Biomedical subjects

M Castel

Publications and source records attributed to M Castel.

At least 19 recordsLinked to original sources

PubMed2001

Cell-specific expression and subcellular localization of neurophysin-CAT-fusion proteins expressed from oxytocin and vasopressin gene promoter-driven constructs in transgenic mice.

The cell-specific expression of both the oxytocin (OT) and vasopressin (VP) genes in magnocellular neurons (MCNs) of the hypothalamus has been proposed to be under the control of cis-elements in an intergenic region downstream of the VP gene. We examined this hypothesis using transgenic mice containing mouse genomic DNA-derived constructs linked to chloramphenicol acetyltransferase (CAT) reporters. VP gene expression was studied using constructs containing 3.8 kbp of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to a CAT reporter. The two VP-transgene constructs differed by the lengths of their VP gene 3' flanking regions (2.1 versus 3.6 kbp). A similar construct for the oxytocin CAT transgene was used which contained the full-length (3.6 kbp) downstream intergenic region between the mouse genes. All three transgenic constructs produced cell-specific expression of the CAT-reporter in the magnocellular neurons as determined by CAT-immunoreactivity. Oxytocin transgene expression was restricted to OT cells in two founders, and the two VP transgenes to VP cells in five founders. Electron microscopic immunocytochemistry showed that the CAT fusion proteins produced from the OT- and VP-transgenes were efficiently trafficked through the regulated secretory pathways in their respective magnocellular neurons, packaged into large dense core vesicles, and transported to nerve terminals in the posterior pituitary.

Amygdala↗

PubMed2000

Morphological heterogeneity of the GABAergic network in the suprachiasmatic nucleus, the brain's circadian pacemaker.

GABA (gamma-amino-butyric acid) is the predominant neurotransmitter in the mammalian suprachiasmatic nucleus (SCN), with a central role in circadian time-keeping. We therefore undertook an ultrastructural analysis of the GABA-containing innervation in the SCN of mice and rats using immunoperoxidase and immunogold procedures. GABA-immunoreactive (GABA-ir) neurons were identified by use of anti-GABA and anti-GAD (glutamic acid decarboxylase) antisera. The relationship between GABA-ir elements and the most prominent peptidergic neurons in the SCN, containing vasopressin-neurophysin (VP-NP) or vasoactive intestinal polypeptide (VIP), was also studied. Within any given field in the SCN, approximately 40-70% of the neuronal profiles were GABA-ir. In GABA-ir somata, immunogold particles were prominent over mitochondria, sparse over cytoplasm, and scattered as aggregates over nucleoplasm. In axonal boutons, gold particles were concentrated over electron-lucent synaptic vesicles (diameter 40-60 nm) and mitochondria, and in some instances over dense-cored vesicles (DCVs, diameter 90-110 nm). GABA-ir boutons formed either symmetric or asymmetric synaptic contacts with somata, dendritic shafts and spines, and occasionally with other terminals (axo-axonic). Homologous or autaptic connections (GABA on GABA, or GAD on GAD) were common. Although GABA appeared to predominate in most neuronal profiles, colocalisation of GABA within neurons that were predominantly neuropeptide-containing was also evident. About 66% of the VIP-containing boutons and 32% of the vasopressinergic boutons contained GABA. The dense and complex GABAergic network that pervades the SCN is therefore comprised of multiple neuronal phenotypes containing GABA, including a wide variety of axonal boutons that impinge on heterologous and homologous postsynaptic sites.

Animals↗

PubMed1998

Prominent neuronal-specific tub gene expression in cellular targets of tubby mice mutation.

The tubby strain of mice exhibits maturity-onset obesity and sensory deficits in vision and hearing. The mutated gene, tub , responsible for this phenotype was identified recently, but the function of the TUB protein has not been deduced from its amino acid sequence. This prompted us to undertake expression mapping studies with the hope that they might help to elucidate the biological role of the TUB protein. We report the tub gene expression pattern in embryonic, fetal and adult mice tissues as determined by northern blots and in situ hybridization, using antisense oligonucleotidic probes. In mouse embryos, tub is expressed selectively in differentiating neurons of the ensemble of central and peripheral nervous systems, starting at 9.5 days after conception. In adult mice, tub is transcribed in several major brain areas, including cerebral cortex, hippocampus, several nuclei of the hypothalamus controlling feeding behavior, in the spiral ganglion of the inner ear and in the photoreceptor cells of the retina. These structures contain potential cellular targets of the tubby mutation-induced pathogenesis. The neuronal-specific tub gene distribution allows the establishment of a genotype-phenotype correlation in the tubby mice. This correlation is reminiscent of that observed in fat/fat mice, whose phenotype, also characterized by obesity, is caused by a null mutation in the carboxypeptidase E (CPE) gene. Our observations highlight similarities between CPE, prohormone convertases, several neuropeptides and tub gene expression patterns during embryogenesis, and may narrow down the avenues to explore in order to determine ultimately the function of the TUB protein.

Adaptor Proteins, Signal Transducing↗

PubMed1997

GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity.

Mammals manifest circadian behaviour timed by an endogenous clock in the hypothalamic suprachiasmatic nucleus (SCN). Considerable progress has been made in identifying the molecular basis of the circadian clock, but the mechanisms by which it is translated into cyclic firing activity, high during the day and low at night, are still poorly understood. GABA (gamma-aminobutyric acid), a common inhibitory neurotransmitter in the central nervous system, is particularly densely distributed within the SCN, where it is located in the majority of neuronal somata and synaptic terminals. Using an in vitro brain-slice technique, we have now studied the effect of bath-applied GABA on adult SCN neurons at various times of the day. We find that GABA acts as an inhibitory neurotransmitter at night, decreasing the firing frequency; but during the day GABA acts as an excitatory neurotransmitter, increasing the firing frequency. We show that this dual effect, which is mediated by GABA(A) receptors, may be attributed to an oscillation in intracellular chloride concentration. A likely explanation is that the amplitude of the oscillation in firing rate, displayed by individual neurons, is amplified by the dual effect of GABA in the SCN's GABAergic network.

Action Potentials↗

PubMed1997

Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types.

Although light is known to regulate the level of c-fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light-microscopic immunocytochemistry and immunoelectron microscopy to detect c-Fos protein in the SCN of Sabra mice exposed to brief nocturnal light pulses at zeitgeber time 15-16. Stimulation with light pulses that saturated the phase-shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo-inducible c-Fos cells at about one-fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin-neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c-Fos-positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.

Animals↗

PubMed1996

Non-synaptic and dendritic exocytosis from dense-cored vesicles in the suprachiasmatic nucleus.

The sites of exocytosis by dense-cored vesicles (DCVs) from neurones in the rat suprachiasmatic nucleus (SCN) were studied at the ultrastructural level. The tannic acid procedure, which stabilizes extruded proteinaceous substances, was used to demonstrate exocytosis of DCV cores. Fresh brian slices containing the SCN were incubated in media containing high levels of potassium (56 mM) or glutamate (10 mM) in the presence of tannic acid. Long-term slice explant cultures of the SCN were similarly treated. Exocytosis from DCVs occurred from axonal terminals, from dendrites, and occasionally from somata. The sites of DCV exocytosis were generally nonsynaptic or para-synaptic, including release immediately adjacent to axo-spinous synaptic densities. These observations show that, in the suprachiasmatic nucleus, neuroactive substances contained in DCVs do not necessarily function at synaptic contract zones, but could also act as neuromodulators at non-synaptic sites.

Animals↗

PubMed1996

The suprachiasmatic nucleus in stationary organotypic culture.

Suprachiasmatic nuclei, derived from neonate rats, were maintained for several weeks in stationary organotypic culture. Hypothalamic slice explants, supported by Millicell filters and incubated in Petri dishes containing serum-based medium, flattened appreciably, yet preserved the organization of the suprachiasmatic nucleus and the surrounding hypothalamic tissue. After two to three weeks, cultures were fixed, and three neuronal sub-populations were identified as vasopressinergic, vasoactive intestinal peptide-containing, or GABA-containing. The GABAergic component of the cultured suprachiasmatic nucleus was particularly profuse, projecting extensively into the hypothalamic slice. Unilateral ablation of the nucleus in the explant dramatically reduced ipsilateral GABA-immunoreactivity in the slice. Explants in which an incision separated the bilateral suprachiasmatic nucleus from the paraventricular nucleus, deprived the latter of its fine-caliber GABA-immunoreactive input. Extra- or intra-cellular electrophysiological recordings from the suprachiasmatic nucleus were obtained in 51 of 58 cultures. The electrical properties of the long-term cultured suprachiasmatic nucleus were similar to those recorded in acute slices from adult rats. In six cultures recordings were extended for up to 10-24 h. Within long-term stationary organotypic cultures of the suprachiasmatic nucleus, sub-populations of neurons, intrinsic to the nucleus in vivo, were identified immunocytochemically. Lesion studies supported the observation that the main source of the GABAergic innervation within the entire hypothalamic slice explant appeared to be the suprachiasmatic nucleus. Electrophysiological studies confirmed the viability of the long-term cultured nucleus and revealed changes in spontaneous electrical activity that may indicate circadian fluctuation.

Animals↗

PubMed1994

[Desensitization to mites: literature review].

It is agreed that efficacy of specific desensitization to mites is proved by objective double-blind studies, in the short term to reduce crises and drug treatment and in the long term to reduce inflammation and in the case of asthma, irreversible broncho-pulmonary blockages. The selected population must contain both children and adults. Well-defined criteria for inclusion and exclusion are essential in making this selection. Evaluation of specific desensitization is made subjectively by clinical assessment and objectively by different scores for symptoms, drug consumption, skin tests lung function and biological assays. A review of the different methods of desensitization will be made, with their results and tolerance.

Adult↗

PubMed1993

Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures.

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18-39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP-containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA-immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker.

Animals↗

PubMed1993

Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus.

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde-coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin--horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H2O2, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti-glutamate and anti-gamma-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per micron2) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non-retinal non-glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was approximately 3 times higher than that over GABAergic terminals, in which glutamate-like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino-recipient region of the suprachiasmatic nucleus there was also a small population of non-retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate-containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles, has still to be addressed.

Animals↗

PubMed1992

Ultrastructural immunolocalization of rat oxytocin-neurophysin in transgenic mice expressing the rat oxytocin gene.

Cell-specific expression of the rat oxytocin (OT)-neurophysin transgene in mice was achieved using a construct containing both OT and vasopressin genes (Young III, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M., Cell-specific expression of the rat oxytocin gene in transgenic mice, J. Neuroendocrinol., 2 (1990) 1-9). The present study describes the distribution of the protein products of these genes in various regions of the cell, and determines whether the transgenic rat and endogenous mouse OT-neurophysins are colocalized within the same neurosecretory granules. Two monoclonal antibodies against OT-neurophysins were used: PS38 which can react with both rat and mouse OT-neurophysin (pan-specific), and PS67 which is specific for rat OT-neurophysin only. Various approaches to double immunolabeling at the ultrastructural level were employed; these included: (1) pre-embedding immunoperoxidase followed by post-embedding immunogold; (2) post-embedding immunolabeling using gold particles of different sizes; and (3) labeling of consecutive ultrathin sections with different antibodies. Results from each of these approaches showed that both in the transgenic mouse and in the rat (used as control), immunocytochemical labeling for both PS38 and PS67 occurred in the same OT-ergic neurosecretory granules. In the control mouse, only PS38 elicited labeling. Hence, it may be concluded that the protein and peptide products of the transgene and the endogenous gene for OT-neurophysin are being processed similarly in the cell and finally concentrated together in the same neurosecretory granules.

Animals↗

PubMed1990

Vasopressinergic innervation of the mouse suprachiasmatic nucleus: an immuno-electron microscopic analysis.

Attempts are being made to unravel the local circuitry of the suprachiasmatic nucleus, with a view toward eventually correlating specific neuronal systems with circadian events. Hence, the vasopressinergic innervation of this nucleus in the laboratory mouse has been analyzed immunocytochemically at the light and electron microscopical levels. Monoclonal antineurophysin and polyclonal antivasopressin were used on aldehyde-fixed brains. Serial vibratome sections of the appropriate forebrain region were prepared for pre-embedding immunoperoxidase staining and/or postembedding immunogold labeling. Immunoreactive somata, processes, varicosities, and synaptic terminals were found throughout the suprachiasmatic nucleus, their ratio and density varying at different locations. The predominant type of vasopressinergic soma was ovoid to rounded (7-10 microns), containing secretory granules (85-120 nm), a large proportion of which were immunoreactive. Axon terminals, both nonimmunoreactive and immunoreactive, impinged upon vasopressinergic somata and processes, often displaying synaptic specializations. Vasopressinergic terminals, containing secretory granules and microvesicles, were found throughout the nucleus, particularly within the dorsomedial neuropil. These labeled terminals varied in size (0.4-3.4 microns 2) and shape, ranging from compact boutons to pleomorphic profiles, some deeply indented by postsynaptic spines, either dendritic or somatic. Approximately 65% of the vasopressin-containing terminals were axodendritic and 30% axosomatic; about 5% appeared to be axoaxonic. At least a quarter of all vasopressinergic synaptic terminals were axospinous. Other forms of interneuronal contact involving vasopressinergic elements (somata, dendrites) included extensive membrane to membrane appositional sites, and multiple puncta adhaerentia. The versatility of interconnections between vasopressin-containing neurons in the mouse suprachiasmatic nucleus suggests a highly active and coordinated network, which contributes substantially to local intranuclear circuitry. In addition, a dense efferent vasopressinergic output is directed dorsally towards the periventricular hypothalamus, where direct associations may be established with diverse hypothalamic neuroendocrine systems.

Animals↗

PubMed1990

Immunocytochemical localization of the GABA transporter in rat brain.

Polyclonal antibodies were raised against the GABA transporter (GABA-Tp) purified from rat brain tissue (Radian et al., 1986) and used for immunocytochemical localization of the antigen in several rat brain areas, including the cerebellum, hippocampus, substantia nigra, and cerebral cortex. Light microscopic studies with the peroxidase-antiperoxidase and biotin-avidin-peroxidase techniques suggested that GABA-Tp is localized in the same types of axons and terminals that contain endogenous GABA, as judged by comparison with parallel sections incubated with antibodies against glutaraldehyde-conjugated GABA. However, as expected from biochemical results, different neurons differed in their relative contents of GABA-Tp and GABA; thus, GABA-Tp was relatively low in striatonigral and Purkinje axon terminals and relatively high in nerve plexus around the bases of cerebellar Purkinje cells and hippocampal pyramidal and granule cells. The GABA-Tp antiserum did not produce detectable labeling of nerve cell bodies. Electron microscopic studies supported the light microscopic observations and provided direct evidence of cellular co-localization of GABA-Tp and GABA (as visualized by the peroxidase-antiperoxidase technique and postembedding immunogold labeling, respectively). The ultrastructural studies indicated the presence of GABA-Tp also in glial processes but not in glial cell bodies. The relative intensity of the neuronal and glial staining varied among regions: glial staining predominated over neuronal staining in the substantia nigra, whereas the converse was true in the cerebellum and hippocampus. The present immunocytochemical data demonstrate directly what has previously been inferred from biochemical and autoradiographic evidence: that the mechanisms for high-affinity GABA uptake is selectively and differentially localized in GABAergic neurons and in glial cells.

Animals↗

PubMed1989

Variability in gold bead density in cells. Quantitative immunocytochemistry.

Variability in gold bead distribution between individual cells was demonstrated in both pituitary melanotrophic cells immunocytochemically reacted for ACTH and in neurohypophysial terminals reacted for oxytocin-neurophysin. Gold beads were confined to the secretory granules compartment of both tissues. Density of gold beads in melanotrophic cells reacted for ACTH varied from 100-480 gold beads/microns 2. A much narrower range of gold beads distribution (460-900 gold beads/microns 2) was observed in axons of the neurohypophysis reacted with anti-oxytocin-neurophysin. These results indicate that the labeling density varies from cell to cell (as well as axon terminals) within morphologically homogeneous populations. Thus, it may reflect certain physiological differences between cells. A suggestion is being made that mean gold bead density coefficient of variation should be calculated by comparison between individual cells.

Adrenocorticotropic Hormone↗

PubMed1988

The neurophysin-containing innervation of the forebrain of the mouse.

The oxytocinergic and vasopressinergic innervation of the forebrain of normal mice was studied immunocytochemically by use of a set of mouse monoclonal anti-neurophysins applied to serial vibratome sections. The extensive hypothalamic and extra-hypothalamic location of these neuropeptides was revealed, with, or without colchicine pretreatment. Magnocellular perikarya immunoreactive for either oxytocin-neurophysin or vasopressin-neurophysin were concentrated mainly: in the anterior commissural nucleus; in various subdivisions of the paraventricular nucleus; in a profuse array in the periventricular region; in the supraoptic nucleus including its retrochiasmatic division; in various accessory nuclei; and as a number of cells scattered throughout the preoptic and hypothalamic regions. Extensive groups of parvocellular neurons, containing only vasopressin-neurophysin, were located in the suprachiasmatic nucleus including a ventromedial division, in the bed nucleus of the stria terminalis, and in the medial amygdaloid nucleus. Perikarya in the magnocellular nuclei were of generally similar size distribution and there was no evidence that distinct populations of magnocellular and parvicellular neurons, separable on the basis of size, had been labelled within these nuclei. Within the paraventricular nucleus, however, neurons in the posterior part were smaller than those located more anteriorly, and the cells containing oxytocin-neurophysin were slightly smaller than those containing vasopressin-neurophysin. Within the generally similar size distribution, magnocellular neurons of the anterior commissural nucleus were the largest. During processing, shrinkage of the tissue and immunolabeled cells had occurred. The immunocytochemical procedure delineated neuronal processes, in particular dendrites, very effectively. The dendrites were shown to project for far greater distances than is generally recognized, some were of a characteristic corkscrew-like morphology, and most were oriented in a well-defined pattern. Many dendrites of paraventricular neurons passed medially than caudally towards and then along the third ventricle. Most dendrites of supraoptic neurons, in particular those containing vasopressin-neurophysin, had an extensive anteroposterior course beneath the pia of the base of the brain. The axons containing oxytocin- and vasopressin-neurophysin were shown to take rather different paths from the paraventricular nucleus towards the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Ultrastructural characterisation of vasopressinergic terminals in the lateral septum of murine brains by use of monoclonal anti-neurophysins.

Synapses in the lateral septum of the murine brain have been investigated by ultrastructural immunocytochemistry, using monoclonal anti-neurophysins in both immunoperoxidase and immunogold techniques. In the region shown by light microscopy to be rich in vasopressinergic innervation, synaptic boutons containing approximately 30 nm clear vesicles and occasional approximately 100 nm dense-cored granules (granules) were stained by pre-embedding immunoperoxidase procedures with antisera to vasopressin-neurophysin, but not oxytocin-neurophysin; reaction product was diffusely distributed in the terminals. Terminals were symmetrical, and both axosomatic and axodendritic in type. Postembedding immunogold procedures by use of anti-vasopressin-neurophysin labeled only the approximately 100 nm diameter granules in the terminals. Sodium meta-periodate treatment 'bleached' immunoreactive granules, indicating the presence of a carbohydrate residue. The quantum of peptide packaged in the granules appears to be smaller than that in magnocellular neurones; nevertheless, the results indicate that, as in the magnocellular neurosecretory system, vasopressin and its neurophysin are packaged exclusively in granules, and that vasopressin in the septum is likely to be derived from a precursor comprising vasopressin, vasopressin-neurophysin and a glycosylated residue.

Animals↗

PubMed1986

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques.

Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling. Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate. The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity. The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.

Animals↗

PubMed1985

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Animals↗