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Notes: Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment. The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall. In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules. The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.

Notes: General Summary The investigations described herein were made primarily to ascertain the effects of dehydration on acid-phosphatase and catheptic-proteinase activities in the hypothalamo-hypophysial neurosecretory system of Zonotrichia leucophrys gambelii, thereby providing a basis for comparison with the previously demonstrated changes in the activities of these enzymes in photoperiodically induced gonadotropic activity. Thus they provide a further test of the hypothesis that the accumulations of neurosecretory material in the pars nervosa and the palisade layer of the median eminence are under essentially independent controls. They also provide further information concerning the usefulness of data on the activities of these enzymes as indicators of the functional state of the hypothalamo-hypophysial neurosecretory system. 1. Dehydration by deprivation of drinking water for 8–32 hours was found to have the following effects: a) Region of supraoptic nucleus. Initial increase in acid-phosphatase activity with return to normal by 24 hours after the beginning of the period without water; increase in proteinase activity that becomes significant between 8 and 32 hours of deprivation from water; increase in nuclear diameter in neurosecretory cells and reduced amounts of coarse, dense aldehyde-fuchsin positive material with an increase in fine granules. Similar, although less extensive changes in nuclear size and neurosecretory material were observed also in the paraventricular nucleus. b) Regions of the median eminence. No clearly significant change in acid phosphatase activity; marked increase in proteinase activity; no change in density of neurosecretory material in the posterior division; an initial increase followed by a subsequent, marked decrease in neurosecretory material in the zona externa of the anterior median eminence (regions I and II). c) Pars nervosa. Marked increase in acid-phosphatase and proteinase activities and in weight of gland; decrease in neurosecretory material. d) Adenohypophysis (pars distalis). No significant change in acid-phosphatase activity; increase in proteinase activity. e) Adrenal gland. Increase in volume of cortical tissue; decrease in sudanophilic material. 2. The results of these experiments, when compared with the results of experiments with photoperiodic stimulation of the hypothalamo-hypophysial neurosecretory system, support the hypothesis of independent functions of the eminential and neurohypophysial components. 3. It appears that dehydration may exert two separate effects on the hypothalamo-hypophysial system of this species: a) The well-known reduction of the density of neurosecretory material in the neurosecretory cells and in the pars nervosa (also in the hypothalamo-hypophysial neurosecretory tract) now associated with the release of antidiuretic hormone (probably arginine vasotocin in birds). b) Possibly also an effect operating through the median eminence and adenohypophysis (pars distalis) that causes an increase in activity in the cortical cells of the adrenal gland and a reduction in the amount of sudanophilic material therein. It is suggested that the latter may be associated with the salt-excreting and/or reabsorbing functions of the kidney.

Notes: Summary Localization of monoamine oxidase (MAO) was investigated essentially according to the method of Glenner et al. (1957) in the hypothalamo-hypophysial system of the tree sparrow and the rat. The hypothalamic neurosecretory cells of both species showed relatively weak MAO activity. A similar localization of MAO activity was observed in the median eminence of both species: (1) slight or no MAO activity was observed in the ependymal layer, (2) relatively strong activity was revealed in the tissue just beneath the ependymal layer, (3) strong activity was revealed in the outer layer, particularly in the tissues surrounding capillary loops of the primary plexus. It is suggested that an adrenergic mechanism functions in the median eminence. In the pars nervosa, strong reaction was observed in the rat, while a weak reaction occurred in the tree sparrow. However, the color and the size of formazan crystals deposited in the rat pars nervosa differed from those in the hypothalamus. As a whole, the distribution of the neurosecretory material differed from the localization of MAO activity in the hypothalamo-hypophysial system. It is discussed that the neurosecretory neuron is not adrenergic but cholinergic.

Notes: Summary By use of a stereotaxic instrument it has been consistently possible to introduce 35S DL-cysteine into the third ventricle of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. Such injections are followed within six hours by a reproducible pattern of accumulation of the isotope in the hypothalamic nuclei around the third ventricle. The nucleus supraopticus, the nucleus paraventricularis magnocellularis, the lamina terminalis, and the area ependymalis vasculosa consistently showed the greatest accumulation of the isotope. In the supraoptic and paraventricular nuclei, conspicuous accumulation of the isotope was consistently observed in association with cells containing aldehyde-fuchsin positive material. The supraoptico-hypophysial tract, the pars nervosa, and the palisade layer of the zona externa of the median eminence showed pronounced accumulations of the isotope. These accumulations were, to a great extent, associated with aldehyde-fuchsin positive neurosecretory material.

Notes: Summary The distribution of monoamine-oxidase and acetylcholinesterase activities in the hypothalamus of the White-crowned Sparrow has been studied in relation to the hypothalamohypophysial neurosecretory system. The enzyme activities, as revealed by the methods employed, are unaffected during photoperiodically induced testicular growth. Monoamine oxidase has a distribution distinctly different from that of the aldehyde-fuchsin positive neurosecretory material in that there is high activity in the peripheral palisade layers of both the anterior and posterior divisions of the median eminence. Intimate contact is made between these areas with the primary vessels of the hypophysial portal system. A second concentration of activity lies in a layer between the ependymal cells and the neurosecretory material of the fiber tract. In general, monoamine oxidase appears to be associated with glial elements and non-neurosecretory axons. The pars nervosa has little or no monoamine-oxidase activity. The distribution of acetylcholinesterase activity in the anterior division of the median eminence is very similar to that of the aldehyde-fuchsin positive neurosecretory neurons; however, acetylcholinesterase also occurs in the posterior division without associated neurosecretory fibers. These distribution of enzyme activities are considered in relation to possible adrenergic and cholinergic mechanisms in the median eminence.

Notes: Summary 1. The metabolic activity of the hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, has been investigated by means of quantitative estimates of acid- and alkaline-phosphatase activity. The activities of samples from the median eminence, samples from the supraoptic region, the adenohypophyses, and the neurohypophyses of photosensitive and refractory birds, both subjected to short and long daily photoperiods, have been compared. Histochemical demonstrations of the distribution of acid- and alkaline-phosphatase activity have been effected to provide a better basis for interpretation of the quantitative data. 2. The photosensitive birds which were subjected to 20-hour daily photoperiods showed the typical photoperiodically induced testicular growth and fat deposition. There was an increase in acid-phosphatase activity in the supraoptic region. This increase in acid-phosphatase activity may be correlated with increased activity of the neurosecretory cells as indicated by cytologic criteria, including an increased rate of formation of aldehyde-fuchsin stainable material. No such increase in acid phosphatase occurred in refractory birds on 20-hour photoperiods. In photosensitive birds, there was also an increase in acid-phosphatase activity in the median eminence, an increase which appears to be attributable primarily to changes in activity of the axons in the glandular layer although a contribution by the nucleus tuberis cannot be excluded. The change in acid-phosphatase activity in the median eminence coincides well with the observed increase in rate of disappearance of aldehyde-fuchsin-positive granules from the axons. Neither an increase in acid phosphatase nor a decrease in granules occurred in the median eminences of refractory birds subjected to 20-hour daily photoperiods. The acid-phosphatase activity of the adenohypophyses of photosensitive birds was greater than in refractory birds although neither was affected by long daily photoperiods. These results are consistent with the hypothesis that the photoperiodic response in Zonotrichia leucophrys gambelii involves, in the supraoptic region, an increase in acid-phosphatase activity associated with increased synthesis of aldehyde-fuchsin-positive material in the neurosecretory cells, and, in the axons of the glandular layer of the median eminence, an essentially simultaneous increase in acid-phosphatase activity associated with a humoral transfer to the capillaries of the hypophysial portal system. Our data suggest that the higher rate of production of gonadotropins in the adenohypophysis by photosensitive birds involves a higher acid-phosphatase activity but that the photoperiodically induced increase in release of these gonadotropins, mediated by a humoral agent in the hypophysial blood, does not involve a detectable further increase in acid-phosphatase activity. 3. The data on alkaline phosphatase in supraoptic-region, median-eminence region, and adenohypophysis give no indication that this enzyme is involved in the photoperiodically induced activity of the hypothalamo-hypophysial system. 4. In agreement with previous investigations in this laboratory (Oksche et al., 1959), the data obtained in this investigation on phosphatase activity indicate no participation by the neurohypophysis in the photoperiodic response. 5. The histochemical observations indicate that the neurosecretory cells of the paraventricular and supraoptic nuclei have acid- and alkaline-phosphatase activities similar to those of ordinary neurones. The axons of neurosecretory cells, except in the median eminence, are not identifiable by histochemical demonstration of phosphatase activity. In the fibers of the glandular layer of the median eminence there is a pronounced acid-phosphatase, but no alkaline-phosphatase reaction, in these fibers. Also the nuclei and nucleoli of cells of the mammillary and tuberal nuclei have a fairly strong acid-phosphatase activity. In the adenohypophysis there is a conspicuous acid-phosphatase and a strong alkalinephosphatase reaction in the nuclei and nucleoli, but only slight activities in the cytoplasm.

Notes: Summary The distribution of an immunoreactive endothelin-1-like peptide was investigated in the nereid, Neanthes diversicolor, using an antiserum raised against synthetic endothelin-1. Immunoreactive perikarya were localized in the brain, and nerve fibers containing endothelin-1-like material were found in the neuropil occupying the central portion of the brain. No immunostained fiber elements were traced in the circumesophageal connectives. Immunoreactive perikarya occurred in the subesophageal ganglion. From this ganglion, specifically stained fibers run posteriorly toward the ventral nerve cord. In each segmental ganglion, immunoreactive neurons were observed in medio-ventral and latero-ventral regions, and one or two marked fibers extended to the parapodium. In the parapodium, small immunoreactive perikarya and fiber elements were visible. Immunolabeled fibers occurred in the stomatogastric nerves, in the wall of the buccal cavity, and in the pharynx, esophagus, intestine and its anal region. Immunoreactive perikarya and nerve fibers were visualized between the circular muscle layer and epithelial cell layer in the esophagus and intestine. The endothelin-1-like substance shown to occur in N. diversicolor appears to function as a neurotransmitter or neuromodulator.

Notes: Abstract. Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.

Notes: Summary The distribution of cholinesterases in hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow has been examined histochemically. The perikarya of the neurosecretory cells of the paraventricular and supraoptic nuclei have a high acetylcholinesterase activity. Acetylcholinesterase activity also occurs in the cells of the infundibular nucleus. The proximal parts of the axons of the cells of the neurosecretory and infundibular nuclei have strong acetylcholinesterase activity and weak non-specific cholinesterase activity. In the median eminence, the activity of acetylcholinesterase is strongest in the palisade layer. In the pars nervosa, there is definite, although weak, acetylcholinesterase activity.

Notes: Summary Mechanisms inducing drinking after water deprivation, and mechanisms terminating drinking after rehydration, were investigated in the quail,Coturnix coturnix japonica. 1. Water intake was induced after 4 h of water deprivation, and the amount of water drunk increased in proportion to the period of water deprivation. Drinking occurred immediately after deprived birds were given access to water, and continued for periods proportional to the period of water deprivation. 2. Plasma angiotensin II concentration increased, as did plasma osmolality and Na+ concentration, and blood volume decreased after water deprivation. The increase in plasma angiotensin II concentration and decrease in blood volume occurred soon after the start of water deprivation, whereas plasma osmolality and Na+ concentration did not increase until at least 4 h after the start of water deprivation. 3. These results indicate that extracellular dehydration and angiotensin II are responsible for the significant drinking that follows 4 h of water deprivation, and that cellular dehydration is also involved in the stimulation of drinking that occurs after longer periods of water deprivation. 4. Plasma osmolality and Na+ concentration in birds deprived of water for 48 h quickly returned to normal levels after the birds were allowed access to water. Plasma angiotensin II levels and blood volume also approached the values measured prior to water deprivation. However, the rate and degree of restoration of normal values were reduced, and normal values were not restored even after 1.5 h of rehydration when drinking terminated. 5. The amount of water drunk over the course of 1.5 h by birds deprived of water for 48 h was much greater than the amount required to restore the changes in plasma osmolality and blood volume to normal, but neither excessive dilution of plasma nor abnormally high blood volumes were observed during drinking or 0.5 h after drinking terminated.