Gustav F. Jirikowski

Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress

In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.

Expression of c-fos protein by immunohistochemically identified oxytocin neurons in the rat hypothalamus upon osmotic stimulation

Double immunostaining for c-fos and oxytocin (OXY) was used to study the topography and time course of the metabolic activation of the hypothalamic oxytocinergic system upon osmotic stress in the male rat. Animals injected i.p. with hypertonic saline expressed c-fos-like immunoreactivity (FLI) in the paraventricular (PVN), periventricular (PEV) and supraoptic (SON) hypothalamic nuclei, and in the preoptic and retrochiasmatic regions, as early as 30 min after stimulation and up to 6 h, while these areas were mostly devoid of staining in isotonic saline-injected animals. The activation of the oxytocinergic system peaked at 30 min and declined at different rates in the PVN and in the SON after 90 min. The maximal percentage of OXY neurons expressing FLI upon osmotic stress was about 80% in the SON, PEV and LSN, 60% in the PVN and 50% in the medial preoptic area. Activated OXY neurons were found in both the magnocellular and parvocellular divisions of the system. These data show that OXY nuclei in the rat hypothalamus are differentially activated by osmotic stress. They also suggest a role of OXY in the central as well as in the humoral response to changes in plasma osmolarity.

Angiotensin II promotes development of neurophysin neurons in dissociated culture

Dissociated cultures of retrochiasmatic hypothalamus of 18-day-old rat embryos were continuously treated with 2 X 10(-9) M angiotensin II (ang II) from the third day in vitro on. Cultivation was terminated at days 9 and 16 in vitro, respectively. Neurons immunoreactive for neurophysin, arg-vasopressin and oxytocin were visualized by immunocytochemistry, using the unlabeled antibody technique, and counted. Large, well-differentiated magnocellular neurons and small, probably immature cells could be distinguished. A certain number of large neurons were, in addition, immunoreactive for either vasopressin or oxytocin whereas the small cells were devoid of such staining. Ang II treatment brought about a modest increase in neurophysin-immunoreactive (NEU-IR) cell numbers at day 9 and a drastic augmentation of both large and small NEU-IR cells at day 16 in vitro, without appreciably affecting the total counts of neurons per culture.

GABAergic neurons in dissociated cultures of rat hypothalamus, septum, and midbrain

SummaryCultures of dissociated tissue blocks from 18 day-old rat embryos containing either septum plus anterior hypothalamus, retrochiasmatic hypothalamus, or midbrain, were maintained for 8–29 days in vitro. GABAergic neurons were visualized by incubation with (3H)γ-aminobutyric acid followed by autoradiography and by immunocytochemistry using an antiserum against glutamic acid decarboxylase. Positive neurons were of variable shapes and sizes. Labeled neurons in the three cultured regions represented 3.4, 1.1 and 1.7%, respectively, of all neurons. Apart from a few exceptions, autoradiographic labeling and immunocytochemical staining coincided. Glial cells did not stain and showed very low grain densities. Radioactivity could be released from preloaded cultures upon depolarization by 50 mM potassium and 100 μM Veratridine. The demonstration of GABAergic neurons in cultures that are known to contain a variety of peptidergic neurons opens up the possibility of studying the interaction between the two classes of neurons in vitro.

Oxytocinergic Neuronal Systems during Mating, Pregnancy, Parturition, and Lactation

In addition to being a posterior lobe hormone, oxytocin (OT) is a central neuroactive substance suspected to be involved in the regulation of reproductive Changes in OT-immunoreactivity in certain hypothalamic regions have been demonstrated by means of radioimmunoassays and light microscopical immunocytochemistry upon treatment with steroid hormones?*5 Increased levels of OT immunoreactivity were observed in the supraoptic (SON) and paraventricular (PVN) nuclei as well as in the lateral septum and other extrahypothalamic sites at around the time of parturition.”* It is likely that these observations reflect changes in synthetic and secretory properties of oxytocinergic hypothalamic systems, modulated in part by changing steroid conditions. We performed extensive light and electron microscopic studies with oxytocin immunocytochemistry under more physiological conditions in order to evaluate the functional significance of previous experiments performed with steroid administration. Since it is known that mating, pregnancy, parturition, and lactation are paralleled by changes in ovarian steroid levels and since oxytocin is well known to be strongly involved in these conditions, we focused our experiments on these physiological changes.

PLASTICITY OF THE INTERFACE BETWEEN OXYTOCIN NEURONS AND THE VASCULATURE IN LATE PREGNANT RATS : AN ULTRASTRUCTURAL MORPHOMETRIC STUDY

Increased numbers of oxytocin-immunoreactive perivascular neurons have been shown to occur in the preoptic region and lateral hypothalamus of female rats around parturition. In the present study, electronmicroscopical immunocytochemistry and morphometry were used to examine such perivascular oxytocinergic neurons as well as those in the classical magnocellular nuclei in late pregnant and lactating rats. In 22 d pregnant animals and in rats killed after 2 d of lactation, numerous oxytocinergic neurons were found in direct apposition to the outer basement membrane of arterioles, venules, and capillaries. The distance between immunoreactive neurons and blood vessels was significantly lower in these animals than in 9 d lactating rats and in ovariectomized controls. It is likely that, around parturition, oxytocinergic perivascular neurons are uncovered by active retraction of glial elements. This plasticity is perhaps facilitated by changing hormonal conditions around parturition. The observed changes seem to be transitory and might reflect altered secretory properties of perivascular oxytocinergic neurons.

Expression of Exogenous Vasopressin mRNA by Magno‐Cellular Neurons of the Supraoptic and Paraventricular Nuclei in Brattleboro Rats

Neurons containing presumptive peptide neurotransmitters have been extensively studied by antibodies for the peptides and their prohormone precursors and by in situ hybridization for the mRNAs encoding these precursors. A basic premise of mammalian neuronal biology is that the mRNA is restricted to the perikaryal and dendritic compartments containing rough endoplasmic reticulum. However, there are multiple reports that mRNAs can be detected in peripheral axons in invertebrate sy~tems.~-~ In the mammalian brain, specific intradendritic mRNAs are seen On the other hand, Guitteny and Bloch,* using autoradiographic detection of vasopressin mRNA hybridization by both light and electron microscopy, reported that hybridization was restricted to perinuclear cytoplasm. Nevertheless, the micrographs presented in this study do not exclude the possibility that vasopressin mRNA is present in more distal neuronal processes. McCabe et aL9 reported the presence of vasopressin mRNA in extracts of the posterior lobe, by solution hybridization. Although they did not directly determine the cellular origin of this mRNA, they suggested that it might arise from pitui~ytes,~ the major cell type in the posterior pituitary, other than the vascular elements.