Colin Ingram

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.

Gonadectomy Reverses The Sexually Diergic Patterns Of Circadian and Stress-Induced Hypothalamic-Pituitary-Adrenal Axis Activity In Male and Female Rats

Enhanced corticosterone release by female compared to male rats under basal and stress conditions is well documented. The demonstration that gonadectomy enhances stress‐induced corticosterone secretion in male rats, but reduces such levels in female rats, suggests a causal association between gonadal steroids and corticosterone release. The present study examined the corticosterone profile of sham gonadectomized and gonadectomized female and male rats under basal and stress conditions. An automated sampling system collected blood from each freely moving, unanaesthetized rat every 10 min (i) over a 24‐h period; (ii) following noise stress; and (iii) following an immune‐mediated stress (lipopolysaccharide, LPS). Plasma was analysed for corticosterone content using radioimmunoassay. Castration resulted in a significant increase in basal corticosterone release compared to the sham‐castrated male rats. Pulsar analysis revealed a significant two‐fold increase in the number of corticosterone pulses over 24 h. Corticosterone increases in response to noise stress and to LPS injection were enhanced following castration. Conversely, ovariectomy resulted in a two‐fold reduction in the number of corticosterone pulses as well as the stress response compared to sham‐ovariectomized female rats. Arginine vasopressin (AVP), corticotrophin‐releasing hormone (CRH) and glucocorticoid receptor mRNAs in the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the anterior pituitary were analysed post‐LPS administration by in situ hybridization. Significantly higher values were found for AVP, CRH and POMC mRNAs examined for sham females and castrated males compared to sham males and ovariectomized females. This study confirms previous reports concerning the influence of gonadal factors in regulating HPA axis activity and stress responsiveness. The present results extend these observations to the regulation of the dynamic pattern of corticosterone release under basal conditions and suggests that this alteration in pulsatility is important for the differences in stress responsiveness when comparing males and females.

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 α1‐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 α1B 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.

Flattening the Corticosterone Rhythm Attenuates 5-HT 1A 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-HT1A receptors, which has been reported in depressed patients. The present study assessed the possibility that this cortisol abnormality is causal in the 5-HT1A 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-HT1A autoreceptors. Corticosterone treatment was found to induce a significant attenuation in the response to 8-OHDPAT, indicating functional desensitization of somatodendritic 5-HT1A 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-HT1A receptors in the brain. These findings suggest that resolution of cortisol abnormalities may be a valuable target for pharmacotherapy in the treatment of depression.

Acute and chronic effects of corticosterone on 5-HT1A receptor-mediated autoinhibition in the rat dorsal raphe nucleus

Corticosteroid modulation of 5-HT(1A) receptor function may contribute to the aetiology of affective disorders. To examine this modulation, the effects of acute and chronic corticosterone administration on 5-HT(1A) autoreceptor function were investigated using in vitro electrophysiology in the rat dorsal raphe nucleus (DRN). The magnitude and time course of the inhibitory response to a submaximal dose of 5-HT was not affected by acute application of either corticosterone (30-200 nM) or dexamethasone (100 nM) in vitro, when tested either in slices from control rats or rats adrenalectomised two weeks prior to recording. For chronic treatment, rats were supplied with drinking water containing corticosterone (50 microg/ml) or ethanol vehicle (0.5%) for 25-31 days. The autoinhibitory response to 5-HT was significantly attenuated in the corticosterone-treated group; vehicle EC(50)=48+/-8 microM vs. corticosterone EC(50)=121+/-20 microM. Furthermore a subpopulation of 5-HT neurones from corticosterone-treated animals exhibited marked insensitivity to 5-HT. In situ hybridisation histochemistry showed that corticosterone did not affect the expression of mRNA encoding the 5-HT(1A) receptor or either the type 1 and type 3 subunits of the G-protein linked inwardly rectifying K+ (GIRK) channel. However, GIRK2 subunit mRNA expression was significantly reduced. Thus, 5-HT(1A) autoreceptor function in the DRN is attenuated following chronic, but not acute, exposure to elevated corticosterone levels, and this effect may involve changes to the receptor-effector coupling mechanism.

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.

5-HT1A receptor-mediated autoinhibition does not function at physiological firing rates: evidence from in vitro electrophysiological studies in the rat dorsal raphe nucleus.

5-HT(1A)-mediated autoinhibition of neurones in the dorsal raphe nucleus (DRN) is considered to be the principal inhibitory regulator of 5-HT neuronal activity. The activation of this receptor by endogenous 5-HT was investigated using electrophysiological recordings from the rat DRN in vitro. At a concentration which blocked the inhibitory effect of exogenous 5-HT, the 5-HT(1A) antagonist WAY 100635 did not alter basal firing rate or modulate the excitatory response to the alpha(1)-agonist phenylephrine. Blockade of 5-HT reuptake by a concentration of fluoxetine, which enhanced the inhibitory effect of exogenous 5-HT, lowered phenylephrine-induced basal firing presumably due to potentiation of the effect of endogenous 5-HT. However, this effect was not firing rate dependent and neither the proportional increase nor the time-course of the response to a higher concentration of phenylephrine were altered in the presence of fluoxetine. These data suggest that the inhibitory 5-HT(1A) receptor on raphe neurones is neither tonically activated nor plays any role in modulating the response to excitatory transmitters. Thus, at physiological firing rates this receptor does not appear to function as an autoreceptor of serotonergic neurones of the DRN.

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.

Anxiety behaviour of the male rat on the elevated plus maze: associated regional increase in c-fos mRNA expression and modulation by early maternal separation

Stressful stimuli cause region-specific increases in c-fos expression within the rat brain. Early maternal separation (EMS) is a model of early life adversity that results in long lasting changes to stress and anxiety responses. This study examined the regional distribution of c-fos mRNA after exposure to the elevated plus-maze (EPM) and how EMS altered this pattern. On each of post-natal days 5–21 pups were separated from the dam for 6 h—control rats remained undisturbed. At 70 days old, male offspring were either exposed to the EPM or left undisturbed in the home cage. After exposure to the EPM, c-fos mRNA expression was significantly increased in specific brain areas, including cingulate cortex, medial amygdala and hippocampus. EMS rats displayed greater anxiety behaviour on the EPM vs. controls. Although EMS caused no overall effect on basal c-fos mRNA, a significant interaction between treatment group and exposure to the EPM occurred in the dentate gyrus and piriform cortex, with lower EPM-induced mRNA levels in EMS rats. The region-specific increase in c-fos mRNA reflects activation of neural circuits associated with EPM-induced anxiety. The effect of EMS on this activation in the two regions suggests these areas may contribute to the differential response to the anxiogenic stress of the EPM.

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.