Localization of neurophysin in the rat supraoptic nucleus

Summary Localization of neurophysin in neurons of the supraoptic nucleus was accomplished using an unlabeled-antibody, post-embedding, immunoperoxidase technique. Neurophysin was exclusively associated with neurosecretory granules within cell bodies of supraoptic neurons and their processes.Supported by U.S.P.H.S. Grant HD-08867     

Contribution of astrocytes to synaptic transmission in the rat supraoptic nucleus

Astrocytes, besides supporting metabolic and scaffolding functions, play a prominent role in the modulation of neuronal communication. In particular, they are responsible for clearing synaptically-released glutamate via highly specific transporters located on their plasma membrane. Since glutamate is the main excitatory neurotransmitter in the central nervous system (CNS), astrocytes are likely to play a central role in the regulation of synaptic processing and overall cellular excitability. We recently investigated the influence of astrocytes on glutamatergic and GABAergic transmission in the rat supraoptic nucleus (SON) of the hypothalamus. This nucleus is part of the hypothalamus-neurohypophysial system (HNS), which constitutes a conspicuous example of activity-dependent neuroglial plasticity, in which certains physiological conditions, such as parturition, lactation, and dehydration are accompanied by a structural remodeling of the neurones, their synaptic inputs and their surrounding glia. The use of pharmacological inhibitors of glutamate transporters on this model, in which a physiological change in the astrocyte environment occurs, has brought new insights on the contribution of astrocytes to both excitatory and inhibitory neurotransmissions. The astrocytic environment of neurons appears to control glutamate uptake and diffusion in the extracellular space. This has direct repercussions on the tonic level of activation of presynaptic glutamate receptors and, as a consequence, on the release of neurotransmitter. This short review summarizes data obtained so far, which clearly support the view that astrocytes are indeed a third partner in synaptic transmission, and which show that the supraoptic nucleus represents a remarkable model to study dynamic physiological interactions between astrocytes and neurons.     

Atrial natriuretic peptide secretion during development of the rat supraoptic nucleus

Since a relationship between atrial natriuretic peptide and oxytocin was recently demonstrated in the heart (Gutkowska et al., 1997), the aim of this study was to determine whether a relationship between the two peptides is present also in the rat hypothalamus. For this purpose, we measured ANP-ontogeny in the rat hypothalamus immunohistochemically and compared it with oxytocin-ontogeny which we previously studied. The results showed that the ANP-peptide and mRNA-ANP start at the 18th day of the fetal life. Our earlier data for oxytocin in the rat hypothalamus showed that only mRNA-oxytocin appeared the 18th day of foetal life (Farina Lipari et al., 2001); thus, at the 18th day of foetal life, mRNA-ANP, ANP-peptide and mRNA-oxytocin are present. We conclude that in the hypothalamus, differently from that in the heart, ANP might play a role on the synthesis of the oxytocin since ANP and its mRNA appear earlier than oxytocin.     

Ultrastructural and morphometric studies of nonsecretory neurons of the rat supraoptic nucleus

Ultrastructural and morphometric studies were made on nonsecretory and complementarily neurosecretory neurons of the rat supraoptic nucleus. 6% of perimeter of nonsecretory neuronal perikarya was covered by presynaptic endings. The value was well in agreement with that of interneurons elsewhere. The covering ratio of neurosecretory neurons was 12%. The perikarya of nonsecretory and neurosecretory neurons bear an average of 14 and 49 axon terminals, respectively. Nonsecretory neurons should be interneurons, receiving much less information than neurosecretory neurons.     

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus

Summary Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Groups of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus undergoes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.     

Choline acetyltransferase immunocytochemical staining of the rat supraoptic nucleus and its surroundings

Summary Two different monoclonal antibodies raised against choline acetyltransferase were used, together with preembedding immunocytochemical techniques, to visualize the possible cholinergic innervation of the supraoptic and paraventricular nuclei of the rat hypothalamus. Light microscopy confirmed the presence of a group of bipolar and multipolar immunoreactive neurones in the hypothalamus dorsolateral to the supraoptic nucleus as well as numerous immunopositive fibers. Electron microscopy showed that the immunopositive cell bodies contained the usual perikaryal organelles while most immunoreactive fibers appeared dendritic; immunonegative terminals made synaptic contact onto these profiles. Immunopositive terminals making synaptic contact onto dendritic profiles were also noted in this area. In contrast, light microscopy showed no immunoreactivity to choline acetyltransferase in the magnocellular nuclei themselves. Electron microscopy revealed some immunopositive profiles along the boundaries of both nuclei, along the optic chiasm adjacent to the supraoptic nucleus and in the ventral glial lamina but not within the nuclei proper. Surprisingly, these immunopositive profiles appeared dendritic and were often contacted by one or more immunonegative synapses. Our observations thus indicate that cell bodies and dendrites in the supraoptic and paraventricular nuclei are not directly innervated by cholinergic synapses. The functional significance of the putative cholinergic dendrites in close proximity to magnocellular neurones remains to be determined.     

Investigating the role of the hypothalamic supraoptic nucleus in nociception in the rat

We investigated the role of the hypothalamic supraoptic nucleus (SON) in nociception in the rat. Electrical stimulation of the SON or microinjection of a small dose of L-glutamate sodium into the SON elevated the nociceptive threshold in a dose-dependent manner, while cauterization of the SON decreased the nociceptive threshold. Pituitary removal did not influence the antinociceptive effect of L-glutamate sodium in the SON. The data suggested that the neurons and not the nervous fibers in the SON played an important role in antinociception.     

Immunocytochemistry and ultrastructure of the neuropil located ventral to the rat supraoptic nucleus

Summary The neuropil located ventral to the SON was investigated by the use of immunoperoxidase staining for neurophysins, oxytocin and vasopressin, and electron miroscopy. The study was performed in six groups of rats: 1) control; 2) infusion of isotonic saline into the CSF; 3) infusion of hypertonic saline into the CSF; 4) drinking hypertonic saline for 4 days; 5) same as group 4 but injection of colchicine into the CSF on second day of dehydration; 6) salt loading for 3 months. In the control rats the ventral neuropil contained a few immunoreactive processes, the general morphology of which was completely different from that of the neurosecretory axons emerging from the SON at its dorsal aspect. In rats of groups 3 to 6 the ventral processes (VP) became loaded with neurosecretory granules, whereas the perikarya and axons were depleted. Based on their general morphology and reactivity pattern it is suggested that the VP are dendrites. Most of these dendrites were embedded in a glial cushion formed by the processes of a particular type of marginal glia. Some of these dendrites enveloped an arteriole penetrating the optic tract. All VP were rich in synaptic contacts. The possibility that the VP of neurosecretory cells may be functionally related to the subarachnoid CSF and the arteriolar blood flow is discussed.Supported by Grant RS-82-18 from Direccíon de Investigaciones, Universidad Austral de Chile     

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10-20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na(+)-K(+)-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.     

Quantitative and morphometric parameters determining the development of the supraoptic nucleus in the albino rat

The principal part of the supraoptic nucleus (SON) of the rat presents specific developmental factors. The parameters selected for their evaluation were volume of the SON, neuronal number and neuronal density. White Wistar rats of the age of 18, 19, 20, 22 and 23 intrauterine days, 15 days and 1, 3, 6, 12, 18 and 30 months were chosen for this study. The rat brains were fixed, cut into 10-micron-thick sections and stained with hematoxylin-eosin. The different measurements were carried out with a semiautomatic IBAS I image analyzer. In all stages of the rat life, an increase in volume and a decrease in neuronal density per surface unit could be observed, but there was a difference in the dynamics of these changes depending on the stage in which the parameters were determined. There were two periods in the life of the rat in which neuronal death was very significant. The first was between the embryonic and juvenile stage and the second between the adult and senile stage. The increase in the volume influences predominantly the value of neuronal density.     

Modifications of neuronal activity in the rat hypothalamic supraoptic nucleus after long-lasting vibrational stimulation

We analyzed the background impulse activity (BIA) generated by neurons of the rat hypothalamic supraoptic nucleus in the norm and under conditions of long-lasting vibrational stimulation (exposure 5, 10, or 15 days). Distributions of neurons by the level of regularity, dynamics of discharge trains, form of histograms of interspike intervals (ISIs), as well as distributions of neurons by the BIA frequency ranges, were studied. We also calculated the mean frequency of impulsation of the neurons under study and the coefficient of variation of ISIs. After vibrational influences, we found modifications of both the internal structure of the recorded spike trains and the mean frequency of impulsation within the entire studied group and different frequency subgroups. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 224–230, May–June, 2006.     

Inhibitory effects of propofol on supraoptic nucleus neurons of rat hypothalamus in vitro

To investigate the effects of novel intravenous general anesthetic propofol on membrane electrophysiological characteristics and action potential (AP) of the supraoptic nucleus (SON) neurons and possible ionic mechanisms, intracellular recordings were conducted in SON neurons from the coronal hypothalamic slice preparation of adult male Sprague Dawley (SD) rats. The results showed that bath application of 0.1 mmol/L propofol induced a significant decline in resting potential (P < 0.01), and higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased time constant and slope resistance of cell membrane (P < 0.01). Under the hyperpolarizing current pulses exceeding 0.5 nA, an anomalous rectification was induced by hyperpolarization-activated cation channel (I(h) channel) in 11 out of 18 tested SON neurons. Bath of propofol reversibly decreased the anomalous rectification. Moreover, 0.1 mmol/L propofol elevated threshold level (P < 0.01) and decreased Max L. slope (P < 0.05) of the spike potential in SON neurons. Interestingly, 0.3 and 1.0 mmol/L propofol nullified APs in 6% (1/18) and 71% (12/17) tested SON neurons, respectively. In the SON neurons where APs were not nullified, propofol (0.3 mmol/L) decreased the amplitude of spike potential (P < 0.05). The higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased firing frequencies evoked by depolarizing current pulses (0.1-0.7 nA), and shifted the current intensity-firing frequency relation curves downward and to the right. These results suggest that propofol decreases the excitability of SON neurons by inhibiting I(h) and sodium channels.