Neuronal subsets express multiple high-molecular-weight cell-surface glycoconjugates defined by monoclonal antibodies Cat-301 and VC1.1.

Cat-301 and VC1.1 are monoclonal antibodies that recognize surface-associated molecules on subsets of mammalian CNS neurons. Earlier work demonstrated that Cat-301 recognizes a 680-kDa chondroitin sulfate proteoglycan (PG). VC1.1 has been shown to recognize 3 polypeptide bands on Western blot analysis; a major band at 95-105 kDa and additional bands at 145 kDa and 170 kDa. In the present report, we show that VC1.1 also reacts with a high-molecular-weight glycoconjugate. Immunoprecipitation experiments and biochemical characterizations indicate that Cat-301 and VC1.1 define at least 3 distinct high-molecular-weight antigens. The VC1.1 antigens react with antikeratan sulfate antibodies, while the Cat-301 antigens do not. By immunodepletion, we show that some VC1.1 antigens are Cat-301 positive, while others are Cat-301 negative. In addition, Cat-301-reactive proteoglycans are heterogeneous with respect to the presence or absence of VC1.1 epitopes. Double-label immunofluorescence studies with these 2 antibodies are consistent with the biochemical results and show that there are 3 classes of immunoreactive neurons in the cat CNS:Cat-301+/VC1.1+, Cat-301-/VC1.1+, and Cat-301+/VC1.1-. These results indicate that structural microheterogeneity exists among Cat-301 and VC1.1 high-molecular-weight glycoconjugates. This heterogeneity may be a reflection of the diverse neuronal phenotypes that are recognized by Cat-301 and VC1.1 in the mammalian CNS.

Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

1B1075: a brain- and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles.

The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.

Molecular and morphological changes in the cat lateral geniculate nucleus and visual cortex induced by visual deprivation are revealed by monoclonal antibodies Cat-304 and Cat-301.

Monoclonal antibody Cat-301 recognizes a surface-associated proteoglycan on subsets of neurons in the mammalian CNS (Hockfield and McKay, 1983). The expression of Cat-301 immunoreactivity on Y cells in the cat LGN is sharply reduced by early visual deprivation (Sur et al., 1988). We employed an immunosuppression strategy (Hockfield, 1987) to further study alterations in the expression of experience-dependent molecules. Newborn BALB/c mice were injected with LGN from dark-reared cats to induce a suppression of the immune response to antigens expressed in visually deprived animals. These mice were then immunized with LGN from normal cats to elicit an immune response to antigens with an expression dependent on normal early visual experience. This strategy permitted the generation of monoclonal antibody Cat-304, which recognizes a surface-associated antigen on neuronal cell bodies and proximal dendrites, and which appears histologically identical to Cat-301. Further analyses show that Cat-304 and Cat-301 recognize different epitopes on the same 680-kDa chondroitin sulfate proteoglycan. We examined the effects of early visual deprivation on Cat-304 immunoreactivity in the LGN and visual cortex of cats. In LGN from normal cats, Cat-304 labels neurons in layers A, A1, and C, in interlaminar zones, and in the medial interlaminar nucleus. In LGN from dark-reared cats, the number of antibody-positive neurons is markedly reduced, and the cross-sectional area of the remaining positive neurons is smaller than normal. In cortical area 17 of normally reared cats, Cat 304-positive neurons are densely distributed in 2 bands, in layers IV and V/VI. Labeled neurons are also present in layers II and III. In area 17 of dark-reared cats, the number of antibody-positive neurons is reduced. The reduction in the number of labeled neurons is most pronounced in layers II/III and V/VI. Antibody-positive neurons are smaller in all cortical layers of dark-reared cats. The changes in the expression of Cat-301 immunoreactivity in dark-reared visual cortex and LGN are identical to those of Cat-304. The laminar differences in the effect of dark rearing on Cat-301 and Cat-304 expression in the visual cortex provides support for the suggestion that layer IV of cortical area 17 may be less susceptible to prolongation of plasticity by dark rearing than layers II/III and V/VI. Further, the biochemical and histological studies reported provide evidence that early visual experience regulates protein expression in the cat LGN and visual cortex.

Synaptic connections of neurones identified by Golgi impregnation: characterization by immunocytochemical, enzyme histochemical, and degeneration methods.

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi-impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi-impregnated neurones and their pre- and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre- and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP-filled neurones and Golgi-impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.

Glucagon-like immunoreactivity in hypothalamic neurons of the rat.

Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.

[Chemical neuro-anatomy of the thalamus: an example of the co-expression of 2 neuropeptides, cholecystokinin and intestinal vasoactive peptide]. / La neuroanatomie chimique du thalamus: exemple de coexpression de deux neuropeptides, cholécystokinine et peptide vasoactif intestinal.

Recent studies using hybridization histochemistry have demonstrated the presence of cholecystokinin (CCK) gene expression in thalamocortical and thalamo-striatal neurons. To further understand the chemical anatomy of the thalamus, we used the same method to study the coexpression of CCK and vasoactive intestinal peptide (VIP) genes in the same neurons. Most of the neurons expressing the VIP gene in the rat dorsal thalamus (found especially in the ventrolateral nucleus) also expressed the CCK gene, as demonstrated by autoradiography on emulsion-coated adjacent sections studied with probes recognizing either mRNA. Some further neurons, located in the reticular nucleus, had VIP mRNA at lower levels of expression but did not appear to contain CCK mRNA.

Ezrin immunoreactivity in neuron subpopulations: cellular distribution in relation to cytoskeletal proteins in sensory neurons.

Ezrin was first identified as a low-abundance phosphoprotein associated with the cytoskeletal core of microvilli, where it may function as a regulatory protein. We report immunocytochemical evidence for expression of ezrin, or an ezrin-like protein of molecular mass near 80 KD, confined to select populations of neurons, including sensory, motor, and autonomic, during chick embryonic development. We have compared the distribution of anti-ezrin staining with that of other major cytoskeletal proteins in sensory neurons in an effort to identify a possible association of the neural homologue of ezrin with the neuronal cytoskeleton. The diffuse distribution of anti-ezrin staining in the cell soma bore little resemblance to the filamentous staining observed with antibodies to the 68 KD neurofilament protein and alpha-tubulin. F-actin staining with fluorescein-conjugated phalloidin was indistinguishable from the anti-ezrin staining pattern in the soma of cultured neurons, including a peak in staining intensity around the periphery of the cell. Microfilaments in growth cones did not stain with the ezrin antibody. A close correspondence between the anti-ezrin and anti-spectrin staining patterns was found on cryostat sections of dorsal root ganglia, but the anti-spectrin staining was weak and could not be demonstrated in culture. Our findings, primarily from cultured neurons, are not inconsistent with ezrin associating with F-actin, although not with microfilaments found in motile structures such as growth cones.

Novel localization of a G protein, Gz-alpha, in neurons of brain and retina.

Recently, a cDNA coding for a novel G protein alpha-subunit, Gz-alpha, was isolated from a human retinal cDNA library and shown by Northern blot analysis to be expressed at high levels in neural tissues. We have prepared affinity-purified antibodies specifically directed against synthetic Gz-alpha peptides and employed immunohistochemical methods to map the localization of Gz-alpha in human, bovine, and murine retina and brain. By light microscopy, Gz-alpha was localized to the cytoplasm of neurons, with predominant reactivity in ganglion cells of the retina, Purkinje cells of the cerebellum, and most neurons of the hippocampus and cerebral cortex. Reactivity was confined to perikaryon, dendrites, and a very short segment of proximal axons, except for the retinal ganglion cells, in which the axons in the nerve fiber layer showed intense Gz-alpha immunoreactivity proximal to the lamina cribrosa. Pre-embedding immunoelectron microscopy demonstrated the presence of focal Gz-alpha immunoreactivity on the nuclear membranes, endoplasmic reticulum, and plasma membranes of Purkinje cell perikarya and in association with microtubules in their proximal dendrites. Subcellular fractionation studies confirmed the association of Gz-alpha with plasma and intracellular membranes. The localization of Gz-alpha and its unique amino acid sequence suggest that it may have a specialized function in neural tissues.

Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations.

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the alpha 3 beta 2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The alpha 2 beta 2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the alpha 1 beta 1 gamma delta muscle subunit combination. The alpha 2 beta 2 and alpha 3 beta 2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog-1, whereas complete blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog-1. The alpha 1 beta 1 gamma delta muscle subunit combination was blocked by the alpha-conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both alpha-conotoxins. Thus, neosurugatoxin and the alpha-conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog-1 distinguish between different neuronal subunit combinations on the basis of differing alpha subunits.

Neuropeptide Y (NPY) in human nasal mucosa.

Neuropeptide Y (NPY), a potent vasoconstrictor peptide found in sympathetic neurons, was analyzed in human inferior turbinate nasal mucosal tissue. NPY content determined by radioimmunoassay was 3.13 +/- 0.79 pmol/g tissue (n = 6) in mucosa extracted with ethanol-acetic acid. NPY-immunoreactive nerves were found around small muscular arteries, arterioles, arteriovenous anastomoses, and as free fibers near arteriolar and venous vessels. They formed a plexus around the arterial vessels, and were also present between vascular smooth muscle cells. Few NPY fibers were present near glands or the epithelium. [125I]NPY binding sites were localized by autoradiography to small muscular arteries, arterioles, and a few venous sinusoids. In explant culture experiments, 4 microM NPY did not stimulate release of [3H]glucosamine-labeled glycoconjugates or lactoferrin (a product of serous cells) from nasal mucosal fragments. Degradation of NPY by a tissue homogenate was rapid (t1/2 = 13.5 +/- 2.3 min). The degradation was inhibited by thiorphan and phosphoramidon, inhibitors of neutral endopeptidase activity. NPY released from sympathetic neurons may play a role as a constrictor of arterial vessels and regulate vasomotor tone in the human nasal mucosa.

Sex differences among oxytocin-immunoreactive neuronal systems in the mouse hypothalamus.

Serial frontal sections of male and female mouse hypothalamus were immunostained with an antiserum to oxytocin, in order to study the topographical distribution of oxytocinergic perikarya and processes. Numbers of immunostained perikarya were counted in various hypothalamic regions. The oxytocin content of microdissected hypothalamic tissue samples was measured in radioimmunoassays. While the overall topographical distribution of oxytocin neurons in the classical magnocellular nuclei was similar in both genders, quantitative differences could be observed. The numbers of immunostained perikarya and the amounts of oxytocin found in females exceeded by far the numbers and amounts found in males. Male mice had fewer oxytocin-immunostained axons, projecting within the brain, than females. This was especially apparent in parts of the limbic system. Oxytocin-immunostained neurons in the perifornical region, the lateral hypothalamus and the ventral ansa lenticularis were mostly absent in males. It is possible that the observed sex differences in oxytocin immunoreactive brain architecture are due to the different hormonal conditions in males and females.

Localization of the NGFI-A protein in the rat brain.

Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.

Met-enkephalin immunoreactivity in the basal ganglia in Parkinson's disease and striatonigral degeneration.

This study concerns the expression of Met-enkephalin (MEnk) in the striatum and the external segment of the globus pallidus proper (GPe) in normal controls, idiopathic Parkinson's disease (PD), and striatonigral degeneration (SND). For this purpose, we developed a sensitive immunoperoxidase technique to visualize MEnk-positive patches in routinely prepared formalin-fixed paraffin-embedded striatal tissues. In comparison with normal controls, MEnk-positive patches and pallidal MEnk-positive axon terminals were strongly present in patients with PD, showing characteristic distribution patterns. By comparison, in SND patients, there was striking diminution of MEnk staining in the putamen and ventrolateral portion of the GPe, while MEnk patches were persistent in the caudate nucleus.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

A monoclonal antibody directed against CLIP (ACTH 18-39). Anatomical distribution of immunoreactivity in the rat brain and hypophysis with quantification of the hypothalamic cell group.

After the recent demonstration of the facilitatory effect exerted by corticotropin-like intermediate lobe peptide (CLIP or adrenocorticotropic hormone (ACTH) 18-39) on paradoxical sleep in the rat (Chastrette and Cespuglio, 1985), we undertook the production of monoclonal antibodies against this peptide. Wistar rats were immunized against CLIP and their spleen cells fused with mouse myeloma cells. After recloning, 25 supernatants were found to give positive immunohistochemical reactions in the rat brain. In immunohistochemical tests performed by preabsorption, the 25 supernatants presented similar properties, i.e. recognized CLIP, ACTH (1-39) and ACTH (25-39), but not ACTH (1-24) and the C-terminal fragment (34-39). We assume that the epitope(s) recognized by the 25 supernatants is (are) located between the amino-acids Asn25 and Ala34 of the CLIP molecule. The immunoreactivity observed in the rat brain and hypophysis with this antibody was distributed with a pattern quite similar to that described for anti-ACTH antibodies. A main group of immunoreactive cell bodies was located in the mediobasal hypothalamus and a small group in the nucleus of the solitary tract. Immunoreactive fibres were distributed from the olfactory nucleus to the spinal cord and formed particularly rich networks in the hypothalamus and preoptic area. Among other locations, immunoreactive axons were also present in the brainstem centres involved in the control of the sleep-waking cycle, which is in accordance with the influence of CLIP on paradoxical sleep. Using Abercrombie's formula, the number of immunoreactive cells in the mediobasal hypothalamus was estimated at about 3000 neurons. We conclude that our monoclonal anti-CLIP antibody can be considered as a good marker of proopiomelanocortin neurons.

Distribution of dopamine-immunoreactive neuronal perikarya and fibres in the brain of a teleost, Gasterosteus aculeatus L. comparison with tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive neurons.

The distribution of dopamine in the brain of the teleost Gasterosteus aculeatus L. was demonstrated with the indirect peroxidase-antiperoxidase immunohistochemical method using highly specific antibodies against a dopamine-glutaraldehyde-thyroglobulin conjugate. Dopamine-immunoreactive (DAir) neuronal somata were observed in all main brain regions. In the forebrain, DAir neurons were located in a continuous cell column extending from the caudal part of the olfactory bulbs to the preoptic area. The neurons lie lateral to the dorsal (and caudally to the subcommissural) portion of the ventral telencephalic area, and ventromedial to the central nuclei of the dorsal area. In the diencephalon, cerebrospinal fluid-contacting neurons were located in the paraventricular organ and in the subependymal layers of the dorsal and caudal zones of the periventricular hypothalamus. Small DAir neurons were observed in the suprachiasmatic nucleus, in the parvocellular preoptic nucleus and in the ventromedial thalamic nucleus, while large perikarya were observed dorsolateral to the dorsal zone of the periventricular hypothalamus ('PVO-accompanying cells'), in the posterior tuberal nucleus and in the most rostral portion of the mammillary bodies. Numerous small DAir neurons were located in the periventricular pretectal nucleus. In the brainstem, DAir neurons were observed in the isthmus region, in the dorsal raphe nucleus and in the lateral parts of the nucleus of the solitary tract. DAir perikarya were also observed in the area postrema. Direct comparison with the distribution of tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactivity (THir and DBHir) gave the following results: THir neurons were found in all areas where DAir neurons were located, except for the paraventricular organ and the dorsal and caudal zones of the periventricular hypothalamus, which were devoid of THir. DBHir (putatively noradrenergic or adrenergic) neurons were observed in the lateral parts of the nucleus of the solitary tract, and in the isthmus region. The DBHir neurons in the isthmus region, which have previously been shown to be noradrenergic, appeared to be identical with the THir and DAir neurons of the same area. DAir axons were found in high numbers in most parts of the brain. Especially dense innervation was found in the ventrolateral and posterior parts of the dorsal telencephalic area, the region surrounding the lateral recesses of the third ventricle, the interpeduncular nucleus, the dorsal and median raphe nuclei (the rostral raphe nuclei), and in the nucleus of the solitary tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y in rat sympathetic neurons is altered by genetic hypertension and by age.

We have used immunocytochemistry to quantitate neuronal neuropeptide Y in superior cervical ganglia of a strain of normotensive Wistar-Otago rats and a related genetically hypertensive strain over the age range 1-60 weeks. The numbers of neuropeptide Y-immunoreactive cells and total ganglionic cell numbers were both greater in ganglia of young normotensive than in those of hypertensive rats. Between 10 and 60 weeks of age, peptide immunoreactivity and total cell numbers both fell in normotensive rat ganglia but remained constant in ganglia from hypertensive rats. Densitometric analysis showed that the concentrations of neuropeptide Y were similar in neurons of age-matched individuals of both strains, but during aging there was a substantial decline in neuronal peptide content that was similar in both strains and that was not accompanied by any decline in neuronal immunoreactivity for tyrosine hydroxylase. Our results suggest that there is a developmental abnormality of neuropeptide Y in sympathetic neurons of this strain of genetically hypertensive rat and that, furthermore, the aging process is accompanied by a selective loss of neuronal neuropeptide Y that is independent of blood pressure status.

Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.