Newell H. McArthur

Effects of ethanol on hypothalamic luteinizing hormone-releasing hormone (LHRH) in the male rat an immunocytochemical study

SummaryThese studies were designed to determine if the acute alcohol-induced decreases in luteinizing hormone (LH) seen in previous studies using rats could be due to an inhibitory effect of ethanol (ETOH) on hypothalamic LHRH release. Thus, effects of multiple injections of ETOH on the relative amount of immunoreactive LHRH fibers in the hypothalamus and median eminence (ME) of castrate and intact male rats were determined immunocytochemically. Brains were removed following cardiac perfusion of 10% phosphate-buffered formalin. A block containing the hypothalamus with the ME was isolated from each brain, then postfixed in Bouins solution. Paraffin sections were rehydrated and stained for LHRH with the peroxidaseantiperoxidase technique using an antiserum to synthetic LHRH conjugated to bovine serum albumen. Differences visualized immunocytochemically between saline-treated intact and castrate rats indicated that the LHRH content of the ME was markedly depleted after castration. Conversely, castrate rats treated with ETOH showed only a slight reduction in immunoreactive LHRH fibers. In ETOH-treated intact animals, the LHRH fiber content of both the hypothalamus and ME appeared to be slightly greater than the saline-treated intact controls. Thus, these data support the hypothesis that ETOH diminishes LHRH release, and hence provides an explanation for the depressed plasma LH levels observed in ETOH-treated intact and castrate rats.

Immunocytochemical Evidence that Suckling Inhibits the Postovariectomy Depletion of Median Eminence Luteinizing Hormone Releasing Hormone

Suckling has been demonstrated to impair the release of pituitary luteinizing hormone (LH) and to prevent the dramatic increase in plasma LH observed following ovariectomy. In the present study, the effect of suckling (10 pups/animal) for either 1 or 3 weeks on the relative amount of luteinizing hormone releasing hormone (LHRH) present in the hypothalamus and preoptic area of ovariectomized and intact rats was examined using immunocytochemical methodology. Controls consisted of nonlactating animals which were either intact (diestrous) or ovariectomized for 1 or 3 weeks. Brains were removed following transcardial perfusion of phosphate-buffered formalin and Bouins fixative. After dehydration, clearing and paraffin embedding, the brains were sectioned and LHRH localized by an indirect immunoperoxidase technique. A positive reaction denoting the presence of immunoreactive LHRH was observed over axons and termini throughout the rostral to caudal extent of the median eminence (ME) and surrounding the organum vasculosum of the lamina terminalis (OVLT) in the preoptic area. Ovariectomy resulted in a progressive decline in the concentration of LHRH within the ME as evidenced by a reduction in the intensity of the staining reaction and in the number of axons over which the reaction was observed. In contrast, brains from ovariectomized rats which had been suckled appeared to have concentrations of LHRH in the ME equal to or greater than that of the diestrous controls. Similarly, the concentrations of LHRH In the ME of intact, suckled rats did not differ significantly from that of the diestrous controls. Neither ovariectomy nor suckling produced any observable change in the relative concentration of LHRH located near the OVLT. These data demonstrate that suckling prevents the depletion of LHRH from the ME following ovariectomy and provide evidence for mechanism by which the suckling stimulus may suppress plasma LH.

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

SummaryThe distribution of gonadotropin-releasing hormone (GnRH) was studied in the brain and infundibulum (INF) or median eminence of sheep utilizing a peroxidase-antiperoxidase immunohistochemical method. This procedure utilized a specific antiserum generated against GnRH conjugated to bovine serum albumin. In the rostral INF, the greatest concentration of GnRH positive axons was found in the medial region, mostly in the external layer dorsal to the hypophysial portal plexus. In the intermediate portion of the INF, the hormone was mainly observed in the external layer at the more dorsolateral areas ventral to the tuberoinfundibular sulcus. GnRH was generally located medially in the caudal portion of the INF and dorsomedially in the rostral infundibular stalk. Substantial amounts of reaction product were also noted in the internal layer throughout the entire rostrocaudal extent of the INF. The hormone was localized in axons throughout the brain from the septal and medial preoptic areas to the mammillary bodies. GnRH-positive perikarya were scattered in various regions of the infundibular (arcuate) and for the first time in the ventromedial nuclei of sheep hypothalamus.Preabsorption of the specific antiserum with synthetic GnRH abolished staining in both axons and perikarya, whereas preabsorption with thyrotropin releasing hormone, oxytocin, arginine-vasopressin, and adrenocorticotrophic hormone did not affect staining intensity.

A histochemical and ultrastructural study of lipofuscin accumulation in thyroid follicular cells of aging domestic cats

Enzyme activity and fine structure of thyroid follicular cells were investigated in domestic cats at ages: 2 and 8 months, and 1, 3, 5 and 8 years. The following major changes in the follicular cells with advancing age were observed: with histochemistry--an increase in acid phosphatase and beta-glucuronidase activities confined to numerous granules (vesicles); with electron microscopy -- an increase in colloid vesicles, the appearance and increase in size and number of lipofuscin vesicles, and the increase in number of follicular cells containing numerous colloid or lipofuscin vesicles. It is our opinion that the numerous enzymatically active granules of the older groups, observed with histochemistry, are synonymous with the lipofuscin vesicles and many of the colloid vesicles observed with electron microscopy. The cellular changes involving increased enzymatic activity and lipofuscin accumulation with advancing age would correlate with decreased thyroid function reported in the literature. These observations, therefore, would suggest increased autophagic activity involving organelle or unneeded product degradation.

Calcium/Calmodulin-Dependent Protein Kinase II Involvement in Release of Gonadotropin-Releasing Hormone

Involvement of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in regulation of GnRH release was tested by determining the effect of CaM kinase II antagonists (KN-62 or KN-93) on GnRH release from rat or cattle infundibular (stalk median eminence) explants. Preincubation of male rat infundibular explants for 30 min with KN-62 (0.5, 1, 5 or 10 µM) 1.5 h prior to the addition of 59.3 mM (high) K+ resulted in a dose-dependent suppression of GnRH release. A longer pretreatment period (2 h) of rat infundibular explants with KN-62 (1 or 10 µM) appeared to enhance the suppressive effect of the CaM kinase II antagonist. Exposure (2 h) of rat infundibular explants to 10 µM, but not 0.1 µM KN-93, resulted in a complete inhibition of high K+-induced GnRH release. Exposure of steer infundibular explant halves to KN-62 (50 or 100 µM) or KN-93 (50 µM) inhibited high K+-induced GnRH release. Likewise, treatment of heifer infundibular explant halves with KN-93 (50 µM) abolished high K+-induced GnRH release. The period of exposure required for KN-62 to elicit its effect was relatively short since exposure of KN-62 (100 µM) for only 91–150 min of incubation was sufficient to block high K+-induced GnRH release from steer infundibular explant halves. In conclusion, these results: (1) support the hypothesis that CaM kinase II is involved in GnRH release from the rat and cattle infundibulum, (2) demonstrate that the effect of CaM kinase II on GnRH release from cattle infundibula is independent of reproductive state, (3) confirm previous reports supporting Ca2+ and CaM involvement in GnRH release from rat and cattle infundibula and (4) establish that infundibular explants incubated in vitro are useful for studying selected mechanisms regulating hypothalamic neurohormone release from neuron terminals.

GnRH localization in the equine brain and infundibulum: An immunohistochemical study

Immunohistochemical localization of the decapeptide gonadotropin releasing hormone (GnRH) in neural structures in the pony brain and infundibulum (INF) was conducted at the light-microscopic level. This procedure utilized an antiserum generated against GnRH conjugated to bovine serum albumin. In the rostral INF, GnRH was distributed mainly in the external layer, with greatest concentrations adjacent to the long capillary loops of the hypophyseal portal system. The intermediate portion of the INF contained the hormone throughout the external layer, especially in the dorsolateral regions just ventral and medial to the tuberoinfundibular sulcus (TIS) with lesser amounts dorsal to the TIS. Caudally, GnRH was very concentrated along the medial border of the Tis, and in small amounts within the medial portion of the INF just rostral to the mammillary bodies. Throughout the INF, reaction product was noted in the internal layer, although the concentration was less than that observed in the external layer. The hormone was localized in axons of the brain from the medial and lateral septal nuclei through the mammillary region. GnRH positive perikarya were localized in the lamina terminalis, infundibular nucleus and the caudal periventricular nucleus. Preabsorption of the specific antiserum with synthetic GnRH abolished staining in both axons and perikarya, whereas preabsorption with other hypothalamic peptides did not affect staining intensity.

Calcium involvement in luteinizing hormone-releasing hormone release from the bovine infundibulum☆

Bovine infundibular (stalk median eminence) explants were incubated in vitro to test the hypothesis that calcium (Ca) is involved in the release of luteinizing hormone-releasing hormone (LHRH) from LHRH neuron terminals in cattle. Right and left infundibular halves from individual heifers and/or steers were randomly assigned to either control or treated (EGTA [a Ca chelator] or verapamil [an L-type Ca channel antagonist]) groups. Each half was incubated in 600 microliters of Krebs-Ringer bicarbonate medium (KRB) in the presence or absence of a treatment agent for 180 min. At 30-min intervals, 500-microliters samples were removed from each incubated and replaced with fresh media. Spontaneous (basal) and depolarization-induced (60 mM potassium) LHRH release was evaluated by radioimmunoassay of the LHRH content in the media incubated from 91 to 120 and 121 to 150 min of culture, respectively. The effect of treatment on depolarization-induced LHRH release was analyzed by comparing the differences between spontaneous and depolarization-induced LHRH release in control and treated groups. Spontaneous LHRH release was not different between control and 1.25 mM EGTA- or 100 microM verapamil-treated halves from steers. In contrast, steer infundibular halves incubated with EGTA (replacing Ca in KRB and chelating any Ca in the media) released less LHRH during depolarization than did control halves. In addition, verapamil-treated (to block Ca uptake by the terminal) infundibular halves from steers or heifers released less LHRH in response to depolarization than did control halves.(ABSTRACT TRUNCATED AT 250 WORDS)

The armadillo infundibulum: Correlative histochemistry, scanning and transmission electron microscopy of the ventricular surface

The ependymal and supraependymal cells of the armadillo infundibulum (INF) were investigated by correlative histochemistry, scanning and transmission electron microscopy. Eighteen armadillos (8 adult females, 6 adult males, 2 immature females and 2 immature males) were examined. The following supraependymal elements were observed: (a) individual pleiomorphic cells made up of neurons, macrophages, and astrocytic-glial cells; (b) numerous spherical blebs of various sizes occurring singly or in clusters; (c) axons, traversing the surface alone or in association with macrophages and other SEC; (d) multicellular clusters containing SEC, macrophages, axons and other cell types. There were neurosecretory axons or blebs on and below the ependymal cell layer and a unique arrangement of multipolar cells and their processes, traversing the INF floor for several millimeters. The presence of neurosecretory axons at the INF ventricular surface, spherical blebs and SEC in contact with one another via long filaments or vast networks of smaller axons on the surface and numerous macrophages in close apposition to possible metabolic and transport sites give evidence of organized activity in a regulatory system.

Epithalamus of the nine-banded armadillo, Dasypus novemcinctus

The epithalamus of embryonic, neonatal and adult nine-banded armadillo (Dasypus novemcinctus) was examined for evidence of pineal-like tissue. The evagination of the diencephalic roof (the anlage of the epiphysis) was not found in any embryonic specimens. In the adult brain, the epithalamus is dominated by the sub-commissural organ (SCO) which is surrounded entirely by the posterior commissure. At the most caudal aspect of the SCO, previous investigators have observed pineal-like tissue. Using pineal-specific staining techniques however, we found no evidence of this tissue. Because the armadillo produces melatonin in a rhythmic manner, exhibits exacting circadian rhythms, and shows altered rhythms when exposed to exogenous melatonin, we believe other organs, perhaps the retina or Harderian gland, must be involved in maintaining the coordinated melatonin titer.