Origin of adenohypophysial lobes and cells from Rathke's pouch in Swiss albino mice. Proliferation and expression of Pitx 2 and Calbindin D28K in corticotropic and somatotropic cell differentiation.

A developmental study of the adenohypophysis of the mouse was carried out in response to several as yet unanswered questions about its organogenesis and differentiation. The main objectives were to establish the origin of adenohypophysial lobes and cells from the Rathke's pouch (RP) and elucidate the mechanisms of development and functional differentiation of the gland. Using diverse techniques, the morphological development, proliferation and differentiation were studied in order to delimit different proliferative regions in the RP, and provide a satisfactory explanation for the distribution of each cell type in the adult gland. Combining the proliferation and differentiation studies, corticotropic and somatotropic cells appear to mainly originate from undifferentiated precursors located within each of these proliferative regions. The involvement of transcription factor Pitx 2 and calcium-binding protein Calbindin D 28K in the differentiation of corticotropic and somatotropic cells is further clarified.

Immunohistochemical localization of hormones and peptides in the human pituitary cells in a case of hypercortisolism by ACTH secreting microadenoma.

This study assesses the action of hypercortisolism on the hormone and peptide periadenoma region of removed ACTH-producing microadenoma. Our findings show that cortisol excess affects both ACTH and GH production, with no immunoreaction for these hormones. The remaining pituitary hormones (TSH, FSH and PRL) and POMC-derived peptides (betaEnd, alphaMSH and betaMSH) were not modified. Likewise, we observed pituitary immunoreactive cells for Neurotensin (NT), Intestinal vasoactive peptide (VIP), Substance P (SP) and Angiotensin-II (Ang-II). The colocalization demonstrated that NT was expressed in thyrotrope and gonadotrope cells, VIP in gonadotrope cells and SP in corticotrope cells. The results about Ang-II were inconclusive. On the other hand, immunoreaction for the NPY and Gal peptides were not present. In the adenomatous cells, the peptide NT is present in ACTH cells as well as SP. These results suggest a peptide regulation of pituitary cells in the pathological state that can differ between normal and tumoural cells of the same pituitary.

An androgen-dependent sexual dimorphism visible at puberty in the rat hypothalamus.

Morphological studies in rodents have well documented the masculinization of the perinatal brain by estradiol derived from aromatized testosterone, and the resulting irreversible quantitative sex-differences generated in cell numbers or expression of chemical phenotypes. Here, using immunohistochemistry, we explored how this applies to the postnatal development and masculinization of the neurokinin B (NKB)-containing system of the arcuate nucleus/median eminence complex (ARC/ME). In adult rats, NKB-immunoreactive neurons exhibit an unusual, qualitative sexual dimorphism of their ventral axonal projections: to the neuropil in females, to capillary vessels in males. In adults, there was no sex-difference in the numbers of NKB-immunoreactive perikarya or capillary vessels in the ARC/ME, suggesting that this sexual dimorphism cannot be explained by the existence of supernumerary structures. At birth (day 0) the NKB system was immature in both sexes, and while its adult features emerged progressively until puberty in females, they did not develop before puberty (day 40) in males, revealing a sexual dimorphism only late postnatally. When males were orchidectomized at day 30, the masculine distribution of NKB-immunoreactive axons expected at day 40 was not seen, while it was apparent after chronic treatment with testosterone or dihydrotestosterone, suggesting a testicular masculinizing action via androgen receptors at puberty. Moreover in these prepubertal-orchidectomized males, the distribution of NKB-immunoreactive axons was surprisingly feminized by chronic estradiol alone, suggesting that NKB neurons are not irreversibly programmed before puberty. Last, in adult females, the distribution of NKB-immunoreactive axons was feminine 30 days after ovariectomy, and it was masculinized after concurrent chronic dihydrotestosterone, suggesting that NKB neurons remain responsive to androgens late in reproductive life. Thus, the sexual differentiation of the hypothalamus proceeds well beyond the perinatal period and includes the epigenetic action of non-aromatizable androgens upon subsets of neurons that have retained bipotent features.

Immunocytochemical detection of cholecystokinin and corticotrophin-releasing hormone neuropeptides in the hypothalamic paraventricular nucleus of the jerboa (Jaculus orientalis): modulation by immobilisation stress.

The hypothalamic response to an environmental stress implicates the corticotrophin-releasing hormone (CRH) neuroendocrine system of the hypothalamic parvicellular paraventricular nucleus (PVN) in addition to other neuropeptides coexpressed within CRH neurones and controlling the hypothalamo-pituitary-adrenal (HPA) axis activity as well. Such neuropeptides are vasopressin, neurotensin and cholecystokinin (CCK). It has previously been demonstrated that the majority of the CRH neuronal population coexpresses CCK after a peripheral stress in rats. In the present study, we explored such neuroendocrine plasticity in the jerboa in captivity as another animal model. In particular, we studied CCK and CRH expression within the hypothalamic PVN by immunocytochemistry in control versus acute immobilisation stress-submitted jerboas. The results show that CCK- and CRH-immunoreactive neuronal systems are located in the hypothalamic parvicellular PVN. The number of CCK-immunoreactive neurones within the PVN was significantly increased (138% increase) in stressed animals compared to controls. Similarly, the number of CRH-containing neurones was higher in stressed jerboas (128%) compared to controls. These results suggest that the neurogenic stress caused by immobilisation stimulates CCK as well as CRH expression in jerboas, which correlates well with previous data obtained in rats using other stressors. The data obtained also suggest that, in addition to CRH, CCK is another neuropeptide involved in the response to stress in jerboa, acting by controlling HPA axis activity. Because CCK is involved in the phenotypical plasticity of CRH-containing neurones in response to an environmental stress, we also explored their coexpression by double immunocytochemistry within the PVN and the median eminence (i.e. the site of CRH and CCK corelease in the rat) following jerboa immobilisation. The results show that CCK is not coexpressed within CRH neurones in either control or stressed jerboa, suggesting differences between jerboas and rats in the neuroendocrine regulatory mechanisms of the stress response involving CRH and CCK. The adaptative physiological mechanisms to environmental conditions might vary from one mammal species to another.

Sexual dimorphism in the organization of the rat hypothalamic infundibular area.

The hypothalamic infundibular area is located outside the blood-brain barrier and includes, the ventromedial arcuate nucleus (vmARC) sensing circulating substances, and the median eminence (ME) where neurohormones are released into the hypothalamo-hypophysial vasculature. This integrated functional unit, pivotal in endocrine control, adjusts neuroendocrine output to feedback information. Despite a differing physiology in males and females, this functional unit has not appeared differently organized between sexes. Using immunocytochemistry, we describe here for the first time in adult rats, a conspicuous sex-difference in its axonal wiring by intrinsic glutamatergic neurons containing the neuropeptides neurokinin B (NKB) and dynorphin. In the male, NKB neurons send axons to capillary vessels of the vmARC and of the ME (only where gonadotropin-releasing hormone (GnRH) axons terminate). Electron microscopy revealed that NKB axons target the barrier of tanycytes around fenestrated capillary vessels (in addition to GnRH axons), suggesting a control of regional bidirectional permeability. In the female, NKB neurons send axons to the neuropile of the vmARC, suggesting a direct control of its sensor neurons. The other projections of NKB neurons, studied by surgical isolation of the ARC-ME complex and confocal microscopy, are not sexually dimorphic and target both integrative and neuroendocrine centers controlling reproduction and metabolism, suggesting a broad influence over endocrine function. These observations demonstrate that the mechanisms subserving hypothalamic permeability and sensitivity to feedback information are sexually dimorphic, making the infundibular area a privileged site of generation of the male-to-female differences in the adult pattern of pulsatile hormonal secretions.

Vasopressin-containing neurons of the hypothalamic parvocellular paraventricular nucleus of the jerboa: plasticity related to immobilization stress.

The corticotropin-releasing hormone (CRH) neurons of the hypothalamic parvocellular paraventricular nucleus (PVN) have a high potential for phenotypical plasticity, allowing them to rapidly modify their neuroendocrine output, depending upon the type of stressors. Indeed, these neurons coexpress other neuropeptides, such as cholecystokinin (CCK), vasopressin (VP), and neurotensin, subserving an eventual complementary function to CRH in the regulation of the pituitary. Unlike in rats, our previous data showed that in jerboas, CCK is not coexpressed within CRH neurons in control as well as stressed animals. The present study explored an eventual VP participation in the phenotypic plasticity of CRH neurons in the jerboa. We analyzed the VP expression within the PVN by immunocytochemistry in male jerboas submitted to acute stress. Our results showed that, contrary to CRH and CCK, no significant change concerned the number of VP-immunoreactive neurons following a 30-min immobilization. The VP/CRH coexpression within PVN and median eminence was investigated by double immunocytochemistry. In control as well as stressed animals, the CRH-immunopositive neurons coexpressed VP within cell bodies and terminals. No significant difference in the number of VP/CRH double-labeled cells was found between both groups. However, such coexpression was quantitatively more important into the posterior PVN as compared with the anterior PVN. This suggests an eventual autocrine/paracrine or endocrine role for jerboa parvocellular VP which is not correlated with acute immobilization stress. VP-immunoreactive neurons also coexpressed CCK within PVN and median eminence of control and stressed jerboas. Such coexpression was more important into the anterior PVN as compared with the posterior PVN. These results showed the occurrence of at least two VP neuronal populations within the jerboa PVN. In addition, the VP expression did not depend upon acute immobilization stress. These data highlight differences in the neuroendocrine regulatory mechanisms of the stress response involving CRH/CCK or VP. They also underline that adaptative physiological mechanisms to stress might vary from one mammal species to another.

Mapping of tachykinins in the cat spinal cord.

Using an indirect immunoperoxidase technique, the location of cell bodies and fibers containing substance P, neurokinin A and neurokinin B was studied in the cat spinal cord. The former two neuropeptides showed a widespread distribution throughout the whole spinal cord, whereas the distribution of neurokinin B was more restricted. Neurokinin A-immunoreactive structures showed a more widespread distribution and a higher density than the immunoreactive structures observed to contain substance P. In the cat spinal cord, we observed cell bodies containing neurokinin A, but no cell bodies containing neurokinin B or substance P were found. These cell bodies were located in laminae V (sacral 1 and 2 levels), VI (sacral 1 and 3), VII (lumbar 7, sacral 1 and 3, caudal 1) and X (sacral 1). Laminae I and II showed the highest density of immunoreactive fibers for each of the three tachykinins studied, being in general lamina IV who showed the lowest number of immunoreactive fibers containing substance P, neurokinin A or B. The anatomical distribution of the three tachykinins studied in the cat spinal cord indicates that the neuropeptides could be involved in the neurotransmission and/or in the neuromodulation of nociceptive information, as well as in autonomic and affective responses to pain. Moreover, the involvement of substance P, neurokinin A or B in other functions unrelated to the transmission of pain is also possible (autonomic and motor functions). The distribution of the neuropeptides studied in the cat is compared with the location of the same neuropeptides in the spinal cord of other species. The possible origin of the tachykinergic fibers in the cat spinal cord is also discussed.

Oestradiol effect on galanin-immunoreactive neurones in the diencephalon of the ewe.

Galanin is a neuropeptide involved in the regulation of numerous functions such as reproduction. In female rats, this peptide stimulates gonadotropin-releasing hormone (GnRH)/luteinizing hormone release and its synthesis is stimulated by oestradiol. It could therefore be an intermediary between the oestrogenic signal from the ovaries and the GnRH neurones (e.g. during the time course leading to the preovulatory GnRH surge). However, although the involvement of galanin is well-known in rodents, it is poorly understood in ewes. Using immunohistochemistry with a specific antigalanin antiserum, we detected the peptide in neurones of two groups of ovariectomized ewes treated for 6 h with subcutaneous implants, either with oestradiol (experimental group) or empty (control group). The galanin-immunoreactive neurones were counted in three areas, the preoptic area, the bed nucleus of the stria terminalis and the infundibular nucleus, using a computerized image analysis system. There was no change in the mean number of galanin-immunoreactive (GAL-ir) neurones in the infundibular nucleus (37 +/- 12 neurones/section in treated animals and 31 +/- 11 in controls) or in the bed nucleus of the stria terminalis (22 +/- 5 neurones/section in treated animals and 16 +/- 4 in controls), but the number of GAL-ir neurones was higher in the preoptic area in treated than in control ewes (35 +/- 4 versus 14 +/- 10, P < 0.001). To determine whether the neurones of the preoptic area were directly sensitive to oestradiol, we performed double immunohistochemical labelling for oestradiol receptor alpha and galanin. More than 50% of the GAL-ir neurones contained the oestradiol receptor alpha and therefore could be directly regulated by oestradiol. These results indicate that oestradiol might act directly on a GAL-ir neuronal population situated in the preoptic area, without any effect on the GAL-ir neurones of the infundibular nucleus or the bed nucleus of the stria terminalis. Because a 6-h oestradiol treatment can induce a preovulatory GnRH surge in ewes, the GAL-ir neuronal population of the preoptic area might be one of the neuronal systems by which oestradiol activates the GnRH neurones. However, although the morphological relationships between galanin and GnRH neurones have been described in rodents, they remain to be demonstrated in the ewe.

An immunocytochemical mapping of methionine-enkephalin-Arg(6)-Gly(7)-Leu(8) in the human brainstem.

Using an indirect immunoperoxidase technique, we studied the distribution of immunoreactive fibers and cell bodies containing methionine-enkephalin-Arg(6)-Gly(7)-Leu(8) in the adult human brainstem. Immunoreactive cell bodies were found in the reticular formation of the medulla oblongata (in which we observed the highest density of immunoreactive cell bodies) and the pons, the solitary nucleus, the hypoglossal nucleus, the medial and spinal vestibular nuclei, the lateral cuneate nucleus, the nucleus prepositus, the central gray of the pons and mesencephalon, the central and pericentral nuclei of the inferior colliculus, the superior colliculus, ventral to the superior olive and in the midline region of the pons and mesencephalon. The highest density of immunoreactive fibers containing methionine-enkephalin-Arg(6)-Gly(7)-Leu(8) was found in the spinal trigeminal nucleus, the central gray and the reticular formation of the medulla oblongata, pons and mesencephalon, the solitary nucleus, the spinal vestibular nucleus, the dorsal accessory olivary nucleus, the raphe obscurus, the substantia nigra and in the interpeduncular nucleus. The widespread distribution of immunoreactive structures containing methionine-enkephalin-Arg(6)-Gly(7)-Leu(8) in the human brainstem indicates that this neuropeptide might be involved in several physiological mechanisms, acting as a neurotransmitter and/or neuromodulator.

Distribution of ACTH immunoreactivity in the diencephalon and the brainstem of the dog.

The present work describes for the first time the anatomical distribution of adrenocorticotropic hormone (ACTH) in the diencephalon and the brainstem of the dog by means of the indirect immunoperoxidase technique. The distribution found in this species agrees well with the pattern found in other mammals and particularly confirms much of the findings reported in the cat. An exception to that concordance is the presence of ACTH perikarya in the nucleus of the solitary tract of the dog, a population that has been described in the rat but not in the cat, and in the ventral mesencephalon. This last population spread across the ventral tegmental area from the raphe to the cerebral peduncle and appeared to be a specific feature of the canine brain. On the other hand, we can not see ACTH fibers in the substantia nigra of the dog which could be a characteristic of the domestic carnivores, opposite to rodents, since these fibers appeared to be also lacking in the cat. Nevertheless, the widespread distribution of ACTH fibers in the brain of the dog included many other nuclei containing monoaminergic neurons which supported a possible role for ACTH in the regulation of these neurotransmitter systems.