pituitrin and lucifer-yellow

This study was carried out to characterize angiotensin II (ANG II) sensitive neurons in the hypothalamic paraventricular nucleus (PVN) of the rat. An approach was chosen in which a combination of an electrophysiological, a morphological, and an immunocytochemical method was focused on one single neuron. The cell's reaction to an application of ANG II and its specific antagonist Losartan (Dup753) was investigated using the technique of intracellular recording inside 450-microm-thick brain slices. A final injection of a fluorescent dye labelled the neurons. Optical sections were taken through the marked cells by a confocal laser-scanning microscope and made into a three-dimensional cell model on a computer. One-micrometer thin sections were cut from the thick slice at the level of the electrophysiologically characterized and marked cell body for immunocytochemical tests with different antibodies. Our results show an example of such a neuron inside the PVN excited by ANG II. It was possible to block this excitation with the specific ANG II receptor subtype 1 (AT1) antagonist Losartan. The result indicated that the ANG II reaction was mediated by the AT1 receptor subtype. Immunocytochemical studies show that this ANG II-sensitive neuron contains ANG II but no vasopressin. The combination of the results enables us to gain improved information on interactions of peptidergic systems.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Cell Size; Fluorescent Dyes; Immunohistochemistry; Isoquinolines; Losartan; Male; Neurons; Paraventricular Hypothalamic Nucleus; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasopressins

Intracellular recording and labeling were combined with neurophysin immunohistochemistry to study neurons in the paraventricular nucleus region of the rat hypothalamus. Neuronal membrane properties were examined in hypothalamic slices, and cells were labeled by injecting biocytin or Lucifer yellow. Slices were then embedded, sectioned, and immunohistochemically processed for neurophysin. Immunoreactivity patterns, and in some cases counterstaining, enabled determinations of the cytoarchitectonic positions of recorded cells to be made. Recorded cells were divided into three types according to their electrophysiological characteristics. The first type lacked low-threshold Ca2+ spikes and displayed linear current-voltage relations, a short time constant, and evidence for an A current. These were relatively large cells that were typically immunoreactive for neurophysin and were situated near other neurophysin-positive neurons. The second type had relatively small low-threshold potentials that did not generate bursts of Na+ spikes. These cells had heterogeneous current-voltage relations and intermediate time constants. They did not label for neurophysin, and most were located in the parvicellular subregion of the paraventricular nucleus. The third type had large low-threshold Ca2- spikes that generated bursts of Na+ spikes, and these cells had nonlinear current-voltage relations and long time constants. These neurons were dorsal or dorsolateral to the paraventricular nucleus and were not immunoreactive for neurophysin. These results indicate that paraventricular magnocellular neurons lack low-threshold potentials, whereas paraventricular parvicellular neurons display low-threshold potentials that generate one or two action potentials. Neurons that fire spike bursts from low-threshold potentials are adjacent to the paraventricular nucleus, confirming earlier reports.

Topics: Action Potentials; Animals; Calcium; Electrophysiology; Immunohistochemistry; In Vitro Techniques; Iontophoresis; Isoquinolines; Male; Neurons; Neurophysins; Oxytocin; Paraventricular Hypothalamic Nucleus; Rats; Rats, Inbred Strains; Sodium; Vasopressins

This study shows that foetal neurons from the suprachiasmatic area, after dissociation and culture, contain in vitro the same characteristics as are found in the in vivo situation. The main peptidergic neurotransmitters present in the suprachiasmatic nucleus in vivo, vasoactive intestinal polypeptide (VIP) and vasopressin, are expressed in vitro while the cytoskeleton of these cells possesses phosphorylated neurofilaments. The exclusive uptake of Lucifer Yellow liposomes by neurons is also refound in suprachiasmatic cultures. The electrophysiological results are in agreement with those characteristics found in vitro and in vivo.

Topics: Action Potentials; Animals; Culture Techniques; Drug Carriers; Fluorescent Dyes; Immunohistochemistry; Intermediate Filaments; Isoquinolines; Liposomes; Rats; Suprachiasmatic Nucleus; Vasoactive Intestinal Peptide; Vasopressins

The origin of the vacuoles that form in the mammalian collecting duct during antidiuretic hormone (ADH)-mediated water reabsorption was examined using two computer-assisted, light microscopic methods. First, differential interference-contrast microscopy was used in combination with a simple morphometric procedure to quantitatively characterize the time course, magnitude, and cell specificity of vacuole formation in the microperfused rabbit cortical collecting tubule. Second, video-intensified fluorescence microscopy was used to visualize the basolateral endocytosis of a fluorescent, fluid-phase marker (i.e., lucifer yellow) during vacuole formation. In the presence of a lumen-to-bath osmotic gradient, ADH addition induced the rapid (less than 10 min) formation of large (1- to 3-micron diam) vacuoles in principal cells and, to a lesser extent, in a subpopulation of intercalated cells. The vacuoles subsequently shrank and disappeared over the course of 60-90 min in the continued presence of the hormone and osmotic gradient. The vacuoles collapsed very slowly after elimination of the osmotic gradient at the peak of the vacuolation response, which implies that these structures are intracellular compartments rather than dilated extracellular spaces. During their formation the vacuoles could be loaded with peritubular (but not luminal) lucifer yellow, which remained trapped within most of these structures well after the dye was removed from the bath (greater than 30 min). These results indicate that most vacuoles that form during ADH-mediated water reabsorption are intracellular, endocytic compartments that communicate with the peritubular space via endocytosis of basolateral cell membrane.

Topics: Animals; Endocytosis; Epithelial Cells; Epithelium; Female; Image Processing, Computer-Assisted; Isoquinolines; Kidney Tubules; Kidney Tubules, Collecting; Microscopy; Organoids; Osmosis; Perfusion; Rabbits; Time Factors; Vacuoles; Vasopressins

Recently published work in the rat has shown that: the incidence of electrical coupling, as measured by dye coupling, is decreased from control levels by 8 days of drinking hypertonic saline; an index of circulating testosterone, seminal vesicle weight, is also decreased by 8 days of saline drinking; and both plasma and urinary vasopressin levels are reduced in castrated males, but can be returned to normal with testosterone replacement. These findings have led to the hypothesis that dye coupling, particularly that involving vasopressinergic cells, may be affected by gonadal steroids. We have investigated the effects of castration and testosterone replacement on the incidence of dye coupling among the neurons of the predominantly vasopressinergic magnocellular lateral paraventricular nucleus in slices of male rat hypothalamus. Incidence of dye coupling in this nucleus of castrated rats was found to be decreased by 67% from sham castrated control levels. Testosterone-filled Silastic capsules (but not empty capsules) implanted subcutaneously at the time of castration abolished the effect of castration on dye coupling. We conclude that testosterone has a powerful influence upon coupling among PVN vasopressinergic neurons and may participate in the control of vasopressin release in intact animals.

Topics: Animals; Electrophysiology; Feedback; In Vitro Techniques; Isoquinolines; Male; Neurons; Orchiectomy; Paraventricular Hypothalamic Nucleus; Rats; Testis; Testosterone; Vasopressins

An immunocytochemical procedure is described for reliably determining the hormone content of magnocellular neuroendocrine neurons that have been injected with Lucifer Yellow in slices of rat hypothalamus. The chief advantages of this procedure over others currently available are: (a) it permits whole mount observation of the tissue, and thus, of the morphology of filled cell(s) as well as of such phenomena as dye-coupling; (b) the reliability of tissue preparation and peptide determination has been optimized so that about 85% of injected cells are identified immunocytochemically; and (c) the final immunostained product is permanent, permitting bright-field examination of the injected cell. Relative advantages and limitations of this and other recently published methods are discussed.

Topics: Animals; Fluorescent Antibody Technique; Fluorescent Dyes; Hypothalamus; Isoquinolines; Neurons; Neurophysins; Oxytocin; Peptides; Rats; Vasopressins

An attempt was made to identify Lucifer Yellow-labeled neurons in the rat supraoptic nucleus as vasopressin-containing neurons, by means of a combination of immunoperoxidase histochemistry and iontophoretic single cell-injection. We came to the conclusion that the fluorescent dye does not diminish the immunoreactivity of vasopressin in the magnocellular neurons. This newly developed method, along with its modifications, should prove to be quite useful for electrophysiological and morphological studies on the neuropeptide-releasing neurons in the mammalian neuroendocrine system.

Topics: Animals; Immunoenzyme Techniques; Isoquinolines; Male; Microscopy, Fluorescence; Rats; Supraoptic Nucleus; Vasopressins

Topics: Arginine Vasopressin; Electric Conductivity; Electric Stimulation; Electrophysiology; Fluorescent Dyes; Hypothalamo-Hypophyseal System; Isoquinolines; Neurons; Organ Culture Techniques; Radioimmunoassay; Supraoptic Nucleus; Vasopressins