Andre L. Curtis

Activation of noradrenergic locus coeruleus neurons by hemodynamic stress is due to local release of corticotropin-releasing factor

The present study was designed to determine whether activation of locus coeruleus (LC) neurons by hemodynamic stress is mediated by local release of corticotropin-releasing factor (CRF) within the LC. The ability of local LC injection of the CRF antagonist, alpha helical CRF9-41, to prevent LC activation elicited by i.v. nitroprusside infusion was investigated in halothane-anesthetized rats. Nitroprusside infusion (10 micrograms/30 microliters/min for 15 min) consistently increased LC spontaneous discharge rate with the mean maximum increase of 32 +/- 5% (n = 8) occurring between 3 and 9 min after the initiation of the infusion. Prior local LC injection of alpha helical CRF9-41 (150 ng), but not of saline (150 nl), prevented LC activation by nitroprusside. Alpha helical CRF9-41 did not alter LC spontaneous discharge rate or LC discharge evoked by repeated sciatic nerve stimulation suggesting that the CRF antagonist selectively attenuates stress-elicited LC activation. In contrast to alpha helical CRF9-41, the excitatory amino acid antagonist, kynurenic acid, did not attenuated LC activation by nitroprusside at a dose (0.5 mumol in 5 microliters, i.c.v.) that prevented LC activation by sciatic nerve stimulation. Taken together, these findings suggest that hemodynamic stress elicited by nitroprusside infusion activates LC neurons by releasing CRF within the LC region. The onset of LC activation by nitroprusside was temporally correlated with electroencephalographic (EEG) activation recorded from the frontal cortex and hippocampus. EEG activation was characterized by a change from low frequency, high amplitude activity to high frequency low amplitude activity recorded from the cortex and theta rhythm recorded from the hippocampus. LC activation usually outlasted the EEG activation. Nitroprusside infusion following local LC injection of alpha helical CRF9-41 was also associated with EEG activation in most rats. However, the duration of hippocampal theta rhythm was shorter in rats administered alpha helical CRF9-41. Thus, LC activation during cardiovascular challenge may play some role in EEG activation but is not necessary for this effect.

Effects of Corticotropin-Releasing Factor on Brain Serotonergic Activity

The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 μg decreasing, and 3.0 μg increasing 5-HT dialysate concentrations. I.C.V. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 μg) and high (3 μg) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated.

Sex differences in corticotropin-releasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology

Although the higher incidence of stress-related psychiatric disorders in females is well documented, its basis is unknown. Here, we show 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.

Locus coeruleus activation by colon distention: role of corticotropin-releasing factor and excitatory amino acids

The present study was designed to elucidate the neurotransmitters involved in activation of the noradrenergic nucleus, locus coeruleus, by distention of the distal colon. Locus coeruleus spontaneous discharge rate was recorded from halothane-anesthetized rats before, during and after distention of the colon produced by inflation of a balloon catheter with varying volumes of water. Locus coeruleus activation by colon distention was volume-dependent and reversible. Activation of cortical electroencephalographic activity was temporally correlated with locus coeruleus activation during colon distention and prolonged distention (greater than 2 min) resulted in tachyphalaxis to both locus coeruleus and cortical electroencephalographic activation. The corticotropin-releasing factor antagonist, DPheCRF(12-41), administered intracerebroventricularly (3 microg) or microinfused into the locus coeruleus (10 ng) significantly attenuated locus coeruleus activation produced by lower, but not higher magnitudes of colon distention, implicating corticotropin-releasing factor afferents to the locus coeruleus in this response. Consistent with this, prior exposure to 30 min of footshock stress, which desensitizes locus coeruleus neurons to corticotropin-releasing factor, produced a similar attenuation of locus coeruleus activation by low, but not high magnitudes of distention. Kynurenic acid, administered intracerebroventricularly (5 micromol), significantly antagonized locus coeruleus activation by all magnitudes of colon distention. However, this excitatory amino acid antagonist was ineffective when administered directly into the locus coeruleus (0.3 nmol). Together, these findings suggest that low magnitudes of colon distention activate the locus coeruleus-noradrenergic system via corticotropin-releasing factor release within the locus coeruleus and that excitatory amino acid neurotransmission at a site distal to the locus coeruleus is necessary for this response. Activation of the locus coeruleus-noradrenergic system during colon distention may serve as a cognitive limb of the peripheral parasympathetic response. This activation may also play a role in disorders characterized by comorbidity of colonic and psychiatric symptoms, such as irritable bowel syndrome.

Sexually dimorphic responses of the brain norepinephrine system to stress and corticotropin-releasing factor.

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.

Selective Activation of Corticotropin-Releasing Factor-2 Receptors on Neurochemically Identified Neurons in the Rat Dorsal Raphe Nucleus Reveals Dual Actions

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 = 34) neurons, whereas a higher dose (30 ng) inhibited 28% and activated 41% (n = 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.

Activation of the Locus Ceruleus Brain Noradrenergic System during Stress: Circuitry, Consequences, and Regulation

Publisher Summary This chapter reviews that physiological evidence supporting the hypothesis that corticotropin-releasing factor (CRF) serves as a neurotransmitter in the locus ceruleus (LC) to mediate its activation by particular stimuli. The chapter also describes potential circuitry underlying CRF-LC interactions, speculates on consequences of LC activation by CRF, and discusses regulation of CRF-LC interactions. Anatomical and physiological findings suggest that endogenous CRF, perhaps acting as a neurotransmitter within the LC, activates the LC-noradrenergic (NE) system under physiological conditions and that this activation is sufficient to impact on LC targets. The hypotensive stress that has been examined is sufficient to elicit CRF release into the hypophyseal portal system and activate the hypothalamic- pituitary-adrenal axis, and, therefore, may be classified as a stressor. The potential circuitry underlying LC activation by hypotension and possible consequences are discussed in the chapter. It has been suggested that CRF-LC interactions and CRF neurohormone activity may be coregulated. Such parallel regulation could underlie the coexistence of neuroendocrine and behavioral dysfunction in certain psychiatric disorders. CRF-LC interactions may be important components of the stress response and serve to link certain peripheral autonomic effects with forebrain activity. Finally, coregulation of CRF-LC interactions and neurohormone CRF may be important in symptoms of stress-related psychiatric disorders.

Corticotropin-Releasing Factor Neurones of the Central Nucleus of the Amygdala Mediate Locus Coeruleus Activation by Cardiovascular Stress

Hypotensive stress engages corticotropin‐releasing factor (CRF) release within the rat locus coeruleus (LC), which activates LC neurones, initiating norepinephrine release in forebrain and activation of forebrain electroencephalographic activity. This study identified CRF afferents to the LC that are engaged during hypotensive stress. One of two potential CRF afferents, the central nucleus of the amygdala (CNA) or bed nucleus of the stria terminalis (BNST), was electrolytically lesioned and LC activation during hypotensive stress was quantified. Neither lesion altered LC spontaneous discharge rate or activation by intra‐LC administered CRF. By contrast, LC activation by hypotensive stress was greatly attenuated in CNA‐lesioned, but not BNST‐lesioned, rats. Hypotensive stress‐induced changes in transcriptional activation were immunohistochemically identified in CRF neurones that were retrogradely labelled from the LC region. c‐fos immunoreactivity was prevalent in the paraventricular nucleus of the hypothalamus (PVN), CNA and BNST. However, only the PVN contained a substantial number of neurones that were doubly immunolabelled for CRF and c‐fos, and few of these were retrogradely labelled from the LC. By contrast, immunoreactivity for the phosporylated form of cyclic AMP response‐element binding protein (PCREB) was prevalent in CRF neurones in the CNA and BNST. Moreover, approximately one‐third of the PCREB‐expressing CRF neurones in the CNA were retrogradely labelled from the LC. These electrophysiological and anatomical findings implicate the CNA as a primary source of CRF that activates the LC during hypotensive stress. Additionally, CREB phosphorylation, rather than c‐fos induction, is associated with hypotensive activation of CRF‐CNA neurones that project to the LC.

Previous stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus

Spontaneous and stress-evoked discharge of locus coeruleus neurons were characterized in rats with a history of stress. Rats exposed to one or five daily 30-min sessions of footshock were anesthetized with halothane and surgically prepared for locus coeruleus single-unit recording immediately following the last session. Locus coeruleus spontaneous discharge rate and discharge evoked by sciatic nerve stimulation were comparable between acutely and repeatedly stressed rats and controls. In contrast, locus coeruleus activation produced by intracerebroventricular administration of corticotropin-releasing factor (3 micrograms) or by hypotensive challenge (which requires endogenous corticotropin-releasing factor release in the locus coeruleus) was greatly attenuated in acutely stressed rats. The corticotropin-releasing factor dose-response curve was shifted to the right in acutely stressed rats compared with controls. In repeatedly stressed rats, the effects of 3 micrograms corticotropin-releasing factor on locus coeruleus discharge were similarly diminished. Although the maximum effect produced by corticotropin-releasing factor was decreased in these rats, the dose-response curve was shifted to the left, indicative of sensitization. Hypotensive challenge, which was ineffective in acutely stressed rats, increased locus coeruleus discharge of repeatedly stressed rats by a similar magnitude as in matched controls. The return of locus coeruleus responsiveness to hypotension in repeatedly stressed rats may be related to the sensitization to corticotropin-releasing factor. Finally, the protocol of repeated stress did not alter the affinity or density of corticotropin-releasing factor receptors in either the frontal cortex or brainstem. Taken together, the results suggest that a history of stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus at the postsynaptic level. However, these effects are not reflected by corticotropin-releasing factor binding kinetics in brainstem. Stress-induced changes in corticotropin-releasing factor neurotransmitter function in the locus coeruleus may play a role in certain symptoms of stress-related psychiatric disorders.

Hemodynamic stress activates locus coeruleus neurons of unanesthetized rats

The effects of hypotensive stress elicited by nitroprusside infusion on discharge activity of noradrenergic locus coeruleus (LC) neurons of unanesthetized rats were characterized. Nitroprusside (75 micrograms/30 microliters/min, 15 min IV infusion) decreased mean arterial pressure of unanesthetized rats by 50 +/- 2 mmHg (n = 5). Simultaneous recordings of LC spontaneous discharge revealed an increase in discharge rate (197 +/- 87%) that was associated with hypotension. A lower concentration of nitroprusside (10 micrograms/30 microliters/min) that decreased blood pressure of halothane-anesthetized rats by 55 +/- 2 mmHg was much less effective in producing hypotension and did not increase LC discharge when administered to unanesthetized rats. Prior administration of the CRF antagonist, alpha helical CRF9-41 (50 micrograms, ICV) greatly attenuated LC activation by nitroprusside. These findings demonstrate that LC activation elicited by nitroprusside is dependent on the magnitude of hypotension. The present results also demonstrate that nitroprusside is a less potent hemodynamic challenge in unanesthetized rats. Finally, LC activation associated with nitroprusside administration to unanesthetized rats is mediated to a large extent by CRF, confirming findings in anesthetized rats.