R. Vázquez

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.

CaBP D-28k and NADPH-diaphorase coexistence in the magnocellular neurosecretory nuclei.

COEXISTENCE of the calcium binding protein calbindin D-28k and NADPH-diaphorase activity was studied in the magnocellular secretory nuclei of the rat hypothalamus using both immunocytochemical and histochemical techniques. Coexistence was found in all the nuclei considered (supraoptic, paraventricular, circularis and fornicals nuclei) with the exception of the hypothalamic area situated between the supraoptic and the paraventricular nuclei. Since both stainings are reliable markers, not based upon the physiological characteristics at a given moment, our study provides a further characterization of the neurons in the magnocellular neurosecretory nuclei.

Immunohistochemical evidence of the presence of aromatase P450 in the rat hypophysis

Abstract In order to analyze whether aromatase is present in the hypophysis of adult rats, we have performed an immunohistochemical study in young adult male and female rats. Our study has revealed that the hypophysis of adult rats contains aromatase, although marked differences are found between the sexes. The hypophyses of male rats have cells immunoreactive for the enzyme, 34.40% of these hypophyseal cells showing reaction. By contrast, cells from female rats show very little reaction, only 0.84% of them being reactive. No significant differences in the percentage of immunoreactive cells between one phase and another are observed during the estrous cycle. Our results point to the immunohistochemical expression of aromatase in the hypophysis of adult rats and at the same time suggest that its expression is sex-dependent. The enzyme may therefore be involved in the regulation of adenohypophyseal cytology by androgens.

Infrequent cellular coexistence of NADPH-diaphorase and calretinin in the neurosecretory nuclei and adjacent areas of the rat hypothalamus

Colocalization of the calcium-binding protein calretinin and NADPH-diaphorase activity at the cellular level was studied in the magnocellular secretory nuclei of the rat hypothalamus using sequential immunocytochemical and histochemical staining of the same sections. A low degree of colocalization of these markers was observed in certain cellular subpopulations within all the areas considered (supra-optic, paraventricular, circular and both fornicals nuclei and in the hypothalamic area located between the supraoptic and paraventricular nuclei). However, since in the paraventricular nucleus both markers were expressed by different neuronal populations, the coexistence was almost non-existent in some subdivisions of this nucleus. This rare coexistence strongly suggests that NADPH-diaphorase and calretinin are related to different functions shared by restricted hypothalamic neuronal populations.

Swim stress enhances the NADPH-diaphorase histochemical staining in the paraventricular nucleus of the hypothalamus.

Regulatory control of the hypothalamic-pituitary-adrenocortical axis (HPA) originates principally from the paraventricular nucleus (PVN), which contains an important population of nitric oxide synthesizing (NOS) neurons. In the present study, the effect of swim stress upon these neurons was investigated by means of the NADPH-diaphorase (ND) histochemical technique. A significant increase in the number of ND-neurons was observed following forced swim, especially after 30 min. These data confirm the involvement of NOS-neurons of the PVN in the response to different types of acute stressors.

Expression of Aromatase P450 is Increased in Spontaneous Prolactinomas of Aged Rats

We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats.

Calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat

The distribution of calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat was studied using the avidin-biotin-immunoperoxidase method and highly specific monoclonal antibodies. Incubation with anticalbindin D-28K-antiserum revealed immunoreactive neurons in the following nuclei: supraoptic, paraventricular (both in the magnocellular and parvicellular regions), circularis, fornicals and medial forebrain bundle. Incubation with parvalbumin antiserum displayed immunoreactive neurons only in the circularis nucleus. Additionally, it was possible to observe scattered calbindin and parvalbumin immunoreactive neurons (which do not form part of the nuclei considered) located in the hypothalamic area between the supraoptic and the paraventricular nuclei, especially for the calbindin D-28K antiserum.

Partial co-existence of NADPH-diaphorase and acetylcholinesterase in the hypothalamic magnocellular secretory nuclei of the rat

Co-localization of NADPH-diaphorase (ND) and acetylcholinesterase (AChE) activities were explored in the magnocellular secretory nuclei of the rat hypothalamus by means of a double histochemical staining of the same sections. Partial co-existence was found in all the nuclei studied (paraventricular, supraoptic, fornicals and circular nuclei). No particular location of the neurons expressing both markers was found, although in the paraventricular nucleus all of them (ND +, AChE + and neurons expressing both markers) were preferentially located in the magnocellular subdivisions whereas in the parvicellular ones only some neurons belonging to all three types were detected, mainly located in the periventricular and medial subdivisions. The lowest degree of co-existence was found at the level of the main magnocellular nuclei (supraoptic and paraventricular) when compared with the accessory magnocellular nuclei, especially the posterior fornical and the circular nuclei. These results extend previous data on the chemical nature of the neurons producing nitric oxide in the neurosecretory nuclei and the possible functional role of this atypical messenger in the hypothalamus.

Reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity in the paraventricular nucleus of the rat hypothalamus is modulated by estradiol

Based on previous studies demonstrating that reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) activity is modulated by estradiol and the discovery of a new subtype of estrogen receptor highly expressed in the paraventricular nucleus of the hypothalamus (PVN), a possible estrogen influence on this activity was investigated in the neuronal populations (magno- and parvicellular) of this nucleus. Cryostat sections were cut and processed for the histochemical detection of the ND activity. Following ovariectomy (14 days), numerical data displayed a slight decrease in the number of ND-neurons, especially in the posterior magnocellular and the medial parvicellular subdivisions, which was reversed after daily treatment with estradiol benzoate. Administration of estradiol benzoate to male rats (14 days) induced a significant increase (P < 0.05) in the number of ND-neurons, mainly at the level of the posterior magnocellular subdivision. These data indicate that paraventricular ND-neurons are influenced by estradiol.

Coexistence of NADPH-diaphorase with tyrosine hydroxylase in hypothalamic magnocellular neurons of the rat

The presence and distribution of NADPH-diaphorase (ND) neurons as well as tyrosine hydroxylase-immunoreactive (TH) neurons in the hypothalamus are well established. Previous studies have shown the coexistence of ND with neuroactive substances such as calbindin, somatostatin, vasopressin and oxytocin in neurons of this region of the brain. As the tópographical patterns of distribution of ND and TH coincide in many cases, the aim of this study was to determine the possible coexistence of both substances in the main hypothalamic magnocellular nuclei of the albino rat. Histochemical-immunocytochemical double labelling was employed on the same sections as well as a morphometric study. NADPH-diaphorase and tyrosine hydroxylase neurons were observed in all the nuclei under study (supraoptic, paraventricular and accessory nuclei), although most neurons showing the coexistence of both substances were mainly located in the supraoptic nucleus, isolated neurons with double labelling being found in the magnocellular parts of the paraventricular nucleus and in some of the accessory nuclei. Although both substances have previously been shown to be modified in hypothalamic neurons after osmotic stimuli, the range of functions of ND in the CNS is only beginning to be understood. Further studies are needed to elucidate the functional role that ND/TH neurons play in the nervous system.