Components of the Basal Lamina and Dystrophin�CDystroglycan Complex in the Neurointermediate Lobe of Rat Pituitary Gland: Different Localizations of ��-Dystroglycan, Dystrobrevins, ��1-Syntrophin, and Aquaporin-4

The so-called neurointermediate lobe is composed of the intermediate and neural lobes of the pituitary. The present immunohistochemical study investigated components of the basal lamina (laminin, agrin, and perlecan), the dystrophin–dystroglycan complex (dystrophin, β-dystroglycan, α1-dystrobrevin, β-dystrobrevin, utrophin, and α1-syntrophin), and the aquaporins (aquaporin-4 and -9). Glia markers (GFAP, S100, and glutamine synthetase) and components of connective tissue (collagen type I and fibronectin) were also labeled. In the neurohypophysis, immunostaining of basal lamina delineated meningeal invaginations. In these invaginations, vessels were seen to penetrate the organ without submerging into its parenchyma. On the parenchymal side of the invaginations, β-dystroglycan was detected, whereas utrophin was detected in the walls of vessels. Immunostaining of α1-dystrobrevin and α1-syntrophin did not delineate the vessels. The cells of the intermediate lobe were fully immunoreactive to α1-dystrobrevin and α1-syntrophin, whereas components of the basal lamina delineated the contours of the cells. GFAP-immunoreactive processes surrounded them. Aquaporin-4 localized at the periphery of the neurohypophysis, mainly adjacent to the intermediate lobe but not along the vessels. It colocalized only partially with GFAP and not at all with α1-syntrophin. Aquaporin-9 was not detected. These results emphasize the possibility that the components of the dystrophin–dystroglycan complex localize differently and raise the question about the roles of dystrobrevins, α1-syntrophin, and aquaporin-4 in the functions of the intermediate and neural lobes, respectively. (J Histochem Cytochem 58:463–479, 2010)     

Vasopressinergic,Oxytocinergic, and Somatostatinergic Neuronal Activity after Adrenalectomy and Immobilization Stress

Twenty days after bilateral adrenalectomy (ADX) or immediately after the last of three 6-h long immobilization periods, the levels of hypothalamic and neurohypophyseal L-[³⁵S]Cys-labeled arginine vasopressin (AVP), oxytocin (OT), and somatostatin-14 (SRIF) (only stressed animals) were measured simultaneously in male Wistar rats, after third ventricular administration of the labeled precursor, via guide-cannulae. The acetic acid-extracted labeled peptide fractions were purified by two sequential HPLC steps. After a 4 h period of labeling, only L-[³⁵S]Cys-AVP was selectively increased in the hypothalami of ADX-ized rats, compared to the sham-operated animals, possibly reflecting a significant activation of the paraventricular parvocellular (PVC) AVP/corticotropin-releasing factor (CRF) neurons. The increased accumulation of neurohypophyseal L-[³⁵S]Cys-labeled AVP and OT in these animals, without changes in the endogenous levels of these peptides, as measured by UV absorbance, also suggests a moderate activation of the magnocellular (MGC) AVP and OT neurons, as a consequence of adrenal insufficiency. In response to immobilization stress, levels of L-[³⁵S]Cys-OT were selectively increased in the hypothalami and corresponding neurohypophyses, 2 h and 4 h after receiving the label, concomitantly with a statistically significant reduction in the stores of OT in the neural lobes. AVP and SRJF biosynthesis remained unaffected by immobilization; the neurohypophyseal AVP stores likewise remained unchanged. These observations suggest the selective activation of MGC-OT neurons in response to chronic immobilization stress. Selective increases in hypothalamic L-[³⁵S]Cys-AVP in ADX-ized rats, and in hypothalamic L-[³⁵S]Cys-OT in chronically stress-immobilized rats, are presented as a measure of PVC-AVP/CRF and MGC-OT neuronal activation, respectively.     

Cellular and Subcellular Aquaporin-4 Distribution in the Mouse Neurohypophysis and the Effects of Osmotic Stimulation

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.     

VIP and PHI in cat neurons: co-localization but variable tissue content possible due to differential processing

The concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with NH2- terminal histidine and COOH-terminal isoleucine (PHI) in various peripheral tissues and some areas in the CNS of the cat were compared with their immunohistochemical localization. The VIP levels in the gastrointestinal tract were 3 to 6 times higher than PHI levels. Much (up to 10-fold) higher VIP than PHI levels were also observed in the genitourinary tract as well as in the lung and heart. In the neurohypophysis, however, the VIP/PHI ratio was close to 1. Gel-permeation chromatography revealed that VIP- and PHI-immunoreactivity (IR) in the intestine, pancreas and brain consisted of three larger molecular forms in addition to the 'standard' peptides. These larger forms which had overlapping elution positions may represent prepro-VIP/PHI forms. The immunohistochemical analysis revealed that VIP- and PHI-IR was present in the same ganglion cells in the intestine, pancreas, uterus and sympathetic ganglia. Furthermore, the terminal networks for these two peptides were very similar in the periphery. In the median eminence of the hypothalamus and in the posterior lobe of the pituitary, considerably more nerves were PHI- than VIP-IR. This observation was in parallel to a low VIP/PHI ratio. In conclusion, VIP and PHI seem to co-exist in most neuronal systems. Although the ratio of VIP and PHI on the precursor gene is 1:1, differences in posttranslational processing may create a considerably higher content of VIP than PHI in most terminal areas.     

Neurophysin and the neurosecretory fibres of the sheep infundibulum

Summary A cross-species reactive antiserum was used to study the cellular localization of neurophysin in the sheep hypothalamus and neurohypophysis. The immunofluorescent technique was used to follow the neurosecretory fibres emanating from the magnocellular nuclei to the lower infundibular stem. It was confirmed that neurophysin was present in both the internal and external zone of the infundibulum.This work was supported by a research grant from the New Zealand Medical Research Council.     

Time course of induction of oxytocin messenger ribonucleic acid levels in the hypothalamic paraventricular nucleus of ovariectomized rats following gonadal steroid administration

The nonapeptide oxytocin (OT) is important for uterine contractility at parturition, milk ejection during lactation, and the induction of maternal behavior. OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of late pregnant and lactating rats and are modulated by the steroid milieu that accompanies these states. Specifically, sequential exposure to estradiol (E₂) and progesterone (P) followed by P withdrawal 48 hrs prior to sacrifice increases PVN, and to a lesser but significant degree, SON OT mRNA. To better define the time course of induction of OT mRNA levels following P withdrawal, ovariectomized Sprague-Dawley rats were treated with empty or steroid-filled capsules. On day 1, animals received an E₂-filled or empty capsule, followed by P-filled or empty capsules on day 3. On day 14, P-filled or empty capsules were removed and animals were sacrificed 24, 36, or 48 hrs later. The hypothalamic PVN were analyzed for OT mRNA by in situ hybridization histochemistry. Significant differences in PVN OT mRNA were found among the groups (P < 0.0001, Kruskal-Wallis). Animals in the 48 hr (P = 0.007) and 36 hr (P = 0.005), but not the 24 hr, steroid-treated groups had significantly increased OT mRNA relative to their respective sham-treated cohorts (Mann-Whitney U test). The relative abundance of PVN OT mRNA differed among the steroid-treated groups (Kruskal-Wallis, P < 0.0003), with highest levels at 48 hr. We conclude that increases in PVN OT mRNA occur by 36 hrs, and are highest at 48 hrs, after P withdrawal in the E₂-primed rat. Future studies will determine if OT-mediated changes in behavior or physiology that surround parturition are related to these changes in OT mRNA.     

Release of neuropeptides does not only occur at nerve terminals

Neurohypophysial hormones are packed in secretory granules which are stored in nerve endings and in dilatations called nerve swellings. Although it was originally believed that the nerve swellings were storage compartments and that release occurred solely from the nerve terminals, the present paper demonstrates that secretion can occur to the same extent from both nerve endings and nerve swellings.     

beta-Endorphin controls vasopressin release during foot shock-induced stress in the rat

This study tested the possibility that beta-endorphin is involved in the regulation of vasopressin release during stress induced by inescapable electric foot shock. To this end, a specific anti-beta-endorphin antiserum or a control serum lacking the specific anti-beta-endorphin antibodies was administered to male rats. Plasma vasopressin concentrations, measured by radioimmunoassay, were not affected by brief foot shock stress in control rats, but were raised significantly by the stress in animals which had received an intracerebroventricular (i.c.v.) injection of the anti-beta-endorphin antiserum. In contrast, when the same volume of the anti-beta-endorphin antiserum was injected into a tail vein, foot shock stress produced only a slight effect on vasopressin release. I.c.v. injection of the antiserum changed neither basal nociceptive threshold nor stress-induced analgesia as revealed by the tail-flick latency. Vasopressin release induced by an osmotic stimulus was not influenced by the anti-beta-endorphin antiserum given i.c.v. The opiate antagonist naloxone or the glucocorticoid dexamethasone raised plasma vasopressin concentration in stressed rats which had received the control serum (i.c.v.); however, after i.c.v. injection of the anti-beta-endorphin antiserum neither naloxone nor dexamethasone elevated the plasma vasopressin concentration beyond the level reached by the anti-beta-endorphin antiserum (i.c.v.) alone. These results suggest that beta-endorphin inhibits the release of vasopressin during foot shock-induced stress in the rat.     

The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.