Although the higher incidence of stress-related psychiatric disorders in females is well documented, its basis is unknown. Here we demonstrate that the receptor for corticotropin-releasing factor (CRF), the neuropeptide that orchestrates the stress response, signals and is trafficked differently in female rats in a manner that could result in a greater response and decreased adaptation to stressors. Most cellular responses to CRF in the brain are mediated by CRF receptor (CRFr) association with the GTP-binding protein, Gs. Receptor immunoprecipitation studies revealed enhanced CRFr-Gs coupling in cortical tissue of unstressed female rats. Previous stressor exposure abolished this sex difference by increasing CRFr-Gs coupling selectively in males. These molecular results mirrored the effects of sex and stress on sensitivity of locus ceruleus (LC)-norepinephrine neurons to CRF. Differences in CRFr trafficking were also identified that could compromise stress adaptation in females. Specifically, stress-induced CRFr association with β-arrestin2, an integral step in receptor internalization, occurred only in male rats. Immunoelectron microscopy confirmed that stress elicited CRFr internalization in LC neurons of male rats exclusively, consistent with reported electrophysiological evidence for stress-induced desensitization to CRF in males. Together, these studies identified two aspects of CRFr function, increased cellular signaling and compromised internalization, which render CRF-receptive neurons of females more sensitive to low levels of CRF and less adaptable to high levels of CRF. CRFr dysfunction in females may underlie their increased vulnerability to develop stress-related pathology, particularly that related to increased activity of the LC-norepinephrine system, such as depression or post-traumatic stress disorder.

Stress-related psychiatric disorders are more prevalent in females than males, and this has been attributed to differences in stress sensitivity. As activation of the locus coeruleus (LC)-norepinephrine (NE) system is an important component of the stress response, this study compared LC responses to stress in female and male rats under different hormonal conditions in the halothane-anesthetized state. The mean basal LC discharge rate was similar between groups. However, the magnitude of LC activation elicited by hypotensive stress was substantially greater in females, regardless of hormonal status. The difference in stress sensitivity could be attributed to the differential postsynaptic sensitivity of LC neurons to corticotropin-releasing factor (CRF), which mediates LC activation by hypotension. CRF was 10-30 times more potent in activating LC neurons in female vs male rats. Interestingly, previous exposure to swim stress differentially regulated LC responses to CRF by sensitizing LC neurons of male, but not female, rats to CRF. The net effect of this was to abolish sex differences in LC sensitivity. Finally, CRF receptor (CRF-R) protein levels in the LC were greater in ovarectomized female vs male rats. This is the first study to demonstrate sex differences in the stress responsiveness of the brain noradrenergic system. Substantial sex differences were apparent in postsynaptic sensitivity to CRF and stress-induced regulation of postsynaptic sensitivity to CRF. These sex differences in the CRF regulation of the LC-NE system translate to a differential response to stress and may play a role in the increased vulnerability of females to stress-related psychiatric disorders.

No abstract

The norepinephrine nucleus, locus coeruleus (LC), has been implicated in cognitive aspects of the stress response, in part through its regulation by the stress-related neuropeptide, corticotropin-releasing factor (CRF). LC neurons discharge in tonic and phasic modes that differentially modulate attention and behavior. Here, the effects of exposure to an ethologically relevant stressor, predator odor, on spontaneous (tonic) and auditory-evoked (phasic) LC discharge were characterized in unanesthetized rats. Similar to the effects of CRF, stressor presentation increased tonic LC discharge and decreased phasic auditory-evoked discharge, thereby decreasing the signal-to-noise ratio of the sensory response. This stress-induced shift in LC discharge towards a high tonic mode was prevented by a CRF antagonist. Moreover, CRF antagonism during stress unmasked a large decrease in tonic discharge rate that was opioid mediated because it was prevented by pretreatment with the opiate antagonist, naloxone. Elimination of both CRF and opioid influences with an antagonist combination rendered LC activity unaffected by the stressor. These results demonstrate that both CRF and opioid afferents are engaged during stress to fine-tune LC activity. The predominant CRF influence shifts the operational mode of LC activity towards a high tonic state that is thought to facilitate behavioral flexibility and may be adaptive in coping with the stressor. Simultaneously, stress engages an opposing opioid influence that restrains the CRF influence and may facilitate recovery towards pre-stress levels of activity. Changes in the balance of CRF:opioid regulation of the LC could have consequences for stress vulnerability.

The dorsal raphe (DR)-serotonin (5-HT) system has been implicated in stress-related psychiatric disorders. Stress may impact on this system through corticotropin-releasing factor (CRF), which densely innervates the DR. CRF binds to CRF-R1 and CRF-R2 receptors in the DR and has complex and opposing effects depending on the dose used and the endpoint examined. To clarify the impact of CRF on the DR-5-HT system, the effects of selectively activating CRF-R2 receptors (the predominant subtype) on extracellular DR neuronal activity were examined in halothane-anesthetized rats. Because the DR is neurochemically heterogeneous, when possible, neurons were labeled with neurobiotin for subsequent neurochemical classification as 5-HT or non-5-HT. Relatively low doses of urocortin II (UII) (0.1-10 ng) injected into the DR inhibited most (79%; n ϭ 34) neurons, whereas a higher dose (30 ng) inhibited 28% and activated 41% (n ϭ 29). An analysis of effects on neurochemically identified neurons revealed that 5-HT neurons were inhibited by 0.1-10 ng of UII and activated by 30 ng of UII. Activation of 5-HT neurons by 30 ng of UII likely resulted from disinhibition because the majority of non-5-HT neurons were inhibited by this dose. Antisauvagine-30, but not antalarmin, antagonized UII, implicating CRF-R2 receptors in the effects. The results suggest that activation of CRF-R2 on DR-5-HT neurons inhibits neuronal activity, whereas activation of CRF-R2 receptors on non-5-HT neurons may indirectly excite DR-5-HT neurons through disinhibition. Importantly, the tone of the DR-5-HT system can be regulated in a dynamic manner through CRF-R2 activation, being either decreased or increased depending on the level of endogenous or exogenous ligand.

Endogenous opioids target noradrenergic locus ceruleus (LC) neurons and potently inhibit LC activity. Nonetheless, it has been difficult to demonstrate functional regulation of the LC-noradrenergic system by endogenous opioids because of the lack of effect of opiate antagonists. The present findings provide evidence that endogenous opioids regulate LC neuronal activity during the termination of a stressor. LC neuronal discharge was recorded from halothane-anesthetized rats before, during, and after hypotensive stress elicited by intravenous nitroprusside infusion. In naive rats, mean arterial blood pressure was temporally correlated with LC activity such that hypotension was associated with increased LC discharge and a return to the normotensive state was associated with a decrease in LC discharge below pre-stress values. After microinfusion of an antagonist of the stress neuropeptide corticotropin-releasing factor (CRF) into the LC, the increase in LC discharge associated with hypotension was prevented, whereas LC inhibition associated with termination of the challenge occurred at an earlier time and was of a greater magnitude. In contrast, microinfusion of naloxone into the LC completely abolished LC inhibition associated with termination of the stressor. Naloxone microinfusion did not prevent LC inhibition associated with hypertension produced by intravenous vasopressin administration, suggesting that endogenous opioids may be selectively engaged during the termination of hypotensive stress. These results provide evidence for a functional release of endogenous opioids within the LC. This action of endogenous opioids may serve to counterbalance excitatory effects of CRF on the LC-norepinephrine system, thereby limiting its activation by stress.

No abstract

The norepinephrine nucleus, locus ceruleus (LC), is activated by diverse stimuli and modulates arousal and behavioral strategies in response to these stimuli through its divergent efferent system. Afferents communicating information to the LC include excitatory amino acids (EAAs), corticotropin-releasing factor (CRF), and endogenous opioids acting at -opiate receptors. Because the LC is also innervated by the endogenous -opiate receptor (-OR) ligand dynorphin and expresses -ORs, this study investigated -OR regulation of LC neuronal activity in rat. Immunoelectron microscopy revealed a prominent localization of -ORs in axon terminals in the LC that also contained either the vesicular glutamate transporter or CRF. Microinfusion of the -OR agonist (trans)-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)-cyclo-hexyl] benzeneacetamide (U50488) into the LC did not alter LC spontaneous discharge but attenuated phasic discharge evoked by stimuli that engage EAA afferents to the LC, including sciatic nerve stimulation and auditory stimuli and the tonic activation associated with opiate withdrawal. Inhibitory effects of the -OR agonist were not restricted to EAA afferents, as U50488 also attenuated tonic LC activation by hypotensive stress, an effect mediated by CRF afferents. Together, these results indicate that -ORs are poised to presynaptically inhibit diverse afferent signaling to the LC. This is a novel and potentially powerful means of regulating the LC-norepinephrine system that can impact on forebrain processing of stimuli and the organization of behavioral strategies in response to environmental stimuli. The results implicate -ORs as a novel target for alleviating symptoms of opiate withdrawal, stress-related disorders, or disorders characterized by abnormal sensory responses, such as autism.