Adaptive changes in basal and stress-induced HPA activity in lactating and post-lactating female rats.

Lactation represents a period of marked adaptation of the hypothalamo-pituitary-adrenal HPA axis. We characterized basal and stress-induced HPA activity during lactation and experimental weaning using dynamic blood sampling in rats. Pulsatile and diurnal corticosterone release occurred at all reproductive stages studied (virgin; day 10 of lactation; 3 and 14 days after experimental weaning on day 10 of lactation). However, in lactating rats the diurnal peak was significantly reduced, resulting in a flattened rhythm, and three days after weaning, basal HPA activity was markedly suppressed: the number of pulses and underlying basal levels of corticosterone were reduced and the diurnal rise phase delayed. Marked changes in the HPA response to 10 min noise stress also occurred at these times: being completely absent in lactating animals, but restored and highly prolonged in early weaned animals. Injection of methylprednisolone (2 mg, iv) was used to determine whether changes in fast glucocorticoid suppression correlated with these adaptive changes. Methylprednisolone induced a rapid suppression of corticosterone in virgin animals, but this effect was markedly attenuated in lactating and early weaned animals and was accompanied by significant changes in relative expression of hippocampal glucocorticoid and mineralocorticoid receptor mRNA. All effects were reversed or partially reversed 14 days after experimental weaning. Thus, the presence of the pups has an important influence on regulation of the HPA axis, and while postpartum adaptations are reversible, acute weaning evokes marked reorganisation of basal and stress-induced HPA activity.

Acute glucocorticoid administration rapidly suppresses basal and stress-induced hypothalamo-pituitary-adrenal axis activity.

Hypothalamo-pituitary-adrenal (HPA) axis activity is subject to negative feedback control by glucocorticoids. Although the rapid component of this feedback is widely considered to contribute to regulation of dynamic HPA activity, few in vivo data exist on the temporal and pharmacological characteristics of this phenomenon. Thus, frequent automated blood sampling was undertaken in rats to determine the effects of acute glucocorticoid administration on basal and stress-induced corticosterone secretion. The glucocorticoid agonist methylprednisolone (5-2000 µg) or dexamethasone (5-500 µg) injected iv at the peak of the diurnal rhythm caused dose-dependent suppression of basal corticosterone secretion, which was attenuated by the glucocorticoid receptor antagonist RU38486. With 50 µg methylprednisolone, the onset of this suppression occurred at 40 min and remained significant for 120 min. However, although higher doses led to a greater and more sustained suppression of endogenous corticosterone, the response was delayed by the emergence of an initial stimulatory response that imposed a finite minimum delay. A corticosterone response to injection of CRH (1 µg, iv) during the period of maximal suppression indicated a suprapituitary site for the inhibitory effect glucocorticoid activation. This mechanism was supported by glucocorticoid injection immediately before a psychological stress (30 min, white noise); methylprednisolone caused dose-dependent attenuation of stress-induced corticosterone release and expression of the activity marker c-fos mRNA in the paraventricular nucleus but did not block the pituitary response to CRH. Thus, in rats, glucocorticoid receptor activation rapidly suppresses basal and stress-induced HPA activity that operates, at least in part, through a central mechanism of action.

Reduced stress responsiveness in pregnancy: relationship with pattern of forebrain c-fos mRNA expression.

Stress-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis is known to be attenuated during late pregnancy and throughout lactation. To investigate the neural basis of this stress hyporesponsiveness we examined the changes in the restraint-induced HPA response and accompanying forebrain expression of c-fos mRNA that occur in rats between days 16 (D16) and 19 (D19) of gestation, times associated with declining levels of progesterone, a potential mediating factor. Compared to D16, the D19 group showed a significantly attenuated release of ACTH following 30min restraint. This reduced HPA response was accompanied by significantly lower levels of restraint-induced c-fos mRNA expression in the hypothalamic paraventricular nucleus. Other areas of the forebrain, including medial amygdala, piriform cortex, and ventrolateral septum, showed low c-fos mRNA expression in non-stressed (control) animals and a large increase following restraint, the magnitude of which was similar between D16 and D19 animals indicating no involvement in the differential HPA response to stress. However, a markedly different pattern of c-fos mRNA expression was observed in other brain areas, including barrel cortex and CA1 ventral and CA3 regions of the ventral hippocampus: D19 animals had low control expression which was increased by restraint, but D16 control animals had raised c-fos mRNA expression which was not further elevated by stress. These data demonstrate that region-specific changes in basal and stress-induced cellular activity occur during a period of late gestation coincident with attenuated HPA responsiveness. These changes in neuronal activity may contribute to the adaptive processes that prepare the mother for parturition and lactation.

Profound changes in dopaminergic neurotransmission in the prefrontal cortex in response to flattening of the diurnal glucocorticoid rhythm: implications for bipolar disorder.

Patients with bipolar disorder have abnormalities in glucocorticoid secretion, dopaminergic neurotransmission, and prefrontal cortical function. We hypothesized that the flattening of the diurnal glucocorticoid rhythm, commonly seen in bipolar disorder, modulates dopaminergic neurotransmission in the prefrontal cortex (PFC) leading to abnormalities in prefrontally mediated neurocognitive functions. To address this hypothesis, we investigated the effects of a flattened glucocorticoid rhythm on (i) the release of dopamine in the PFC and (ii) the transcription of genes in the ventral tegmental area (VTA) coding for proteins involved in presynaptic aspects of dopaminergic neurotransmission. Male rats were treated for 13-15 days with corticosterone (50 microg/ml in the drinking water) or vehicle (0.5% ethanol). Corticosterone treatment resulted in marked adrenal atrophy and flattening of the glucocorticoid rhythm as measured by repeated blood sampling. Animals treated with corticosterone showed markedly enhanced basal dopamine release in the PFC as measured by microdialysis in the presence of a dopamine reuptake inhibitor. Depolarization-evoked release was also enhanced, suggesting that the corticosterone effect on basal release did not result from an increase in the neuronal firing rate. Local blockade of terminal D(2) autoreceptors failed to normalize release to control values, suggesting that the enhanced release was not because of reduced autoreceptor sensitivity. In situ hybridization histochemistry showed that mRNAs coding tyrosine hydroxylase and the vesicular monoamine transporter 2 were elevated in the VTA of corticosterone-treated rats. Our data show that flattening of the glucocorticoid rhythm increases dopamine release in the PFC possibly as a result of increased synthesis and vesicular storage. This provides a mechanistic explanation for prefrontal dysfunction in bipolar and other affective disorders associated with glucocorticoid dysrhythmia.

Fluoxetine inhibits corticotropin-releasing factor (CRF)-induced behavioural responses in rats.

Corticotropin-releasing factor (CRF) is a potent neuromodulator of stress-related behaviour but the neural mechanisms underlying these effects are not clear. Studies were designed to test the hypothesis that CRF-induced behavioural arousal involves interactions with brainstem serotonergic systems. To examine interactions between CRF and serotonergic systems in the regulation of behaviour, CRF (1 microg, intracerebroventricular (i.c.v.)) or vehicle was infused in the presence or absence of the selective serotonin re-uptake inhibitor fluoxetine (0, 0.1, 1 or 10 mg/kg, intravenous (i.v.)). Fluoxetine was used at these doses because it is known to decrease serotonin cell firing rates while increasing extracellular serotonin concentrations in select forebrain regions. We then measured behavioural, neurochemical and endocrine responses. CRF increased locomotion and spontaneous non-ambulatory motor activity (SNAMA) in the home cages. Fluoxetine decreased tissue 5-hydroxyindoleacetic acid concentrations, a measure of serotonin metabolism, in specific limbic brain regions of CRF-treated rats (nucleus accumbens shell region, entorhinal cortex, central nucleus of the amygdala). Furthermore, fluoxetine inhibited CRF-induced SNAMA. CRF and fluoxetine independently increased plasma corticosterone concentrations, but the responses had distinct temporal profiles. Overall, these data are consistent with the hypothesis that CRF-induced facilitation of behavioural activity is dependent on brainstem serotonergic systems. Therefore, fluoxetine may attenuate or alleviate some behavioural responses to stress by interfering with CRF-induced responses.

Glucocorticoid receptor antagonists hasten and augment neurochemical responses to a selective serotonin reuptake inhibitor antidepressant.

BACKGROUND: Selective serotonin reuptake inhibitor (SSRI) antidepressant drugs have a delayed onset and commonly produce an incomplete therapeutic response. The therapeutic actions of SSRIs are thought to depend on increased forebrain extracellular serotonin (5-HT), after desensitization of somatodendritic 5-HT(1A) autoreceptors. Here we determined whether concurrent glucocorticoid receptor (GR) blockade enhances these neurochemical responses to the SSRI fluoxetine. METHODS: Male rats were treated (3, 7, or 14 days) with either fluoxetine (10 mg/kg IP) or vehicle once daily, in combination with either a GR antagonist (Org 34850 15 mg/kg SC or Org 34517 25 mg/kg SC) or vehicle twice daily. After treatment, 5-HT in the medial prefrontal cortex was measured by microdialysis. RESULTS: Chronic fluoxetine treatment (14 days) raised basal 5-HT and also attenuated the fall in 5-HT after acute systemic administration of fluoxetine (10 mg/kg IP), indicating desensitization of 5-HT(1A) autoreceptors. Concurrent chronic administration (14 days) of Org 34850 or Org 34517 enhanced the fluoxetine-induced increase in basal 5-HT. Org 34850 also hastened the 5-HT(1A) autoreceptor desensitization induced by chronic fluoxetine treatment. Org 34850 alone (14 days) failed to alter basal 5-HT or 5-HT(1A) autoreceptor desensitization. CONCLUSIONS: Antidepressant response is proposed to depend on 5-HT(1A) autoreceptor desensitization and elevation of forebrain 5-HT. These data suggest adjunctive GR antagonists might both hasten and enhance antidepressant responses to SSRIs.

The progesterone metabolite allopregnanolone potentiates GABA(A) receptor-mediated inhibition of 5-HT neuronal activity.

The dorsal raphe nucleus (DRN) is the origin of much of the 5-HT innervation of the forebrain. The activity of DRN 5-HT neurons is regulated by a number of receptors including GABA(A) and 5-HT(1A) inhibitory receptors and by excitatory alpha(1)-adrenoceptors. Using in vitro electrophysiological recording we investigated the action of progesterone and its metabolite, allopregnanolone on receptor-mediated responses of DRN 5-HT neurons. Neither allopregnanolone nor progesterone affected the alpha(1)-adrenoceptor agonist-induced firing. Allopregnanolone also had no effect on the inhibitory response to 5-HT. However, allopregnanolone significantly potentiated the inhibitory responses to GABA(A) receptor agonists. Progesterone did not enhance GABA(A) receptor-meditated inhibitory responses. Thus, the neuroactive metabolite of progesterone, allopregnanolone, has the ability to cause potentiation of GABA(A)-mediated inhibition of DRN 5-HT neurons. This effect on 5-HT neurotransmission may have relevance for mood disorders commonly associated with reproductive hormone events, such as premenstrual dysphoric disorder and postpartum depression.

Gonadal steroid modulation of stress-induced hypothalamo-pituitary-adrenal activity and anxiety behavior: role of central oxytocin.

Intracerebroventricular administration of oxytocin reduces anxiety behavior and hypothalamo-pituitary-adrenal (HPA) responses to stress in female rats. Similar changes are seen in late-pregnant rats, and oxytocin-sensitive pathways may mediate these effects. This study investigated anxiety behavior and stress responses using a gonadal steroid model of late pregnancy, which is known to increase endogenous oxytocin expression. Compared with continuous progesterone treatment, 3-d withdrawal of progesterone after 11-d treatment of ovariectomized rats with estradiol and progesterone resulted in increased binding of the oxytocin receptor ligand [(125)I]d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)(9)]ornithine vasotocin in selective forebrain regions, including the ventrolateral septum and ventromedial hypothalamus. Behavior in the elevated plus-maze indicated that progesterone withdrawal had an anxiolytic effect, and this was associated with lower levels of c-fos mRNA expression in the ventral hippocampus, an area previously shown to be sensitive to oxytocin. In other groups of animals, the plasma corticosterone response to a psychological stress (10 min of 114 dB white noise) was significantly attenuated by this steroid manipulation. Furthermore, simultaneous infusion of the selective oxytocin receptor antagonist desGlyNH(2), d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT during the period of progesterone withdrawal reversed this attenuation of noise-induced HPA activation, indicating a role for endogenous oxytocin in this effect. Thus, mimicking the steroid profile of late pregnancy leads to a reduction in anxiety behavior and attenuates HPA activity induced by mild stress. These effects appear to be mediated through the involvement of central oxytocin neurotransmission.

Moderate differences in circulating corticosterone alter receptor-mediated regulation of 5-hydroxytryptamine neuronal activity.

Circulating glucocorticoid levels vary with stress and psychiatric illness and play a potentially important role in regulating transmitter systems that regulate mood. To determine whether chronic variation in corticosterone levels within the normal diurnal range altered the control of 5-hydroxytryptamine (5-HT) neuronal activity, male rats were adrenalectomized and implanted with either a 2% or 70% corticosterone/cholesterol pellet (100 mg). Two weeks later, the regulation of 5-HT neuronal activity in the dorsal raphe nucleus was studied by in vitro electrophysiology. At this time, serum corticosterone levels approximated the low-point (2%) and mid-point (70%) of the diurnal range. The excitatory response of 5-HT neurones to the alpha1-adrenoceptor agonist phenylephrine (1-11 microM) was significantly greater in the 2% group compared to the 70% group. By contrast, the inhibitory response to 5-HT (10-50 microM) was significantly lower in the 2% group compared to the 70% group. Thus, chronic variation in circulating corticosterone over a narrow part of the normal diurnal range causes a shift in the balance of positive and negative regulation of 5-HT neurones, with increased alpha 1-adrenoceptor-mediated excitation and reduced 5-HT-mediated autoinhibition at lower corticosterone levels. This shift would have a major impact on control of 5-HT neuronal activity.

GABA receptor modulation of 5-HT neuronal firing: characterization and effect of moderate in vivo variations in glucocorticoid levels.

Evidence from electrophysiological studies suggests that 5-HT neuronal firing in the dorsal raphe nucleus (DRN) may be regulated by both GABA(A) and GABA(B) receptors. Here, we addressed the question of whether the activity of individual 5-HT neurons is regulated by both GABA(A) and GABA(B) receptors. In addition, we examined the concentration-response relationships of GABA(A) and GABA(B) receptor activation and determined if GABA receptor regulation of 5-HT neuronal firing is altered by moderate alterations in circulating corticosterone. The activity of 5-HT neurons in the DRN of the rat was examined using in vitro extracellular electrophysiology. The firing of all individual neurons tested was inhibited by both the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol hydrochloride (THIP) (25 microM) and the GABA(B) receptor agonist baclofen (1 microM). Responses to THIP (5, 10, 25 microM) and baclofen (1, 3, 10 microM) were concentration dependent and attenuated by the GABA(A) and GABA(B) receptor antagonists, bicuculline (50 microM) and phaclofen (200 microM), respectively. To examine the effects of corticosterone on the sensitivity of 5-HT neurons to GABA receptor activation, experiments were conducted on adrenalectomized animals with corticosterone maintained for two weeks at either a low or moderate level within the normal diurnal range. These changes in corticosterone levels had no significant effects on the 5-HT neuronal response to either GABA(A) or GABA(B) receptor activation. The data indicate that the control of 5-HT neuronal activity by GABA is mediated by both GABA(A) and GABA(B) receptors and that this control is insensitive to moderate changes in circulating glucocorticoid levels.

Oxytocin attenuates stress-induced c-fos mRNA expression in specific forebrain regions associated with modulation of hypothalamo-pituitary-adrenal activity.

We reported previously that the neuropeptide oxytocin attenuates stress-induced hypothalamo-pituitary-adrenal (HPA) activity and anxiety behavior. This study sought to identify forebrain target sites through which oxytocin may mediate its anti-stress effects. Ovariectomized, estradiol-treated rats received intracerebroventricular infusions of oxytocin (1 or 10 ng/hr) or vasopressin (10 ng/hr), and the patterns of neuronal activation after restraint stress were determined by semiquantitative mapping of c-fos mRNA expression. Oxytocin administration significantly attenuated the release of ACTH and corticosterone and the increase in corticotropin-releasing factor mRNA expression in the hypothalamic paraventricular nucleus (PVN) in response to 30 min restraint. Restraint also induced the expression of c-fos mRNA in selective regions of the forebrain, including the PVN, paraventricular thalamic nucleus, habenula, medial amygdala, ventrolateral septum (LSV), most subfields of the dorsal and ventral hippocampus, and piriform and endopiriform cortices. In most cases, this level of gene expression was unaffected by concomitant administration of oxytocin. However, in the PVN, LSV, and throughout all subfields of the dorsal hippocampus, restraint evoked no detectable increase in c-fos mRNA in animals treated with either dose of oxytocin. Vasopressin had no effects on either HPA axis responses or neuronal activation in response to restraint, indicating that the effects were highly peptide selective. These data show that central oxytocin attenuates both the stress-induced neuroendocrine and molecular responses of the HPA axis and that the dorsal hippocampus, LSV, and PVN constitute an oxytocin-sensitive forebrain stress circuit.

Early life adversity programs changes in central 5-HT neuronal function in adulthood.

Early life adversity is associated with an increased incidence of psychiatric illness in adulthood. Although the mechanisms underlying this association are unclear, one possible substrate is brain 5-hydroxytryptamine neurotransmission, which is reportedly abnormal in several psychiatric disorders. This study examined the effect of a rat model of early life adversity, early maternal separation, on 5-hydroxytryptamine neurotransmission in adulthood. In vitro electrophysiological experiments revealed that, in early maternal separation rats compared with controls, the sensitivity of alpha1-adrenoceptors on 5-hydroxytryptamine neurons in the dorsal raphe nucleus was significantly reduced, whilst the sensitivity of 5-hydroxytryptamine1A receptors showed a nonsignificant trend to reduction. In in vivo microdialysis experiments, the 5-hydroxytryptamine1A receptor agonist-induced suppression of 5-hydroxytryptamine release in the frontal cortex was reduced in early maternal separation animals, suggesting desensitization of 5-hydroxytryptamine1A autoreceptors. There was no increase in basal 5-hydroxytryptamine in the frontal cortex as measured by microdialysis and a nonsignificant trend towards increased basal firing activity of classical (non-bursting) 5-hydroxytryptamine neurons in the dorsal raphe nucleus measured by in vivo electrophysiology. Finally, early maternal separation failed to alter expression of messenger ribonucleic acids coding for 5-hydroxytryptamine1A or alpha1B receptors in the dorsal raphe nucleus as measured by in situ hybridization histochemistry, suggesting that functional changes in receptor sensitivity observed are not due to changes in receptor gene transcription. The findings demonstrate that early life adversity programs changes in sensitivity of the two principal regulators of 5-hydroxytryptamine neuronal activity. Similar effects in humans may contribute to the increased incidence of psychiatric illness in individuals exposed to early life adversity.

Anatomical and functional evidence for a stress-responsive, monoamine-accumulating area in the dorsomedial hypothalamus of adult rat brain.

The dorsomedial hypothalamus (DMH) plays an important role in relaying information to neural pathways mediating neuroendocrine, autonomic, and behavioral responses to stress. Evidence suggests that the DMH is a structurally and functionally diverse integrative structure that contributes to both facilitation and inhibition of the hypothalamo-pituitary-adrenal axis, depending on the nature of the stimulus and the specific neural circuits involved. Previous studies have determined that stress or stress-related stimuli elevate tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine, and noradrenaline selectively within the DMH. In order to determine the specific region of the rat DMH involved, we used high-performance liquid chromatography with electrochemical detection to measure tissue concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline within five different subregions of the DMH in adult female Lewis and Fischer rats immediately or 4 h following a 30-min period of restraint stress. Compared to unrestrained control rats, restrained rats had elevated concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline immediately after a 30-min period of restraint and had elevated concentrations of 5-HT 4 h following the onset of a 30-min period of restraint stress. These effects were confined to a specific region that included medial portions of the dorsal hypothalamic area and dorsal ependymal, subependymal, and neuronal components of the periventricular nucleus. Furthermore, these effects were observed in Lewis rats, but not Fischer rats, two closely related rat strains with well-documented differences in neurochemical, neuroendocrine, autonomic, and behavioral responses to stress. These data provide support for the existence of a stress-responsive, amine-accumulating area in the DMH that may play an important role in the differential stress responsiveness of Lewis and Fischer rats.

Flattening the corticosterone rhythm attenuates 5-HT1A autoreceptor function in the rat: relevance for depression.

Depression is associated with glucocorticoid abnormalities, in particular a flattening of the diurnal cortisol rhythm. Recent data suggest that an important factor in the aetiology of depression may be a deficit in the function and expression of 5-HT(1A) receptors, which has been reported in depressed patients. The present study assessed the possibility that this cortisol abnormality is causal in the 5-HT(1A) receptor deficits. First, a rat model of flattened glucocorticoid rhythm was developed. Controlled release corticosterone pellets implanted for 14 days flattened the corticosterone rhythm and maintained levels constant midway between the nadir and zenith levels observed in sham-operated rats. Secondly, using microdialysis to assess 5-HT release in the hippocampus, the inhibitory response to 8-OHDPAT was measured to determine the sensitivity of somatodendritic 5-HT(1A) autoreceptors. Corticosterone treatment was found to induce a significant attenuation in the response to 8-OHDPAT, indicating functional desensitization of somatodendritic 5-HT(1A) autoreceptors. There was no effect of corticosterone treatment on basal extracellular 5-HT levels. The data suggest that the glucocorticoid abnormalities associated with depression may impact on the functioning of 5-HT(1A) receptors in the brain. These findings suggest that resolution of cortisol abnormalities may be a valuable target for pharmacotherapy in the treatment of depression.