neurons. These findings, together with the findings that motor endplates in urethral sphincter muscle contain PAG and PAG-like immunostaining in dmnX motoneurons was decreased after axotomy, suggest that glutamate is a co-transmitter of acetylcholine in motoneurons. Brainstem motoneurons were moderately stained, while somatic motoneurons in the spinal cord other than DL, showed very weak staining for PAG. However, they showed intense PAG-like immunoreactivity at their premature stage, suggesting that glutamate has some effects on the maturation of these neurons. A variety of functional roles of glutamate in motoneurons is discussed.     

Electrophysiological studies on the cerebellocerebral projection in the rat

Cerebellocerebral responses in the rat were investigated by laminar field potential analysis in the cerebral cortex under Nembutal anesthesia. Stimulation of the three cerebellar nuclei induced conspicuous responses in the sensory-motor cortex only on the contralateral side, particularly in the forelimb and vibrissae areas of the motor cortex. Laminar field potential analysis and unitary recordings performed extra- and intracellularly from pyramidal tract neurons revealed that the evoked potential was composed of two kinds of responses. One was due to the deep thalamocortical (TC) response (superficial positive-deep negative potentials) which was ascribed to excitatory postsynaptic potentials (EPSPs) generated in the deep cortical layers (somata and dendrites near the somata of pyramidal neurons), and the other was due to the superficial TC response (superficial negative-deep positive potentials) which was ascribed to EPSPs in the superficial cortical layers (upper parts of apical dendrites of pyramidal neurons). Comparison of the responses in the cortex induced by stimulation of the cerebellar and thalamic nuclei confirmed that the ventrolateral complex of the thalamus is the relay portion of the cerebellocerebral responses in the rat. The results of the present study are compared with those of the cat and monkey.     

Branched connections to the septum and to the entorhinal cortex from the hippocampus, amygdala, and diencephalon in the rat

Neuronal cell populations giving origin to bifurcating projections to the septum and the entorhinal cortex were studied in the rat by means of double retrograde labeling using the fluorescent tracers Fast Blue and Diamidino Yellow. Double labeled pyramidal neurons were consistently detected in the temporal level of the CA1 area and subiculum of the hippocampal formation, where they represented at least 50% of the cells retrogradely labeled from the entorhinal injections. Double labeled neurons were also detected in the amygdala, where they prevailed in the basal complex. Scattered double labeled neurons were observed in a number of hypothalamic nuclei, with a slight predominance in the preoptic region. Finally, a few double labeled cells were detected in the midline thalamus, and especially in the thalamic paraventricular nucleus. In all these structures, double labeled neurons were located ispilaterally to the injection sites. The present data indicate that the septum and entorhinal cortex are tightly interconnected by axonal bifurcations deriving from a variety of telencephalic and diencephalic sources.     

Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody.

Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT(1)( )MF-2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2-CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence-labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R-immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs.     

Neuronal populations stained with the monoclonal antibody Cat-301 in the mammalian cerebral cortex and thalamus

The monoclonal antibody Cat-301 was used to examine neurons in the cerebral cortex and dorsal thalamus of several mammalian species, including Old World monkeys, cats, bush babies, guinea pigs, and rats. In each species, subpopulations of cortical and thalamic neurons are stained along the surfaces of their somata and proximal dendrites. Cat-301-positive cortical neurons include specific groups of pyramidal cells (e.g., corticospinal but not corticobulbar or callosal neurons in the monkey sensory-motor areas) and certain GABA-immunoreactive nonpyramidal cells. In the thalamus, the relay neurons projecting to the cortex and not the intrinsic neurons are stained. The Cat-301-positive neurons are nonhomogeneously distributed in the cat and monkey cortex and thalamus. In the cortex, they are densely packed in 2 bands that in most areas include layers III and V, but that in primary sensory areas include layers IV and VI. Because the density of stained neurons, their distribution, and the intensity of their staining vary among cortical areas, the borders between neighboring areas can often be detected by the differences in Cat-301 staining. Broader, regional differences are also readily apparent, for areas in the parietal and occipital lobes contain large numbers of intensely stained cells, but most areas in the frontal and temporal lobes contain fewer, more lightly stained neurons. The same broad differences are seen within the thalamus: only those nuclei reciprocally connected with intensely stained cortical areas contain large numbers of Cat-301-positive neurons. Differences among species include variations in cell density and distribution when a given cortical area or thalamic nucleus is compared between cats and monkeys. Greater differences are seen among the other species. Immunoreactive neurons in the cerebral cortex are sparse and lightly stained in guinea pigs, are restricted to the hippocampal formation in rats, and are very rare and isolated in bush babies. Similarly, Cat-301-positive thalamic neurons are restricted to only one or 2 nuclei in the guinea pig and rat and are extremely rare in the bush baby. Cat-301 stains organized groups of neurons in the cat and monkey cortex and thalamus. In addition to the laminar organization of stained cells in all cortical areas (see above), the Cat-301-positive neurons of monkey areas 17 and 18 are grouped into radial arrays. In area 17, clusters of stained cells are present in layers above and below layer IVC. These clusters lie at the centers of ocular dominance columns, within patches stained for cytochrome oxidase (CO). Most of these cells are also GABA-immunoreactive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential Calcium Binding Protein Immunoreactivity Distinguishes Classes of Relay Neurons in Monkey Thalamic Nuclei

The distributions of neurons displaying immunoreactivity for two calcium binding proteins, parvalbumin and 28Kd calbindin, were studied in the thalamus of M. fascicularis. Colocalization experiments were carried out to determine the extent to which parvalbumin- and calbindin-like immunoreactivity was found in the same cells and the extent to which either was localized in GABAergic interneurons. Anterograde and retrograde tracing experiments involving the fluorescent tracer, fast blue, were also used to determine that cells expressing the calcium binding proteins projected upon the cerebral cortex. In the dorsal thalamus, nuclei are distinguished by different patterns of parvalbumin-like and calbindin-like immunoreactivity. In certain nuclei, for example the lateral dorsal and anterior pulvinar, neurons express immunoreactivity for only one of the calcium binding proteins. In others, neurons in different layers, for example the dorsal lateral geniculate nucleus, or in different compartments, for example the intralaminar nuclei, express immunoreactivity for either parvalbumin or calbindin; in other nuclei, for example the ventral group, neurons are mixed and immunoreactivity for parvalbumin and calbindin is commonly colocalized. In the ventral thalamus and epithalamus, similar patterns are observed. Colocalization of parvalbumin- and GABA-immunoreactivity is found in all cells of the reticular nucleus but only in certain cells in selected nuclei of the dorsal thalamus, namely the dorsal lateral geniculate and magnocellular medial geniculate. No calbindin-positive cells are also GABA-positive. Most parvalbumin and/or calbindin positive cells in the dorsal thalamus project to the cerebral cortex, as indicated by the retrograde tracing studies, and many parvalbumin positive fibres entering the cerebral cortex could also be shown to contain fast blue anterogradely transported from a thalamic injection. Most of the major sensory and motor pathways entering the dorsal thalamus express parvalbumin immunoreactivity. The optic tract also expresses calbindin immunoreactivity but most other calbindin positive fibres entering the thalamus ascend in the midbrain tegmentum. The differential distributions of parvalbumin and calbindin implied by these results suggest that thalamic cells belonging to different functional systems and projecting differentially upon the cerebral cortex can be distinguished by differential expression of these or closely related calcium binding proteins. This may yield clues to their differential responsivity to afferent driving.     

Disinhibition of the mediodorsal thalamus induces Fos-like immunoreactivity in both pyramidal and GABA-containing neurons in the medial prefrontal cortex of rats,but does not affect prefrontal extracellular GABA levels

Stimulation of the mediodorsal and midline thalamic nuclei excites cortical neurons and induces c-fos expression in the prefrontal cortex. Data in the literature data suggest that pyramidal neurons are the most likely cellular targets. In order to determine whether cortical interneurons are also impacted by activation of mediodorsal/midline thalamic nuclei, we studied the effects of thalamic stimulation on (1) Fos protein expression in γ-aminobutyric acid (GABA)-immunoreactive neurons and on (2) extracellular GABA levels in the prefrontal cortex of rats. Perfusion of the GABA-A receptor antagonist bicuculline for 20 minutes through a dialysis probe implanted into the mediodorsal thalamus induced Fos-like immunoreactivity (IR) approximately 1 hour later in the thalamus and in the medial prefrontal cortex of freely moving rats. Immunohistochemical double-labeling for Fos-like IR and GABA-like IR showed that about 8% of Fos-like IR nuclei in the prelimbic and infralimbic areas were located in GABA-like IR neurons. Fos-like IR was detected in three major subsets of GABAergic neurons defined by calbindin, parvalbumin, or vasoactive intestinal peptide (VIP)-like IR. Dual probe dialysis showed that the extracellular levels of GABA in the prefrontal cortex did not change in response to thalamic stimulation. These data indicate that activation of thalamocortical neurons indeed affects the activity of GABAergic neurons as shown by the induction of Fos-like IR but that these metabolic changes are not reflected in changes of extracellular GABA levels that are sampled by microdialysis. Synapse 30:156–165, 1998. © 1998 Wiley-Liss, Inc.     

Expression of kalirin, a neuronal GDP/GTP exchange factor of the trio family, in the central nervous system of the adult rat

Kalirin is a multifunctional protein identified by its interaction with peptidylglycine alpha-amidating monooxygenase, an enzyme essential for neuropeptide biosynthesis. Several forms of Kalirin exist, all containing spectrin-like repeats, a Dbl homology (DH) domain, and an adjacent pleckstrin homology (PH) domain; several different COOH-termini provide additional DH/PH domains and a putative protein kinase. Kalirin binds Rac1 and affects cytoskeletal organization, neuropeptide secretion, and iNOS activity. By in situ hybridization, the highest levels of Kalirin mRNA were found in the cerebral cortex, hippocampal formation, and Purkinje cells, with high levels also in thalamus, caudate putamen, septal nucleus, nucleus accumbens, amygdala, and anterior olfactory nucleus. Low levels of Kalirin mRNA were detected in the paraventricular, supraoptic, and reticular thalamic nuclei and in the ventromedial hypothalamic nucleus. Brain areas with high levels of Kalirin mRNA showed strong Kalirin-like immunoreactivity. Pyramidal neurons with strongly staining soma and long dendrites were observed primarily in layer 5 of the cerebral cortex. In the hippocampus, a uniform distribution of neurons with fine dendritic staining was observed in the pyramidal cell layer, in the granule cell layer, and in the hilar cells of the dentate gyrus as well as in isolated interneurons. Cerebellar Purkinje neurons exhibited intense staining in the soma and in extensive dendritic arbors extending to the surface of the molecular layer. During embryonic development, Trio, the Drosophila orthologue of Kalirin, plays an essential role in axon guidance; localization of Kalirin to the somatodendritic region of adult neurons provides the basis for future studies of regulation and function.     

Thalamic connectivity of the primary motor cortex of normal and reeler mutant mice

Reeler, an autosomal recessive mutation in mice, causes cytoarchitectonic abnormalities of the cerebral cortex, which are characterized by malposition of neurons. Retrograde and anterograde transport of horseradish peroxidase (HRP) was employed to examine the reciprocal connectivity between the hindlimb area of the primary motor cortex (MI) and thalamus of normal and reeler mutant mice. In the normal mouse, most of the cells labelled after HRP injection into the hindlimb area of MI were located in the ventrolateral nucleus, the lateral division of the ventrobasal nucleus, the central lateral, paracentral and central intralaminar nuclei, and the medial division of the posterior complex. HRP reaction product anterogradely transported was also observed in the same nuclei and in the thalamic reticular nucleus. In the reeler mutant mouse, retrogradely labelled neurons and anterogradely labelled terminals were again found in the nuclei referred to above, and the distribution pattern and morphology of HRP-filled neurons were also similar to those of normal controls. The present results suggest therefore that the normal reciprocal connectivity between MI (hindlimb representation) and thalamus is preserved in the reeler mouse. That is to say, dislocated cortical neurons appropriately project to their target nuclei of the thalamus, and conversely, thalamic neurons send their axons precisely to their target cortical areas of the radially disorganized cortex.     

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia

Polyclonal antisera were generated against two identical regions of rat and human A₁ adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A₁ receptors. Immunohistochemistry of CHO cells transfected with the rat or human A₁ adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A_2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A₁ adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A₁ receptor expression and support the concept of cellular specificity of A₁ adenosine receptor action.     

The contribution of the dorsal lateral geniculate nucleus to the total pattern of thalamic terminations in striate cortex of the Virginia opossum

These studies were carried out to show the manner of projection of the dorsal lateral geniculate nucleus and other thalamic nuclei to striate cortex in the Virginia opossum. In order to demonstrate these projections, lesions were made in the dorsal lateral geniculate nucleus, in the ventral lateral geniculate nucleus, in most of the thalamus on one side except for the dorsal lateral geniculate nucleus, and in the entire unilateral thalamus. Following various survival times, usually seven days, the brains were appropriately prepared and stained with procedure I of the Fink-Heimer technique. Dorsal lateral geniculate neurons project in a topographical manner only to certain layers of striate cortex. These projections from the lateral geniculate are compared with the same system in other mammals, and it is concluded that it is similar in all mammals studied, except for the cat. In the cat the lateral geniculate projects beyond the border of striate cortex, but even in the cat the layers of termination within striate cortex are apparently similar. The ventral lateral geniculate nucleus does not project to visual cortex. Dorsal thalamic nuclei other dian the lateral geniculate project to peristriate cortex and to layers VI and I of striate cortex. The finding that thalamic nuclei, other than the lateral geniculate nucleus, project to striate cortex has never been described as part of the visual pathways in other mammals. It is suggested that these additional projections arise mainly from the lateral nuclear group of the thalamus in the opossum, and must be considered in relation to any response characteristics and organization of striate cells determined from physiological studies. These multiple thalamic projections can provide the substrate for more than one representation or “map” of sensory information in striate cortex.     

Chondroitin sulfate proteoglycan surrounds a subset of human and rat CNS neurons

Chondroitin sulfate proteoglycan (CS-PG) bearing glycosaminoglycan (GAG) chains containing unsulfate (COS) and 6-sulfate (C6S) disaccharides was immunolocalized in rat and human CNS by using monoclonal antibodies (MAb) specific for the two disaccharides. The immunostaining with both MAb was restricted to the periphery of a neuronal subset in rat and human CNS. Double immunofluorescence showed codistribution of the antigens around the same neuronal population. The staining with anti-COS MAb was stronger than with anti-C6S MAb, suggesting that the proteoglycan (PG) contains mainly COS disaccharides. In different rat cortical areas, 40-60/mm2 positive interneurons were found, the visual cortex showing the highest value. In human cortex, positivity was also observed around the soma of some pyramidal cells. In the rat, positive neurons were also localized in deep cerebellar nuclei, reticular nucleus of the thalamus, and other structures of the midbrain and hindbrain. CA3 region of hippocampus and the external layer of pyriform cortex were characterized by positivity of the neuropil. Immunoelectronmicroscopy showed the antigens in the extracellular space around the neuronal soma, the synaptic elements and the cell processes of the neuropil. The neuronal surface of the soma and of the proximal dendrites were positive, but the pre- and postsynaptic membranes and clefts were negative.     

Ephrin-B3-EphA4 interactions regulate the growth of specific thalamocortical axon populations in vitro

The role was studied of ephrin-B3, a ligand of the Eph family of tyrosine kinase receptors, in the formation of cortical connectivity. In situ hybridization and immunohistochemistry showed that EphA4, a receptor of ephrin-B3, was expressed in the lateral thalamus (visual and somaotosensory thalamus) of the developing rat brain, but not in the medial thalamic nuclei which project to the limbic cortex. Correspondingly, ephrin-B3 was expressed strongly in the developing limbic cortex including amygdala, entorhinal cortex and hippocampus. To examine the action of ephrin-B3 on thalamic axons, either lateral or medial thalamic explants were cultured on membranes obtained from ephrin-B3-expressing COS cells. Axonal growth was inhibited for cells from the lateral thalamus but not from the medial thalamus. These results suggest that ephrin-B3 contributes to regional specificity by suppressing axonal growth of lateral thalamic neurons.