Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 h for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis, however these were in CRH negative neurons.

Regulation of corticotropin-releasing factor concentration and overflow in the rat central nervous system

Examines alterations in corticotropin-releasing factor concentration and nerve release during maturation and aging in the rat brain. Release of neuropeptide Y was also measured. These studies may provide information leading to the effective treatment of age-related disorders.

Evidence that the bed nucleus of the stria terminalis contributes to the modulation of hypophysiotropic corticotropin-releasing factor cell responses to systemic interleukin-1β

Systemic infection activates the hypothalamic-pituitary-adrenal (HPA) axis, and brainstem catecholamine cells have been shown to contribute to this response. However, recent work also suggests an important role for the central amygdala (CeA). Because direct connections between the CeA and the hypothalamic apex of the HPA axis are minimal, the present study investigated whether the bed nucleus of the stria terminalis (BNST) might act as a relay between them. This was done by using an animal model of acute systemic infection involving intravascular delivery of the proinflammatory cytokine interleukin-1β (IL-1β, 1 μg/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1β-induced increases in Fos immunoreactivity in corticotropin-releasing factor (CRF) cells of the hypothalamic paraventricular nucleus (PVN) and corresponding increases in adrenocorticotropic hormone (ACTH) secretion. Similar lesions had no effect on CRF cell responses to physical restraint, suggesting that the effects of BNST lesions were not due to a nonspecific effect on stress responses. In further studies, we examined the functional connections between PVN, BNST, and CeA by combining retrograde tracing with mapping of IL-1β-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1β administration recruits ventral BNST cells that project directly to the PVN. In the case of the CeA, the results obtained were consistent with an arrangement whereby lateral CeA cells recruited by systemic IL-1β could regulate the activity of medial CeA cells projecting directly to the BNST. In conclusion, the present findings are consistent with the hypothesis that the BNST acts as a relay between the CeA and PVN, thereby contributing to CeA modulation of hypophysiotropic CRF cell responses to systemic administration of IL-1β.

Isolation and restraint stress results in differential activation of corticontrophin-releasing hormone and arginine vasopressin neurones in sheep

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep : a sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis

Seasonal differences in the effect of isolation and restraint stress on the luteinizing hormone response to gonadotropin-releasing hormone in hypothalamopituitary disconnected, gonadectomized rams and ewes

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

The existence of gonadotropin-releasing hormone-like peptides in the neural ganglia and ovary of the abalone, Haliotis asinina L.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the presence and distribution of two isoforms of GnRH-like peptides in neural ganglia and ovary of reproductively mature female abalone, Haliotis asinina, using immunohistochemistry. We found significant immunoreactivities (ir) of anti-lamprey(I) GnRH-III and anti-tunicate(t) GnRH, but with variation of labeling intensity by each anti-GnRH type. IGnRH-III-ir was detected in numerous type1 neurosecretory cells (NS1) throughout the cerebral and pleuropedal ganglia, whereas tGnRH-I-ir was detected in only a few NS1 cells in the dorsal region of cerebral and pleuropedal ganglia. In addition, a small number of type2 neurosecretory cells (NS2) in cerebral ganglion showed lGnRH-III-ir. Long nerve fibers in the neuropil of ventral regions of the cerebral and pleuropedal ganglia showed strong tGnRH-I-ir. In the ovary, lGnRH-III-ir was found primarily in oogonia and stage I oocytes, whereas tGnRH-ir was observed in stage I oocytes and some stage II oocytes. These results indicate that GnRH produced in neural ganglia may act in neural signaling. Alternatively, GnRH may also be synthesized locally in the ovary where it could induce oocytes development.

Stimulatory effects of egg-laying hormone and gonadotropin-releasing hormone on reproduction of the tropical abalone, Haliotis asinina linnaeus

Egg-laying hormone (ELH) is a neuropeptide hormone that stimulates ovulation of gastropods, including Aplysia californica and Lymnaea stagnalis. Other neuropeptides, gonadotropin releasing hormones (GnRHs), also play important roles in controlling reproduction in both vertebrates and invertebrates. In the current study, the effects of abalone ELH (aELH) and several GnRHs on somatic growth, sex differentiation, gonad maturation, and spawning of Haliotis asinina were investigated in 3 experiments. In experiment 1, groups of 4-mo-old juveniles (11.8 ±  0.03 mm shell length (SL) and 0.33 ± 0.04 g body weight (BW)) were injected with aELH and GnRHs, including buserelin (mammalian GnRH analogue), octopus GnRH (octGnRH), and tunicate GnRH-I (tGnRH-I), at doses of 20 ng/g BW and 200 ng/g BW. The aELH induced early sex differentiation with a bias toward females, but with normal somatic growth, whereas the different isoforms of GnRH had no effect on sexual differentiation or somatic growth. In experiment 2, groups of 1-y-old-abalone (SL, 4.04 ± 0.02 cm; BW, 20.15 ± 0.25 g) were injected with aELH and the 3 isoforms of GnRH including buserelin, octGnRH, and lamprey GnRH (1GnRH-I) at doses of 500 ng/g BW and 1,000 ng/g BW, and all produced stimulatory effects. For each peptide treatment, the gonads reached full maturation within 5- 6 wk and spawning occurred, whereas control groups took 8 wk to reach maturity. In experiment 3, injections of ripe abalone with aELH stimulated spawning of both sexes in a dose-dependent manner. Buserelin had a lesser effect on inducing spawning, and octGnRH had no apparent effect. The gametes released from induced spawnings by aELH and GnRH showed normal fertilization and development of larvae. Altogether, these findings provide further knowledge on manipulating abalone reproduction, which is important in improving abalone aquaculture.

Medullary neurones regulate hypothalamic corticotropin-releasing factor cell responses to an emotional stressor

Hypothalamic–pituitary–adrenal axis activation is a hallmark of the stress response. In the case of physical stressors, there is considerable evidence that medullary catecholamine neurones are critical to the activation of the paraventricular nucleus corticotropin-releasing factor cells that constitute the apex of the hypothalamic–pituitary–adrenal axis. In contrast, it has been thought that hypothalamic–pituitary–adrenal axis responses to emotional stressors do not involve brainstem neurones. To investigate this issue we have mapped patterns of restraint-induced neuronal c-fos expression in intact animals and in animals prepared with either paraventricular nucleus-directed injections of a retrograde tracer, lesions of paraventricular nucleus catecholamine terminals, or lesions of the medulla corresponding to the A1 or A2 noradrenergic cell groups. Restraint-induced patterns of neuronal activation within the medulla of intact animals were very similar to those previously reported in response to physical stressors, including the fact that most stressor-responsive, paraventricular nucleus-projecting cells were certainly catecholaminergic and probably noradrenergic. Despite this, the destruction of paraventricular nucleus catecholamine terminals with 6-hydroxydopamine did not alter corticotropin-releasing factor cell responses to restraint. However, animals with ibotenic acid lesions encompassing either the A1 or A2 noradrenergic cell groups displayed significantly suppressed corticotropin-releasing factor cell responses to restraint. Notably, these medullary lesions also suppressed neuronal responses in the medial amygdala, an area that is now considered critical to hypothalamic–pituitary–adrenal axis responses to emotional stressors and that is also known to display a significant increase in noradrenaline turnover during restraint.

We conclude that medullary neurones influence corticotropin-releasing factor cell responses to emotional stressors via a multisynaptic pathway that may involve a noradrenergic input to the medial amygdala. These results overturn the idea that hypothalamic–pituitary–adrenal axis response to emotional stressors can occur independently of the brainstem.

Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1beta-induced hypothalamic pituitary adrenal axis responses

Medial parvocellular paraventricular corticotropin-releasing hormone (mPVN CRH) cells are critical in generating hypothalamic-pituitary-adrenal (HPA) axis responses to systemic interleukin-1beta (IL-1beta). However, although it is understood that catecholamine inputs are important in initiating mPVN CRH cell responses to IL-1beta, the contributions of distinct brainstem catecholamine cell groups are not known. We examined the role of nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) catecholamine cells in the activation of mPVN CRH, hypothalamic oxytocin (OT) and central amygdala cells in response to IL-1beta (1 microg/kg, i.a.). Immunolabelling for the expression of c-fos was used as a marker of neuronal activation in combination with appropriate cytoplasmic phenotypic markers. First we confirmed that PVN 6-hydroxydopamine lesions, which selectively depleted catecholaminergic terminals, significantly reduced IL-1beta-induced mPVN CRH cell activation. The contribution of VLM (A1/C1 cells) versus NTS (A2 cells) catecholamine cells to mPVN CRH cell responses was then examined by placing ibotenic acid lesions in either the VLM or NTS. The precise positioning of these lesions was guided by prior retrograde tracing studies in which we mapped the location of IL-1beta-activated VLM and NTS cells that project to the mPVN. Both VLM and NTS lesions reduced the mPVN CRH and OT cell responses to IL-1beta. Unlike VLM lesions, NTS lesions also suppressed the recruitment of central amygdala neurons. These studies provide novel evidence that both the NTS and VLM catecholamine cells have important, but differential, contributions to the generation of IL-1beta-induced HPA axis responses.