Mona Castel

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‐γ‐aminobutyric acid (GABA). Data were analysed by computer‐assisted image analysis. Density of glutamate labelling (gold particles per μm2) 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 ∼ 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.

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 tight 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.

Cell-Specific Expression of the Rat Oxytocin Gene in Transgenic Mice

The genes for the posterior pituitary hormones oxytocin (OT) and vasopressin (VP) are expressed in magnocellular neurons of the hypothalamus. Previous attempts to obtain cell‐specific expression of OT and VP transgenes in mice have been unsuccessful using constructs containing only the OT or VP genes. As the endogenous genes are located near each other on the same chromosome, we investigated four transgenic mouse lines incorporating a single 5.2 k bp construct of rat genomic deoxyribonucleic acid with 1.63 k bp and 3.55 k bp of the OT and VP genes, respectively. Rat OT transcripts were detected only in transgenic mouse OT neurons of the paraventricular and supraoptic nuclei in two lines. The levels of endogenous and transgene OT transcripts increased in response to 10 days of lactation. Furthermore, rat OT‐associated neurophysin immunoreactivity was detected in magnocellular cell bodies as well as in the posterior pituitary of the transgenic but not the control mice. Cell‐specific rat VP expression was not detected in these transgenic mice.

Changes in hypothalamic and extra-hypothalamic vasopressin content of water-deprived rats.

SummaryA correlative radioimmunoassay (RIA) and immunocytochemical (ICC) study was carried out on vasopressin (VP) distribution and content in brains of normal and 3-day water-deprived rats. By RIA there were statistically significant differences in brain VP per pg/mg between normal and osmotically stressed specimens in hypothalamus (338.4 versus 134.4), thalamus (4.8 versus 0.9), septum (18.0 versus 3.4), striatum (1.6 versus 0.7) and amygdala (17.3 versus 1.3), but not in other brain regions measured. Pituitary VP decreased from 71.1 to 8.7 ng/mg, and plasma VP rose from 3.6 to 19.3 pg/ml during water deprivation. Application of the peroxidase-anti-peroxidase ICC method of Sternberger to vibratome sections showed that VP-immunoreactivity in dehydrated specimens decreased in perikarya of paraventricular nucleus and suprachiasmatic nucleus, while intrahypothalamic immunoreactive magnocellular fibers appeared more conspicuous due to proliferation of large Herring bodies. In extrahypothalamic sites VP-immunoreactivity in water-deprived rats was visibly reduced in periventricular thalamus and septum. Thus it is apparent that both intra- and extrahypothalamic VP are affected by osmotic stress, and these results are discussed within the context of current ideas relating to co-activation of neurosecretory cells that project to different sites.

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

SummaryUltrastructural 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.

Ultrastructural immunohistochemical localization of vasopressin in the hypothalamic-neurohypophysial system of three murids.

SummaryVasopressin was immunohistochemically localized at the electron microscopic (EM) level in the hypothalamic-neurohypophysial system (HNS) of three murids. Antiserum to vasopressin was produced in rabbits injected with lysine vasopressin (LVP) conjugated to egg albumin (EA), anti-EA being precipitated prior to staining. Sternbergers unlabeled antibody peroxidase technique was employed, immunoreactivity being designated by peroxidase anti-peroxidase (PAP) molecules and electron opacity. Immunoreactive neurosecretory granules (NSG) were found in the perikarya of the supraoptic nucleus (SON) in all three murids investigated, although far more profusely in the two wild strains. Immunoreactive axonal NSG were observed in the inner and outer zones of the median eminence (ME), and within most of the axons and terminals in the neurohypophysis. The concentration of primary serum effective for staining the SON (1∶10–1∶50) was far higher than that required for the ME and the neurohypophysis (1:500–1:1,200). AntiLVP also induced electron opacity of granules in cells of the pars intermedia (PI). Discussion centers on the significance of immunoreactive NSG in the neurosecretory (NS) perikarya, on the possibility of an extragranular pool of hormone, and on speculation about the electron opacity of the PI granules.

Immunocytochemical identification of dynorphin-containing vesicles in brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.

Ultrastructure of the anuran pars intermedia following severance of hypothalamic connection

SummaryThe ultrastructure of the pars intermedia of Rana catesbeiana tadpoles was studied following isolation from the hypothalamus, in vivo after sectioning of the pituitary stalk, and in vitro after implantation of the pituitary into a piece of tail fin. Both experimental procedures were followed by rapid and sustained skin darkening. Pituitaries from normal light and dark adapted tadpoles served as controls.In 4-hour disinhibited glands, melanotrophs revealed hyperactive Golgi bodies, colloid vesicles (1–2 microns) in close proximity to axon terminals, and no apparent loss of secretory granules. At 24 hours extracellular colloid adjacent to axon terminals was found, and extensive arrays of RER appeared in the melanotrophs. Obvious granule loss from secretory cells occurred within a week, by which time the cytoplasm was occupied by large cisterns of SER and RER and abundant free ribosomes. Dense core vesicles (600–900 Å) in aminergic nerve terminals disappeared shortly after isolation of the pituitary from the hypothalamus, and only decreasing numbers of translucent vesicles (200–300 Å) were found.The functional significance of these changes is discussed, with particular emphasis on the mode of acute hormone release.

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.

Pseudopodia formation by neurosecretory granules.

SummaryUltrastructural studies of the mouse neurohypophysis, under various experimental conditions, revealed a number of neurosecretory granules (NSG) bearing single pseudopodia-like protrusions. Some NSG adhered to the axolemma via pseudopodia; other NSG, distant from the axolemma, budded electron lucent microvesicles from the tip of the pseudopod.Pseudopodia counts were made on electron micrographs, and calculated as a percentage of the NSG population. In neural lobes from intact mice, small numbers of pseudopodia were observed (0.3%); the count increased significantly after injections of large doses of horseradish peroxidase (HRP) (9.4–14.5%); hypertonic saline augmented the count, as did histamine.In vitro incubation experiments with isolated neural lobes in Krebs Ringer revealed concomitant pseudopodia formation and elevated vasopressin release (measured by antidiuretic bioassay) in the presence of HRP and di-butyryl cyclic AMP respectively. Histamine and excess potassium also increased hormone secretion, but did not induce pseudopodia formation in vitro; pseudopodia were observed neither in controls, nor in the presence of ineffective secretagogues.It is suggested that the pseudopod may represent the active site on the granule membrane. Different ultrastructural images of granule release suggest that several modes of hormone release may be operative in the neurohypophysis. The role of HRP in pseudopodia formation and vasopressin release is enigmatic.