Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and in medullary noradrenergic cell groups

It has been hypothesized that the brain categorizes stressors and utilizes neural response pathways that vary in accordance with the assigned category. If this is true, stressors should elicit patterns of neuronal activation within the brain that are category-specific. Data from previous Immediate-early gene expression mapping studies have hinted that this is the case, but interstudy differences in methodology render conclusions tenuous. In the present study, immunolabelling for the expression of c-fos was used as a marker of neuronal activity elicited in the rat brain by haemorrhage, immune challenge, noise, restraint and forced swim. All stressors elicited c-fos expression in 25-30% of hypothalamic paraventricular nucleus corticotrophin-releasing-factor cells, suggesting that these stimuli were of comparable strength, at least with regard to their ability to activate the hypothalamic-pituitary-ad renal axis. In the amygdala, haemorrhage and immune challenge both elicited c-fos expression in a large number of neurons in the central nucleus of the amygdala, whereas noise, restraint and forced swim primarily elicited recruitment of cells within the medial nucleus of the amygdala. In the medulla, all stressors recruited similar numbers of noradrenergic (A1 and A2) and adrenergic (C1 and C2) cells. However, haemorrhage and immune challenge elicited c-fos expression In subpopulations of A1 and A2 noradrenergic cells that were significantly more rostral than those recruited by noise, restraint or forced swim. The present data support the suggestion that the brain recognizes at least two major categories of stressor, which we have referred to as 'physical' and 'psychological'. Moreover, the present data suggest that the neural activation footprint that is left in the brain by stressors can be used to determine the category to which they have been assigned by the brain.

Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1 beta-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-1 beta (IL-1 beta). However, although it is understood that catecholamine inputs are important in initiating mPVN CRH cell responses to IL-1 beta, 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-1 beta (1 mug/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-1 beta -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-1 beta -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-1 beta. 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-1 beta -induced HPA axis responses. Copyright (C) 2001 S. Karger AG, Basel.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1beta, or air puff. The D-1 antagonist, SCH23390, and the D-2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

The insulin hypoglycemia test: Hypoglycemic criteria and reproducibility

The insulin hypoglycemia test (IHT) is widely regarded as the 'gold standard' for dynamic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to investigate the temporal relationship between a rapid decrease in plasma glucose and the corresponding rise in plasma adenocorticotropic hormone (ACTH), and to assess the reproducibility of hormone responses to hypoglycemia in normal humans. Ten normal subjects underwent IHTs, using an insulin dose of 0.15 U/kg. Of these, eight had a second IHT (IHT2) and three went on to a third test (IHT3). Plasma ACTH and cortisol were measured at 15-min intervals and, additionally, in four IHT2s and the three IHT3s, ACTH was measured at 2.5- or 5-min intervals. Mean glucose nadirs and mean ACTH and cortisol responses were not significantly different between IHT1, IHT2 and IHT3. Combined data from all 21 tests showed the magnitude of the cortisol responses, but not the ACTH responses, correlated significantly with the depth and duration of hypoglycemia. All subjects achieved glucose concentrations of of less than or equal to 1.6 mmol/l before any detectable rise in ACTH occurred. In the seven tests performed with frequent sampling, an ACTH rise never preceeded the glucose nadir, but occurred at the nadir, or up to 15 min after. On repeat testing, peak ACTH levels varied markedly within individuals, whereas peak cortisol levels were more reproducible (mean coefficient of variation 7%). In conclusion, hypoglycemia of less than or equal to 1.6 mmol/l was sufficient to cause stimulation of the HPA axis in all 21 IHTs conducted in normal subjects. Nonetheless; our data cannot reveal whether higher glucose nadirs would stimulate increased HPA axis activity in all subjects. Overall, the cortisol response to hypoglycemia is more reproducible than the ACTH response but, in an individual subject, the difference in peak cortisol between two IHTs may exceed 100 nmol/l.

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-1 (IL-1, 1 g/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1-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-1-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1 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-1 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-1.

Performance and endocrine responses of group housed weaner pigs exposed to the air quality of a commercial environment

The growth performance and endocrine responses of male weaner pigs (3 to 8 weeks of age) was evaluated in two different environments (clean and dirty) and housing (single or groups of 10 pigs/pen) conditions. The dirty environment contained significantly elevated ammonia, carbon dioxide and dust levels compared with the clean environment. Pigs grew faster and consumed more feed in the clean environment and this was associated with reduced plasma cortisol concentrations compared with pigs in the dirty environment. Pigs housed in groups in the dirty environment had increased β-endorphin and decreased IGF-I concentrations compared to group housed pigs in the clean environment. Feed conversion efficiency did not differ due to environment or group housing. Plasma concentration of cortisol, p-endorphin, IGF-I and IGF-II did not differ between single and group housed pigs. Activity of the hypothalamic-pituitary-adrenal (HPA) axis was greater in response to environmental conditions than group housing, and this was associated with reduced growth in weaner pigs. © 2004 Elsevier B.V. All rights reserved.

Role of circumventricular organs in pro-inflammatory cytokine-induced activation of the hypothalamic-pituitary-adrenal axis

1. The past 15 years has seen the emergence of a new field of neuroscience research based primarily on how the immune system and the central nervous system can interact. A notable example of this interaction occurs when peripheral inflammation, infection or tissue injury activates the hypothalamic- pituitary-adrenal axis (HPA). 2. During such assaults, immune cells release the pro- inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor-alpha into the general circulation. 3. These cytokines are believed to act as mediators for HPA axis activation. However, physical limitations of cytokines impede their movement across the blood-brain barrier and, consequently, it has been unclear as to precisely how and where IL-1beta signals cross into the brain to trigger HPA axis activation. 4. Evidence from recent anatomical and functional studies suggests two neuronal networks may be involved in triggering HPA axis activity in response to circulating cytokines. These are catecholamine cells of the medulla oblongata and the circumventricular organs (CVO). 5. The present paper examines the role of CVO in generating HPA axis responses to pro-inflammatory cytokines and culminates with a proposed model based on cytokine signalling primarily involving the area postrema and catecholamine cells in the ventrolateral and dorsal medulla.

Endothelin axis expression is markedly different in the two main subtypes of renal cell carcinoma

BACKGROUND. The endothelin axis has been implicated in cancer growth, angiogenesis, and metastasis, but to the authors' knowledge the expression of endothelin genes has not been defined in renal cell carcinoma (RCC). METHODS. Tissue specimens were harvested from both normal and tumor-affected regions at the time of radical nephrectomy from 35 patients with RCC (22 with clear cell RCC [ccRCC] and 13 with papillary RCC [PRCC]). Real-time reverse transcriptase-polymerase chain reaction analysis determined the expression profile of the preproendothelins (PPET-1, PPET-2, and PPET-3), the endothelin receptors (ETA and ETB), and the endothelin-converting enzymes (ECE-1 and ECE-2). RESULTS. PPET-1 was found to be up-regulated in ccRCC tumor specimens and down-regulated in PRCC tumor specimens. ETA was significantly down-regulated in PRCC tumor specimens. ECE-1 was expressed in all tissue specimens at comparable levels, with moderate but significant elevation in normal tissue specimens associated with PRCC. Of the other genes, PPET-2 and ETB were expressed in all tissue specimens and no differences were observed between tumor subtypes or tumor-affected and normal tissue specimens, whereas PPET-3 and ECE-2 were present in all tissue specimens but were barely detectable. CONCLUSIONS. The endothelin axis was expressed differently in the two main subtypes of RCC and appeared to match macroscopic features commonly observed in these tumors (i.e., high expression of PPET-I in hypervascular ccRCC contrasted against low PPET-1 and ETA expression in hypovascular PRCC). The presence of ECE-1 mRNA in these tissue specimens suggested that active endothelin ligands were present, indicating endothelin axis activity was elevated in ccRCC compared with normal kidney, but impaired in PRCC. The current study provided further evidence that it is not appropriate to consider ccRCC and PRCC indiscriminately in regard to treatment. (C) 2004 American Cancer Society.

PACAP stimulates dopamine neuronal activity in the medial basal hypothalamus and inhibits prolactin

In sheep intracerebroventricular injection of PACAP (10 nmol) significantly (P < 0.01) stimulated the levels of the dopamine metabolite DOPAC within the medial basal hypothalamus las measured by in vivo microdialysis) and this effect was temporally correlated with a significant (P < 0.05) suppression in peripheral prolactin concentrations. This result is in accord with the hypothesis that PACAP suppresses prolactin secretion from the anterior pituitary gland by stimulating dopamine release from tuberoinfundibular dopaminergic neurons. (C) 1998 Elsevier Science B.V.

Processing of urinary pheromones in Antechinus stuartii (Marsupialia: Dasyuridae): Functional magnetic resonance imaging of the brain

Little is known of the neural mechanisms of marsupial olfaction. However, functional magnetic resonance imaging (fMRI) has made it possible to visualize dynamic brain function in mammals without invasion. In this study, central processing of urinary pheromones was investigated in the brown antechinus, Antechinus stuartii, using fMRI. Images were obtained from 18 subjects (11 males, 7 females) in response to conspecific urinary olfactory stimuli. Significant indiscriminate activation occurred in the accessory olfactory bulb, entorhinal, frontal, and parietal cortices in response to both male and female urine. The paraventricular nucleus of hypothalamus, ventrolateral thalamic nucleus, and medial preoptic area were only activated in response to male urine. Results of this MRI study indicate that projections of accessory olfactory system are activated by chemo-sensory cues. Furthermore, it appears that, based on these experiments, urinary pheromones may act on the hypothalamo-pituitary-adrenocortical axis via the paraventricular nucleus of the hypothalamus and may play an important role in the unique life history pattern of A. stuartii. Finally, this study has demonstrated that fMRI may be a powerful tool for investigations of olfactory processes in mammals.

Effect of hypothalamo-pituitary disconnection on the development of anterior pituitary cells in late gestation ovine fetuses

The specific role of the hypothalamus in regulating the developmental profile of anterior pituitary (AP) cells remains largely unknown. The present study evaluated hypothalamic contributions to AP cell development, utilizing the technique of hypothalamo-pituitary disconnection (HPD). HPD of fetal sheep or sham surgery was performed at 110 days gestation (d) (n=6 each group; term ~ 147d). Fetuses were removed and pituitaries collected at 110d (no surgery; n=6) or 141d (sham and HPD groups). The impact of HPD on AP cell development was assessed by single-labeled immunofluorescence for five hormones to identify proportions of AP cells expressing each hormone. HPD was associated with a 70% increase (P

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. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males

GH is being used by elite athletes to enhance sporting performance. To examine the hypothesis that exogenous 22-kDa recombinant human GH (rhGH) administration could be detected through suppression of non-22-kDa isoforms of GH, we studied seventeen aerobically trained males (age, 26.9 +/- 1.5 yr) randomized to rhGH or placebo treatment (0.15 IU/kg/day for 1 week). Subjects were studied at rest and in response to exercise (cycle-ergometry at 65% of maximal work capacity for 20 min). Serum was assayed for total GH (Pharmacia IRMA and pituitary GH), 22-kDa GH (2 different 2-site monoclonal immunoassays), non-22-kDa GH (22-kDa GH-exclusion assay), 20-kDa GH, and immunofunctional GH. In the study, 3 h after the last dose of rhGH, total and 22-kDa GH concentrations were elevated, reflecting exogenous 22-kDa GH. Non-22-kDa and 20-kDa GH levels were suppressed. Regression of non-22-kDa or 20-kDa GH against total or 22-kDa GH produced clear separation of treatment groups. In identical exercise studies repeated between 24 and 96 h after cessation of treatment, the magnitude of the responses of all GH isoforms was suppressed (P < 0.01), but the relative proportions were similar to those before treatment. We conclude: 1) supraphysiological doses of rhGH in trained adult males suppressed exercise-stimulated endogenous circulating isoforms of GH for up to 4 days; 2) the dearest separation of treatment groups required the simultaneous presence of high exogenous 22-kDa GH and suppressed 20-kDa or non-22-kDa GH concentrations; and 3) these methods may prove useful in detecting rhGH abuse in athletes.

Adrenocorticotropin stimulation tests in patients with hypothalamic-pituitary disease: low dose, standard high dose and 8-h infusion tests

Objectives Low doses of ACTH [1-24] (0.1, 0.5 and 1.0 mug per 1.73 m(2)) may provide a more physiological level of adrenal stimulation than the standard 250 mug test, but not all studies have concluded that the 1.0 fig is a more sensitive screening test for central hypoadrenalism. Eight-hour infusions of high dose ACTH [1-24] have also been suggested as a means of assessing the adrenals' capacity for sustained cortisol secretion. In this study, we compared the diagnostic accuracy of three low dose ACTH tests (LDTs) and the 8-h infusion with the standard 250 jig test (HDT) and the insulin hypoglycaemia test (IHT) in patients with hypothalamic-pituitary disease. Subjects and design Three groups of subjects were studied. A healthy control group (group 1, n=9) and 33 patients with known hypothalamic or pituitary disease who were divided into group 2 (n=12, underwent IHT) and group 3 (n=21, IHT contraindicated). Six different tests were performed: a standard IHT (0.15 U/kg soluble insulin); a 60-minute 250 mug HDT; three different LDTs using 0.1 mug, 0.5 mug and 1.0 mug (all per 1.73 m(2)); and an 8-h infusion test (250 mug ACTH [1-24] at a constant rate over 8 h). Results Nine out of the 12 patients in group 2 failed the IHT. Three out of 12 patients from group 2 who clearly passed the IHT, also passed all the ACTH [1-24] stimulation tests. Seven of the 9 patients who failed the lHT, failed by a clear margin (peak cortisol