Beverly A. S. Reyes

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.

Severe stress switches CRF action in the nucleus accumbens from appetitive to aversive

Stressors motivate an array of adaptive responses ranging from ‘fight or flight’ to an internal urgency signal facilitating long-term goals. However, traumatic or chronic uncontrollable stress promotes the onset of major depressive disorder, in which acute stressors lose their motivational properties and are perceived as insurmountable impediments. Consequently, stress-induced depression is a debilitating human condition characterized by an affective shift from engagement of the environment to withdrawal. An emerging neurobiological substrate of depression and associated pathology is the nucleus accumbens, a region with the capacity to mediate a diverse range of stress responses by interfacing limbic, cognitive and motor circuitry. Here we report that corticotropin-releasing factor (CRF), a neuropeptide released in response to acute stressors and other arousing environmental stimuli, acts in the nucleus accumbens of naive mice to increase dopamine release through coactivation of the receptors CRFR1 and CRFR2. Remarkably, severe-stress exposure completely abolished this effect without recovery for at least 90 days. This loss of CRF’s capacity to regulate dopamine release in the nucleus accumbens is accompanied by a switch in the reaction to CRF from appetitive to aversive, indicating a diametric change in the emotional response to acute stressors. Thus, the current findings offer a biological substrate for the switch in affect which is central to stress-induced depressive disorders.

Hypothalamic projections to locus coeruleus neurons in rat brain

Locus coeruleus (LC) neurons respond to autonomic and visceral stimuli and discharge in parallel with peripheral sympathetic nerves. The present study characterized the synaptic organization of hypothalamic afferents with catecholaminergic neurons in the LC using electron microscopy. Peroxidase labeling of axon terminals that were anterogradely labeled from the paraventricular nucleus (PVN) was combined with gold‐silver labeling of tyrosine hydroxylase in the LC. Approximately 19% of the anterogradely labeled axon terminals formed synaptic specializations with tyrosine hydroxylase‐immunoreactive dendrites in the LC. Retrograde transport from the LC combined with immunocytochemical detection of enkephalin and corticotropin‐releasing factor (CRF) suggested that most of the LC‐projecting PVN neurons (30%) were CRF immunoreactive and few (2%) were enkephalin immunoreactive. Finally, dual retrograde tracing from the LC and median eminence revealed that PVN neurons that project to the LC are a population distinct from that projecting to the median eminence. The present data suggest that a population of hypothalamic neurons is poised to directly modulate the activity of LC neurons and may integrate autonomic responses in brain by influencing LC neurons. Moreover, PVN neurons that use CRF as a neurohormone are distinct from those that use CRF as a neuromodulator to impact on the LC.

Agonist‐induced internalization of corticotropin‐releasing factor receptors in noradrenergic neurons of the rat locus coeruleus

Corticotropin‐releasing factor (CRF) acts within the locus coeruleus (LC), to modulate activity of the LC‐norepinephrine (NE) system. Combining molecular and cellular approaches, we demonstrate CRF receptor (CRFr) mRNA expression in Sprague–Dawley rat LC and provide the first in vivo evidence for agonist‐induced internalization of CRFr. CRFr mRNA was detected in LC micropunches by RT‐PCR. In dual labelling immunofluorescence studies, tyrosine hydroxylase (TH) containing neurons exhibited CRFr labelling. At the ultrastructural level, immunogold‐silver labelling for CRFr was localized to the plasma membrane of TH‐immunoperoxidase labelled dendrites. CRF (100 ng) injection into the LC produced a robust neuronal activation that peaked 10–15 min after injection and was maintained for the duration of the recording. This was associated with CRFr internalization in LC neurons that was apparent at 5 and 30 min after injection. By 5 min after injection the ratio of cytoplasmic to total dendritic CRFr‐labelling was 0.81 ± 0.01 in rats injected with CRF and 0.59 ± 0.02 in rats injected with artificial cerebrospinal fluid (ACSF; P < 0.0001). Enhanced internalization of CRFr was maintained at 30 min after CRF injection, with the ratio being 0.86 ± 0.02 for CRF‐injected cases and 0.57 ± 0.03 for ACSF‐injected cases (P < 0.0001). Internalized CRFr was associated with early endosomes, indicative of degradation or recycling. Agonist‐induced CRFr internalization in LC neurons may underlie acute desensitization to CRF or stress. This process may be a pivotal target by which stressors or pharmacological agents regulate the sensitivity of the LC‐NE system to CRF and subsequent stressors.

HIV-1 gp120-Induced Injury to the Blood-Brain Barrier: Role of Metalloproteinases 2 and 9 and Relationship to Oxidative Stress

Blood-brain barrier (BBB) disruption occurs during human immunodeficiency virus encephalopathy, but the mechanisms involved are not understood. We studied how acute and ongoing exposure to human immunodeficiency virus 1 envelope gp120 alters BBB structure and permeability. Intravenous Evans blue, given before stereotaxic gp120 injection into the caudate putamen of rats, was rapidly extravasated. Gelatinolytic activity, studied by in situ zymography, was increased after gp120 administration and was localized within cerebralvessel walls. The gp120 increased the expression of matrix metalloproteinases (MMPs) 2 and 9. Laminin and claudin-5, key BBB components and targets of both MMPs, were greatly reduced upon gp120 administration. The gp120 increased lipid peroxidation in the vascular endothelium and in neurons. Prior administration of rSV40 vectors carrying the antioxidant enzymes Cu/Zn superoxide dismutase or glutathione peroxidase protected from gp120-induced BBB damage. N-methyl-D-aspartate receptor activation upregulated pro-MMP-9 and increased MMP-9 gelatinase activity, and memantine, an N-methyl-D-aspartate receptor blocker, mitigated gp120-induced BBB abnormalities. Using intra-caudate putamen SV(gp120) to test the effects of chronic exposure to expressed gp120, we determined that oxidant stress and increased BBB permeability occurred as in acute exposure. These data indicate that both direct administration and cellular expression of gp120 lead to disruption of the BBB by increasing MMPs and reducing vascular tight junction proteins via mechanisms involving reactive oxygen species generation and oxidant injury.

The locus coeruleus: a key nucleus where stress and opioids intersect to mediate vulnerability to opiate abuse

The interaction between the stress axis and endogenous opioid systems has gained substantial clinical attention as it is increasingly recognized that stress predisposes to opiate abuse. For example, stress has been implicated as a risk factor in vulnerability to the initiation and maintenance of opiate abuse and is thought to play an important role in relapse in subjects with a history of abuse. Numerous reports indicating that stress alters individual sensitivity to opiates suggest that prior stress can influence the pharmacodynamics of opiates that are used in clinical settings. Conversely, the effects of opiates on different components of the stress axis can impact on individual responsivity to stressors and potentially predispose individuals to stress-related psychiatric disorders. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC), the major brain norepinephrine (NE)-containing nucleus. This review summarizes our current knowledge regarding the anatomical and neurochemical afferent regulation of the LC. It then presents physiological studies demonstrating opposing interactions between opioids and stress-related neuropeptides in the LC and summarizes results showing that chronic morphine exposure sensitizes the LC-NE system to corticotropin releasing factor and stress. Finally, new evidence for novel presynaptic actions of kappa-opioids on LC afferents is provided that adds another dimension to our model of how this central NE system is co-regulated by opioids and stress-related peptides.

Presynaptic inhibition of diverse afferents to the locus ceruleus by kappa-opiate receptors: a novel mechanism for regulating the central norepinephrine system.

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 μ-opiate receptors. Because the LC is also innervated by the endogenous κ-opiate receptor (κ-OR) ligand dynorphin and expresses κ-ORs, this study investigated κ-OR regulation of LC neuronal activity in rat. Immunoelectron microscopy revealed a prominent localization of κ-ORs in axon terminals in the LC that also contained either the vesicular glutamate transporter or CRF. Microinfusion of the κ-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 κ-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 κ-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 κ-ORs as a novel target for alleviating symptoms of opiate withdrawal, stress-related disorders, or disorders characterized by abnormal sensory responses, such as autism.

Dynorphin and stress related peptides in rat locus coeruleus: contribution of amygdalar efferents

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress‐related peptide corticotropin‐releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN‐ and CRF‐containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA‐positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA‐containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light‐level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN‐ and CRF‐labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel. J. Comp. Neurol. 508:663–675, 2008.

Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence

BackgroundOpioid agonist drugs produce analgesia. However, long-term exposure to opioid agonists may lead to opioid dependence. The analgesic and addictive properties of opioid agonist drugs are mediated primarily via the mu-opioid receptor (MOR). Opioid agonists appear to alter neuronal morphology in key brain regions implicated in the development of opioid dependence. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid dependence.ResultsGPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was identified as a MORIP in a modified split ubiquitin yeast two-hybrid screen. GPR177 is an evolutionarily conserved protein that plays a critical role in mediating Wnt protein secretion from Wnt producing cells. The MOR/GPR177 interaction was validated in pulldown, coimmunoprecipitation, and colocalization studies using mammalian tissue culture cells. The interaction was also observed in rodent brain, where MOR and GPR177 were coexpressed in close spatial proximity within striatal neurons. At the cellular level, morphine treatment caused a shift in the distribution of GPR177 from cytosol to the cell surface, leading to enhanced MOR/GPR177 complex formation at the cell periphery and the inhibition of Wnt protein secretion.ConclusionsIt is known that chronic morphine treatment decreases dendritic arborization and hippocampal neurogenesis, and Wnt proteins are essential for these processes. We therefore propose that the morphine-mediated MOR/GPR177 interaction may result in decreased Wnt secretion in the CNS, resulting in atrophy of dendritic arbors and decreased neurogenesis. Our results demonstrate a previously unrecognized role for GPR177 in regulating cellular response to opioid drugs.

Increased vulnerability of the brain norepinephrine system of females to corticotropin-releasing factor overexpression

Stress-related psychiatric disorders are more prevalent in women than men. As hypersecretion of the stress neuromediator, corticotropin-releasing factor (CRF) has been implicated in these disorders, sex differences in CRF sensitivity could underlie this disparity. Hyperarousal is a core symptom that is shared by stress-related disorders and this has been attributed to CRF regulation of the locus ceruleus (LC)-norepinephrine arousal system. We recently identified sex differences in CRF1 receptor (CRF1) signaling and trafficking that render LC neurons of female rats more sensitive to CRF and potentially less able to adapt to excess CRF compared with male rats. The present study used a genetic model of CRF overexpression to test the hypothesis that females would be more vulnerable to LC dysregulation by conditions of excess CRF. In both male and female CRF overexpressing (CRF-OE) mice, the LC was more densely innervated by CRF compared with wild-type controls. Despite the equally dense CRF innervation of the LC in male and female CRF-OE mice, LC discharge rates recorded in slices in vitro were selectively elevated in female CRF-OE mice. Immunoelectron microscopy revealed that this sex difference resulted from differential CRF1 trafficking. In male CRF-OE mice, CRF1 immunolabeling was prominent in the cytoplasm of LC neurons, indicative of internalization, a process that would protect cells from excessive CRF. However, in female CRF-OE mice, CRF1 labeling was more prominent on the plasma membrane, suggesting that the compensatory response of internalization was compromised. Together, the findings suggest that the LC-norepinephrine system of females will be particularly affected by conditions resulting in elevated CRF because of differences in receptor trafficking. As excessive LC activation has been implicated in the arousal components of stress-related psychiatric disorders, this may be a cellular mechanism that contributes to the increased incidence of these disorders in females.