Being suckled in a large litter mitigates the effects of early-life stress on hypothalamic-pituitary-adrenal axis function in the male rat.

The perinatal environment influences stress responses in the long-term, as does body composition. Male rats suckled in large litters, where they have reduced access to milk and attention from the dam, are less anxious and have attenuated hypothalamic-pituitary-adrenal (HPA) axis responses to stress compared to rats from control litters. In the present study, we investigated whether this early-life environment can also ameliorate anxiety and HPA axis function in rats prone to be stress-sensitive. We conducted these experiments in male rats from control litters (n = 12) or large litters (n = 20). Half were given 24 h of maternal separation on postnatal day 10 to induce HPA axis hyperactivity; the remainder staying undisturbed with their dam. When the rats reached adulthood, we examined behavioural indices of anxiety (elevated plus maze) and depression (Porsolt's forced swim test) under basal conditions and after 15 min of restraint stress. We also examined neuronal activation in the paraventricular nucleus of the hypothalamus (PVN) as an index of HPA axis function. Being suckled in a large litter led to a significantly attenuated PVN response to stress in adulthood. Maternal separation strongly exacerbated the stress-induced increase in PVN neuronal activation in control rats but did not affect the PVN response in large-litter rats. Immobility in the forced swim after restraint was also exacerbated in neonatally maternally separated control rats but not in those from large litters. Our findings show that being suckled in large litters mitigates the effects of early-life stress on HPA axis function and indices of depression in the rat.

Neonatal hypoxia-ischaemia disrupts descending neural inputs to dorsal raphé nuclei.

Neuronal losses have been shown to occur in the brainstem following a neonatal hypoxic-ischaemic (HI) insult. In particular serotonergic neurons, situated in the dorsal raphé nuclei, appear to be vulnerable to HI injury. Nonetheless the mechanisms contributing to losses of serotonergic neurons in the brainstem remain to be elucidated. One possible mechanism is that disruption of neural projections from damaged forebrain areas to dorsal raphé nuclei may play a role in the demise of serotonergic neurons. To test this, postnatal day 3 (P3) rat pups underwent unilateral common carotid artery ligation followed by hypoxia (6% O2 for 30 min). On P38 a retrograde tracer, fluorescent-coupled choleratoxin b, was deposited in the dorsal raphé dorsal (DR dorsal) nucleus or the dorsal raphé ventral (DR ventral) nucleus. Compared to control animals, P3 HI animals had significant losses of retrogradely labelled neurons in the medial prefrontal cortex, preoptic area and lateral habenula after tracer deposit in the DR dorsal nucleus. On the other hand, after tracer deposit in the DR ventral nucleus, we found significant reductions in numbers of retrogradely labelled neurons in the hypothalamus, preoptic area and medial amygdala in P3 HI animals compared to controls. Since losses of descending inputs are associated with decreases in serotonergic neurons in the brainstem raphé nuclei, we propose that disruption of certain descending neural inputs from the forebrain to the DR dorsal and the DR ventral nuclei may contribute to losses of serotonergic neurons after P3 HI. It is important to delineate the phenotypes of different neuronal networks affected by neonatal HI, and the mechanisms underpinning this damage, so that interventions can be devised to target and protect axons from the harmful effects of neonatal HI.

Ibuprofen inhibits neuroinflammation and attenuates white matter damage following hypoxia-ischemia in the immature rodent brain.

Damage to major white matter tracts is a hallmark mark feature of hypoxic-ischemic (HI) brain injury in the preterm neonate. There is, however, no therapeutic intervention to treat this injury. Neuroinflammation is thought to play a prominent role in the pathogenesis of the HI-induced white matter damage but identification of the key mediators that constitute the inflammatory response remain to be fully elucidated. Cyclooxygenase enzymes (COX-1 and COX-2) are candidate neuroinflammatory mediators that may contribute to the HI-induced demise of early oligodendrocyte progenitors and myelination. We investigated whether ibuprofen, a non-steroidal anti-inflammatory drug that inhibits COX enzymes, can attenuate neuroinflammation and associated white matter damage incurred in a rodent model of preterm HI. On postnatal day 3 (P3), HI was produced (right carotid artery ligation and 30 min 6% O(2)). An initial dose of ibuprofen (100mg/kg, s.c.) was administered 2h after HI followed by a maintenance dose (50mg/kg, s.c.) every 24h for 6 days. Post-HI ibuprofen treatment significantly attenuated the P3 HI-induced increases in COX-2 protein expression as well as interleukin-1beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α) levels in the brain. Ibuprofen treatment also prevented the HI-induced loss O4- and O1-positive oligodendrocyte progenitor cells and myelin basic protein (MBP)-positive myelin content one week after P3 HI. These findings suggest that a repeated, daily, ibuprofen treatment regimen administered after an HI insult may be a potential therapeutic intervention to prevent HI-induced damage to white matter progenitors and early myelination in the preterm neonate.

Efficacy of post-insult minocycline administration to alter long-term hypoxia-ischemia-induced damage to the serotonergic system in the immature rat brain.

Neuroinflammation is a key mechanism contributing to long-term neuropathology observed after neonatal hypoxia-ischemia (HI). Minocycline, a second-generation tetracycline, is a potent inhibitor of neuroinflammatory mediators and is successful for at least short-term amelioration of neuronal injury after neonatal HI. However the long-term efficacy of minocycline to prevent injury to a specific neuronal network, such as the serotonergic (5-hydroxytryptamine, 5-HT) system, is not known. In a post-natal day 3 (P3) rat model of preterm HI we found significant reductions in 5-HT levels, 5-HT transporter expression and numbers of 5-HT-positive dorsal raphé neurons 6 weeks after insult compared to control animals. Numbers of activated microglia were significantly elevated in the thalamus and dorsal raphé although the greatest numbers were observed in the thalamus. Brain levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were also significantly elevated on P45 in the thalamus and frontal cortex. Post-insult administration of minocycline for 1 week (P3-P9) attenuated the P3 HI-induced increases in numbers of activated microglia and levels of TNF-α and IL-1ß on P45 with concurrent changes in serotonergic outcomes. The parallel prevention of P3 HI-induced serotonergic changes suggests that inhibition of neuroinflammation within the first week after P3 HI injury was sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still evident at 6 weeks. Thus early, post-insult administration of minocycline may target secondary neuroinflammation and represent a long-term therapy to preserve the integrity of the central serotonergic network in the preterm neonate.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1beta responses using c-fos expression in the rat brain.

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1beta (IL-1beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge.

A critical role for the parabrachial nucleus in generating central nervous system responses elicited by a systemic immune challenge.

Using Fos immunolabelling as a marker of neuronal activation, we investigated the role of the parabrachial nucleus in generating central neuronal responses to the systemic administration of the proinflammatory cytokine interleukin-1beta (1 microg/kg, i.a.). Relative to intact animals, parabrachial nucleus lesions significantly reduced the number of Fos-positive cells observed in the central amygdala (CeA), the bed nucleus of the stria terminalis (BNST), and the ventrolateral medulla (VLM) after systemic interleukin-1beta. In a subsequent experiment in which animals received parabrachial-directed deposits of a retrograde tracer, it was found that many neurons located in the nucleus tractus solitarius (NTS) and the VLM neurons were both retrogradely labelled and Fos-positive after interleukin-1beta administration. These results suggest that the parabrachial nucleus plays a critical role in interleukin-1beta-induced Fos expression in CeA, BNST and VLM neurons and that neurons of the NTS and VLM may serve to trigger or at least influence changes in parabrachial nucleus activity that follows systemic interleukin-1beta administration.

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.

Systemic apomorphine alters HPA axis responses to interleukin-1 beta administration but not sound stress.

Apomorphine is a dopamine receptor agonist that was recently licensed for the treatment of erectile dysfunction. However, although sexual activity can be stressful, there has been little investigation into whether treatments for erectile dysfunction affect stress responses. We have examined whether a single dose of apomorphine, sufficient to produce penile erections (50 microg/kg, i.a.), can alter basal or stress-induced plasma ACTH levels, or activity of central pathways thought to control the hypothalamic-pituitary-adrenal axis in rats. An immune challenge (interleukin-1 beta, 1 microg/kg, i.a.) was used as a physical stressor while sound stress (100 dB white noise, 30 min) was used as a psychological stressor. Intravascular administration of apomorphine had no effect on basal ACTH levels but did substantially increase the number of Fos-positive amygdala and nucleus tractus solitarius catecholamine cells. Administration of apomorphine prior to immune challenge augmented the normal ACTH response to this stressor at 90 min and there was a corresponding increase in the number of Fos-positive paraventricular nucleus corticotropin-releasing factor cells, paraventricular nucleus oxytocin cells and nucleus tractus solitarius catecholamine cells. However, apomorphine treatment did not alter ACTH or Fos responses to sound stress. These data suggest that erection-inducing levels of apomorphine interfere with hypothalamic-pituitary-adrenal axis inhibitory feedback mechanisms in response to a physical stressor, but have no effect on the response to a psychological stressor. Consequently, it is likely that apomorphine acts on a hypothalamic-pituitary-adrenal axis control pathway that is unique to physical stressors. A candidate for this site of action is the nucleus tractus solitarius catecholamine cell population and, in particular, A2 noradrenergic neurons.

Descending pathways from the paraventricular nucleus contribute to the recruitment of brainstem nuclei following a systemic immune challenge.

Hypothalamic nuclei, particularly the paraventricular nuclei (PVN), are important brain sites responsible for central nervous system responses during an immune challenge. The brainstem catecholamine cells of the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) have been shown to play critical roles in relaying systemic immune signals to the PVN. However, whilst it is well recognised that PVN divisions also innervate the NTS and VLM, it is not known whether descending PVN pathways can modulate the recruitment of brainstem cells during an immune challenge. Using systemic administration of the proinflammatory cytokine interleukin-1beta, in combination with Fos immunolabelling, we firstly investigated the effect of PVN lesions on NTS and VLM catecholamine and non-catecholamine cell responses. We found that ibotenic acid lesions of the PVN significantly reduced numbers of Fos-positive non-catecholamine, noradrenergic and adrenergic cells observable in the VLM and NTS after interleukin-1beta administration. We then investigated the origins of descending inputs to the VLM and NTS, activated by systemic interleukin-1beta, by mapping the distribution of Fos-positive retrogradely-labelled cells in divisions of the PVN after iontophoretically depositing choleratoxin-b subunit into the NTS or VLM one week prior to interleukin-1beta administration. We found that, after either NTS or VLM deposits, the majority of retrogradely-labelled Fos-positive cells activated by interleukin-1beta were localised in the medial and lateral parvocellular PVN divisions. Retrogradely-labelled Fos-positive cells were also observed in the NTS after VLM deposits, and in the VLM after NTS tracer deposits, suggesting reciprocal communication between these two nuclei after systemic interleukin-1beta. Thus the present study shows that the PVN has the capacity to modulate NTS and VLM responses after an immune challenge and that these may result from descending projections arising in the medial and lateral PVN divisions. These findings suggest that central nervous system responses to an immune challenge are likely to involve complex reciprocal connections between the PVN and the brainstem as well as between brainstem nuclei themselves.

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-adrenal 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.

Peripheral withdrawal recruits distinct central nuclei in morphine-dependent rats.

This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis, accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways.

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.

The central nucleus of the amygdala; a conduit for modulation of HPA axis responses to an immune challenge?

Physical stressors such as infection, inflammation and tissue injury elicit activation of the hypothalamic-pituitary-adrenal (HPA) axis. This response has significant implications for both immune and central nervous system function. Investigations in rats into the neural substrates responsible for HPA axis activation to an immune challenge have predominantly utilized an experimental paradigm involving the acute administration of the pro-inflammatory cytokine interleukin- 1ß (IL-1ß). It is well recognized that medial parvocellular corticotrophin-releasing factor cells of the paraventricular nucleus (mPVN CRF) are critical in generating HPA axis responses to an immune challenge but little is known about how peripheral immune signals can activate and/or modulate the mPVN CRF cells. Studies that have examined the afferent control of the mPVN CRF cell response to systemic IL-1ß have centred largely on the inputs from brainstem catecholamine cells. However, other regulatory neuronal populations also merit attention and one such region is a component of the limbic system, the central nucleus of the amygdala (CeA). A large number of CeA cells are recruited following systemic IL-lß administration and there is a significant body of work indicating that the CeA can influence HPA axis function. However, the contribution of the CeA to HPA axis responses to an immune challenge is only just beginning to be addressed. This review examines three aspects of HPA axis control by systemic IL-1ß: (i) whether the CeA has a role in generating HPA axis responses to systemic IL-1ß, (ii) the identity of the neural connections between the CeA and mPVN CRF cells that might be important to HPA axis responses and(iii) the mechanisms by which systemic IL-Iß triggers the recruitment of CeA cells.

Effects of chronic oestrogen replacement on stress-induced activation of hypothalamic-pituitary-adrenal axis control pathways.

Oestrogen replacement therapy reportedly suppresses hypothalamic-pituitary-adrenal (HPA) axis responses to an emotional stressor in postmenopausal women. However, most studies in the rat suggest a facilitatory role for oestrogen in the control of HPA axis function. One explanation for this difference may be the regimen of oestrogen replacement: during oestrogen replacement therapy, oestrogen levels are low and constant whereas most animal studies examined the HPA axis response when oestrogen levels are rising. In the present study, we assessed HPA axis stress responses in mature ovariectomized rats after plasma oestrogen levels had been maintained at physiological levels for a prolonged period (25 or 100 pg/ml for 7 days). In the case of both an emotional stressor (noise) and a physical stressor (immune challenge by systemic interleukin-1beta administration), oestrogen replacement suppressed stress-related Fos-like immunolabelling, in hypothalamic neuroendocrine cells and plasma adrenocorticotropin hormone responses. From the present data, and past reports, it appears unlikely that these effects of oestrogen are due to a direct action on corticotropin-releasing factor or oxytocin cells. Therefore, to obtain some indication of oestrogen's possible site(s) of action, Fos-like immunolabelling was mapped in the amygdala and in brainstem catecholamine groups, which are neuronal populations demonstrating substantial evidence of involvement in the generation of HPA axis stress responses. In the amygdala, oestrogen replacement suppressed central nucleus responses to immune challenge, but not to noise. Amongst catecholamine cells, oestrogen replacement was more effective against responses to noise than immune challenge, suppressing A1 and A2 (noradrenergic) and C2 (adrenergic) responses to noise, but only A1 responses to immune challenge. These data suggest that, as in postmenopausal women on oestrogen replacement therapy, chronic low-level oestrogen replacement can suppress HPA axis stress responses in the rat. Moreover, oestrogen appears to exert effects at multiple sites within putative HPA axis control pathways, even though most of the relevant neuronal populations do not contain genomic receptors for this gonadal steroid and the pattern of oestrogen action differs for an emotional vs a physical stressor.

Opposite effects of short and continuous oestradiol replacement on CNS responses to hypoxic stress.

Oestrogen can alter neuroendocrine responses to stress but the evidence is conflicting. We examined the effects of a short-term 17beta-oestradiol surge versus a continuous 17beta-oestradiol replacement regime on neuroendocrine cell responses to hypoxia in the ovariectomized rat. Short-term oestradiol replacement significantly increased the number of Fos-positive SON and PVN OT cells and VLM A1 and C1 cells following hypoxia. In contrast, continuous oestradiol replacement significantly decreased the number of hypoxia-induced Fos-positive mPVN, PVN OT and VLM A1 and C1 and NTS C2 cells. We propose that the effects of oestradiol replacement on stress-induced neuroendocrine responses may be dependent on whether oestrogen levels are rising rapidly or remaining constant over a relatively long period.

The central amygdala modulates hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta administration.

In the present study we examined the role of the central nucleus of the amygdala in hypothalamic-pituitary-adrenal axis responses to an immune challenge in the form of systemic administration of the proinflammatory cytokine interleukin-1beta (1 microg/kg). We found that bilateral ibotenic acid lesions of the central amygdala substantially reduced adrenocorticotropin hormone release and hypothalamic corticotropin-releasing factor and oxytocin cell c-fos expression responses to interleukin-1,8 suggesting a facilitatory role for this structure in the generation of hypothalamic-pituitary-adrenal axis responses to an immune challenge. Since only a small number of central amygdala cells project directly to the paraventricular nucleus, we then examined the effect of central amygdala lesions on the activity of other brain nuclei that might act as relay sites in the control of the hypothalamic-pituitary-adrenal axis function. We found that bilateral central amygdala lesions significantly reduced interleukin-1beta-induced c-fos expression in cells of the ventromedial and ventrolateral subdivisions of the bed nucleus of the stria terminalis and brainstem catecholamine cell groups of the nucleus tractus solitarius (A2 noradrenergic cells) and ventrolateral medulla (A1 noradrenergic and C1 adrenergic cells). These findings, in conjunction with previous evidence of bed nucleus of the stria terminalis and catecholamine cell group involvement in hypothalamic-pituitary-adrenal axis regulation, suggest that ventromedial and ventrolateral bed nucleus of the stria terminalis cells and medullary catecholamine cells might mediate the influence of the central amygdala on hypothalamic-pituitary-adrenal axis responses to an immune challenge. Thus these data establish that the central amygdala influences hypothalamic-pituitary-adrenal axis responses to a systemic immune challenge but indicate that it primarily acts by modulating the activity of other control mechanisms.

Differential recruitment of hypothalamic neuroendocrine and ventrolateral medulla catecholamine cells by non-hypotensive and hypotensive hemorrhages.

We performed c-fos expression experiments in conscious rats to quantify the threshold and extent of activation of hypothalamic neuroendocrine cells in response to non-hypotensive and hypotensive hemorrhages allowing us to assess whether their pattern of recruitment corresponded to known oxytocin, vasopressin and ACTH release patterns. Also, because previous studies have implicated ventrolateral medulla catecholamine cells in the generation of certain hypothalamic neuroendocrine cell responses, we examined the response of ventrolateral medulla catecholamine cells to non-hypotensive and hypotensive hemorrhages and directly tested their role in regulating neuroendocrine cell responses to hypotensive hemorrhage. Animals were subjected to hemorrhages of 0, 4, 8, 12 or 16 ml/kg BW, the latter two levels being hypotensive. We found that only supraoptic nucleus vasopressin cells were significantly activated by the smallest non-hypotensive hemorrhage (4 ml/kg), which corresponds to reports that only vasopressin is released into the plasma after a small hemorrhage. Hypotensive hemorrhages resulted in significant recruitment of paraventricular and supraoptic oxytocin and vasopressin cells and parvocellular cells of the medial division of the paraventricular nucleus. Vasopressin cells were recruited in much greater numbers than oxytocin cells, which is in agreement with previous findings that there is a greater release of vasopressin than oxytocin into the plasma after hypotensive hemorrhage. In addition, medial parvocellular cells of the paraventricular nucleus, most likely to be tuberoinfundibular-projecting corticotropin-releasing factor cells, were activated by hypotensive hemorrhage only when arterial pressure dropped below 60 mmHg which also corresponds well with the plasma release response of ACTH. Ventrolateral medulla catecholamine cells were only recruited by hypotensive hemorrhages. While caution must be exercised in interpreting an absence of response, this certainly suggests that catecholamine cells are unlikely to have a role in the activation of supraoptic neurosecretory cells in response to non-hypotensive hemorrhages. Unilateral lesions of the ventrolateral medulla catecholamine cell column, corresponding primarily to the location of A1 noradrenergic cells, significantly reduced the hypotensive hemorrhage-induced activation of hypothalamic vasopressin, oxytocin and medial parvocellular paraventricular nucleus cells. This suggests that A1 noradrenergic cells contribute to the activation of these neuroendocrine cell populations, including oxytocin cells, which is an unexpected finding. More significantly, however, because the reduction in responsiveness after A1 lesions was similar for all cell categories, it seems likely that other factors must determine the differential recruitment of hypothalamic neuroendocrine cells in response to a hypotensive hemorrhage.

Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala.

The amygdala plays a pivotal role in the generation of appropriate responses to emotional stimuli. In the case of emotional stressors, these responses include activation of the hypothalamic-pituitary-adrenal (HPA) axis. This effect is generally held to depend upon the central nucleus of the amygdala, but recent evidence suggests a role for the medial nucleus. In the present study, c-fos expression, amygdala lesion and retrograde tracing experiments were performed on adult rats in order to re-evaluate the role of the central as opposed to the medial amygdala in generating neuroendocrine responses to an emotional stressor. Brief restraint (15 min) was used as a representative emotional stressor and was found to elicit c-fos expression much more strongly in the medial than central nucleus of the amygdala; relatively few Fos-positive cells were seen in other amygdala nuclei. Subsequent experiments showed that ibotenic acid lesions of the medial amygdala, but not the central amygdala, greatly reduced restraint-induced activation of cells of the medial paraventricular nucleus, the site of the tuberoinfundibular corticotropin-releasing factor cells that constitute the apex of the HPA axis. Medial amygdala lesions also reduced the activation of supraoptic and paraventricular nucleus oxytocinergic neurosecretory cells that commonly accompanies stress-induced HPA axis activation in rodents. To assess whether the role of the medial amygdala in the control of neuroendocrine cell responses to emotional stress might involve a direct projection to such cells, retrograde tracing of amygdala projections to the paraventricular nucleus was performed in combination with Fos immunolabelling. This showed that although some medial amygdala cells activated by exposure to an emotional stressor project directly to the paraventricular nucleus, the number is very small. These findings provide the first direct evidence that it is the medial rather than the central amygdala that is critical to hypothalamic neuroendocrine cell responses during an emotional response, and also provide the first evidence that the amygdala governs oxytocin as well as HPA axis responses to an emotional stressor.

NTS catecholamine cell recruitment by hemorrhage and hypoxia.

Immunolabelling for Fos and tyrosine hydroxylase was used to determine the patterns of activation of nucleus tractus solitarius catecholamine cells in response to graded levels of hemorrhage (0, 4, 8, 12 and 16 ml/kg) and systemic hypoxia (21, 14, 12, 10 and 8% O2) in conscious rats. Both stimuli elicited graded catecholamine cell recruitment with thresholds of 8 ml/kg and 12% O2. The majority of responsive neurons were A2 noradrenergic rather than C2 adrenergic cells. After hemorrhage most Fos-positive catecholamine cells were found below obex whereas most hypoxia-responsive cells were rostral to obex. These distinctive patterns of catecholamine cell recruitment may explain the differences in neuroendocrine responses to these stimuli.