A photoswitchable GPCR-based opsin for presynaptic inhibition.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Surgical implantation of wireless, battery-free optoelectronic epidural implants for optogenetic manipulation of spinal cord circuits in mice.

The use of optogenetics to regulate neuronal activity has revolutionized the study of the neural circuitry underlying a number of complex behaviors in rodents. Advances have been particularly evident in the study of brain circuitry and related behaviors, while advances in the study of spinal circuitry have been less striking because of technical hurdles. We have developed and characterized a wireless and fully implantable optoelectronic device that enables optical manipulation of spinal cord circuitry in mice via a microscale light-emitting diode (µLED) placed in the epidural space (NeuroLux spinal optogenetic device). This protocol describes how to surgically implant the device into the epidural space and then analyze light-induced behavior upon µLED activation. We detail optimized optical parameters for in vivo stimulation and demonstrate typical behavioral effects of optogenetic activation of nociceptive spinal afferents using this device. This fully wireless spinal µLED system provides considerable versatility for behavioral assays compared with optogenetic approaches that require tethering of animals, and superior temporal and spatial resolution when compared with other methods used for circuit manipulation such as chemogenetics. The detailed surgical approach and improved functionality of these spinal optoelectronic devices substantially expand the utility of this approach for the study of spinal circuitry and behaviors related to mechanical and thermal sensation, pruriception and nociception. The surgical implantation procedure takes ~1 h. The time required for the study of behaviors that are modulated by the light-activated circuit is variable and will depend upon the nature of the study.

Battery-free, fully implantable optofluidic cuff system for wireless optogenetic and pharmacological neuromodulation of peripheral nerves.

Studies of the peripheral nervous system rely on controlled manipulation of neuronal function with pharmacologic and/or optogenetic techniques. Traditional hardware for these purposes can cause notable damage to fragile nerve tissues, create irritation at the biotic/abiotic interface, and alter the natural behaviors of animals. Here, we present a wireless, battery-free device that integrates a microscale inorganic light-emitting diode and an ultralow-power microfluidic system with an electrochemical pumping mechanism in a soft platform that can be mounted onto target peripheral nerves for programmed delivery of light and/or pharmacological agents in freely moving animals. Biocompliant designs lead to minimal effects on overall nerve health and function, even with chronic use in vivo. The small size and light weight construction allow for deployment as fully implantable devices in mice. These features create opportunities for studies of the peripheral nervous system outside of the scope of those possible with existing technologies.

Differential Regulation of Bladder Pain and Voiding Function by Sensory Afferent Populations Revealed by Selective Optogenetic Activation.

Bladder-innervating primary sensory neurons mediate reflex-driven bladder function under normal conditions, and contribute to debilitating bladder pain and/or overactivity in pathological states. The goal of this study was to examine the respective roles of defined subtypes of afferent neurons in bladder sensation and function in vivo via direct optogenetic activation. To accomplish this goal, we generated transgenic lines that express a Channelrhodopsin-2-eYFP fusion protein (ChR2-eYFP) in two distinct populations of sensory neurons: TRPV1-lineage neurons (Trpv1Cre;Ai32, the majority of nociceptors) and Nav1.8+ neurons (Scn10aCre;Ai32, nociceptors and some mechanosensitive fibers). In spinal cord, eYFP+ fibers in Trpv1Cre;Ai32 mice were observed predominantly in dorsal horn (DH) laminae I-II, while in Scn10aCre;Ai32 mice they extended throughout the DH, including a dense projection to lamina X. Fiber density correlated with number of retrogradely-labeled eYFP+ dorsal root ganglion neurons (82.2% Scn10aCre;Ai32 vs. 62% Trpv1Cre;Ai32) and degree of DH excitatory synaptic transmission. Photostimulation of peripheral afferent terminals significantly increased visceromotor responses to noxious bladder distension (30-50 mmHg) in both transgenic lines, and to non-noxious distension (20 mmHg) in Scn10aCre;Ai32 mice. Depolarization of ChR2+ afferents in Scn10aCre;Ai32 mice produced low- and high-amplitude bladder contractions respectively in 53% and 27% of stimulation trials, and frequency of high-amplitude contractions increased to 60% after engagement of low threshold (LT) mechanoreceptors by bladder filling. In Trpv1Cre;Ai32 mice, low-amplitude contractions occurred in 27% of trials before bladder filling, which was pre-requisite for light-evoked high-amplitude contractions (observed in 53.3% of trials). Potential explanations for these observations include physiological differences in the thresholds of stimulated fibers and their connectivity to spinal circuits.

Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain.

Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

Anti-vascular endothelial growth factor treatment decreases bladder pain in cyclophosphamide cystitis: a Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network animal model study.

OBJECTIVE: To investigate whether treatment with anti-vascular endothelial growth factor (VEGF)-neutralizing antibodies can reduce pain and voiding dysfunction in the cyclophosphamide (CYP) cystitis model of bladder pain in mice. MATERIALS AND METHODS: Adult female mice received anti-VEGF-neutralizing antibodies (10 mg/kg i.p. B20-4.1.1 VEGF mAb) or saline (control) pre-treatment, followed by CYP (150 mg/kg i.p.) to induce acute cystitis. Pelvic nociceptive responses were assessed by applying von Frey filaments to the pelvic area. Spontaneous micturition was assessed using the void spot assay. RESULTS: Systemic anti-VEGF-neutralizing antibody treatment significantly reduced the pelvic nociceptive response to CYP cystitis compared with control (saline). In the anti-VEGF pre-treatment group, there was a significant increase in pelvic hypersensitivity, measured by the area under the curve (AUC) using von Frey filaments at 5 h post-CYP administration (P = 0.004); however, by 48 h and 96 h post-CYP administration, pelvic hypersensitivity had reduced by 54% and 47%, respectively, compared with the 5 h post-CYP administration time point, and were no longer significantly different from baseline (P = 0.22 and 0.17, respectively). There was no difference in urinary frequency and mean voided volume between the two pre-treatment groups. CONCLUSION: Systemic blockade of VEGF signalling with anti-VEGF-neutralizing antibodies was effective in reducing pelvic/bladder pain in the CYP cystitis model of bladder pain. Our data support the further investigation of the use of anti-VEGF antibodies to manage bladder pain or visceral pain.

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors.

We introduce a strategy for preclinical research wherein promising targets for analgesia are tested in rodent and subsequently validated in human sensory neurons. We evaluate group II metabotropic glutamate receptors, the activation of which is efficacious in rodent models of pain. Immunohistochemical analysis showed positive immunoreactivity for mGlu2 in rodent dorsal root ganglia (DRG), peripheral fibers in skin, and central labeling in the spinal dorsal horn. We also found mGlu2-positive immunoreactivity in human neonatal and adult DRG. RNA-seq analysis of mouse and human DRG revealed a comparative expression profile between species for group II mGluRs and for opioid receptors. In rodent sensory neurons under basal conditions, activation of group II mGluRs with a selective group II agonist produced no changes to membrane excitability. However, membrane hyperexcitability in sensory neurons exposed to the inflammatory mediator prostaglandin E2 (PGE2) was prevented by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC). In human sensory neurons from donors without a history of chronic pain, we show that PGE2 produced hyperexcitability that was similarly blocked by group II mGluR activation. These results reveal a mechanism for peripheral analgesia likely shared by mice and humans and demonstrate a translational research strategy to improve preclinical validation of novel analgesics using cultured human sensory neurons.

Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics.

Optogenetics allows rapid, temporally specific control of neuronal activity by targeted expression and activation of light-sensitive proteins. Implementation typically requires remote light sources and fiber-optic delivery schemes that impose considerable physical constraints on natural behaviors. In this report we bypass these limitations using technologies that combine thin, mechanically soft neural interfaces with fully implantable, stretchable wireless radio power and control systems. The resulting devices achieve optogenetic modulation of the spinal cord and peripheral nervous system. This is demonstrated with two form factors; stretchable film appliqués that interface directly with peripheral nerves, and flexible filaments that insert into the narrow confines of the spinal epidural space. These soft, thin devices are minimally invasive, and histological tests suggest they can be used in chronic studies. We demonstrate the power of this technology by modulating peripheral and spinal pain circuitry, providing evidence for the potential widespread use of these devices in research and future clinical applications of optogenetics outside the brain.

ERK2 Alone Drives Inflammatory Pain But Cooperates with ERK1 in Sensory Neuron Survival.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are highly homologous yet distinct components of signal transduction pathways known to regulate cell survival and function. Recent evidence indicates an isoform-specific role for ERK2 in pain processing and peripheral sensitization. However, the function of ERK2 in primary sensory neurons has not been directly tested. To dissect the isoform-specific function of ERK2 in sensory neurons, we used mice with Cre-loxP-mediated deletion of ERK2 in Nav1.8(+) sensory neurons that are predominantly nociceptors. We find that ERK2, unlike ERK1, is required for peripheral sensitization and cold sensation. We also demonstrate that ERK2, but not ERK1, is required to preserve epidermal innervation in a subset of peptidergic neurons. Additionally, deletion of both ERK isoforms in Nav1.8(+) sensory neurons leads to neuron loss not observed with deletion of either isoform alone, demonstrating functional redundancy in the maintenance of sensory neuron survival. Thus, ERK1 and ERK2 exhibit both functionally distinct and redundant roles in sensory neurons. SIGNIFICANCE STATEMENT: ERK1/2 signaling affects sensory neuron function and survival. However, it was not clear whether ERK isoform-specific roles exist in these processes postnatally. Previous work from our laboratory suggested either functional redundancy of ERK isoforms or a predominant role for ERK2 in pain; however, the tools to discriminate between these possibilities were not available at the time. In the present study, we use new genetic knock-out lines to demonstrate that ERK2 in sensory neurons is necessary for development of inflammatory pain and for postnatal maintenance of peptidergic epidermal innervation. Interestingly, postnatal loss of both ERK isoforms leads to a profound loss of sensory neurons. Therefore, ERK1 and ERK2 display both functionally distinct and redundant roles in sensory neurons.

A simple and inexpensive method for determining cold sensitivity and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity. Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above. This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo.

The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature set point. The adjustment of this set point requires transient receptor potential cation channel, subfamily member 8 (TRPM8), but not transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC-mediated hydrolysis of phosphoinositol 4,5-bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli.

A technique to measure cold adaptation in freely behaving mice.

BACKGROUND: Adaptation to environmental temperature is essential for survival in seasonal areas. The mechanisms of adaptation have been studied in vitro, but it has not been quantified in vivo. NEW METHOD: The extended Cold Plantar Assay (eCPA) cools the entire testing environment. Once the desired environmental temperature has been reached, a separate focal cold stimulus is applied to the hindpaw and the latency to withdrawal is recorded as a proxy for cold sensitivity. RESULTS: Using this technique, we can test the cold responsiveness of freely behaving mice at ambient temperatures ranging from 5°C to 30°C. The responses are consistent and unambiguous, and the environmental temperatures generated are reproducible. We are also able to measure cold responsiveness as animals are in the process of adapting to cold environments. COMPARISON WITH EXISTING METHOD(S): Existing methods, such as the dynamic cold plate and the 2-plate preference assay test how mice respond to cold environments, but cannot assess how the thresholds for response are changed by acclimation in cold environments. Additionally, the eCPA requires very little specialized equipment, can test many mice at the same time on one apparatus, and has an objective readout. CONCLUSIONS: The extended Cold Plantar assay is a significant methodological improvement, allowing the assessment of cold responsiveness in freely behaving mice at a wide range of environmental temperature conditions and during cold adaptation.

Central amygdala metabotropic glutamate receptor 5 in the modulation of visceral pain.

Painful bladder syndrome is a debilitating condition that affects 3-6% of women in the United States. Multiple lines of evidence suggest that changes in CNS processing are key to the development of chronic bladder pain conditions but little is known regarding the underlying cellular, molecular, and neuronal mechanisms. Using a mouse model of distention-induced bladder pain, we found that the central nucleus of the amygdala (CeA) is a critical site of neuromodulation for processing of bladder nociception. Furthermore, we demonstrate that metabotropic glutamate receptor 5 (mGluR5) activation in the CeA induces bladder pain sensitization by increasing CeA output. Thus, pharmacological activation of mGluR5 in the CeA is sufficient to increase the response to bladder distention. Additionally, pharmacological blockade or virally mediated conditional deletion of mGluR5 in the CeA reduced responses to bladder distention suggesting that mGluR5 in the CeA is also necessary for these responses. Finally, we used optogenetic activation of the CeA and demonstrated that this caused a robust increase in the visceral pain response. The CeA-localized effects on responses to bladder distention are associated with changes in extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation in the spinal cord. Overall, these data demonstrate that mGluR5 activation leads to increased CeA output that drives bladder pain sensitization.

Metabotropic glutamate receptor 5 (mGluR5) regulates bladder nociception.

BACKGROUND: Interstitial cystitis/painful bladder syndrome (IC/PBS), is a severely debilitating chronic condition that is frequently unresponsive to conventional pain medications. The etiology is unknown, however evidence suggests that nervous system sensitization contributes to enhanced pain in IC/PBS. In particular, central nervous system plasticity of glutamatergic signaling involving NMDA and metabotropic glutamate receptors (mGluRs) has been implicated in a variety of chronic pain conditions. Here, we test the hypothesis that mGluR5 mediates both non-inflammatory and inflammatory bladder pain or nociception in a mouse model by monitoring the visceromotor response (VMR) during graded bladder distention. RESULTS: Using a combination of genetic and pharmacologic approaches, we provide evidence indicating that mGluR5 is necessary for the full expression of VMR in response to bladder distention in the absence of inflammation. Furthermore, we observed that mice infected with a uropathogenic strain of Escherichia coli (UPEC) develop inflammatory hyperalgesia to bladder distention, and that the selective mGluR5 antagonist fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl) urea], reduces the VMR to bladder distention in UPEC-infected mice. CONCLUSIONS: Taken together, these data suggest that mGluR5 modulates both inflammatory and non-inflammatory bladder nociception, and highlight the therapeutic potential for mGluR5 antagonists in the alleviation of bladder pain.

Temporal and regional regulation of gene expression by calcium-stimulated adenylyl cyclase activity during fear memory.

BACKGROUND: The Ca2+-stimulated adenylyl cyclases (ACs), AC1 and AC8, are key components of long-term memory processing. AC1 and AC8 double knockout mice (Adcy1(-/-)Adcy8(-/-); DKO) display impaired fear memory processing; the mechanism of this impairment is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that the Ca2+-stimulated ACs modulate long-lasting transcriptional changes essential for fear memory consolidation and maintenance. Here, we report a genome-wide study of gene expression changes associated with conditioned fear (CF) memory in wild-type and DKO mice to identify AC-dependent gene regulatory changes that occur in the amygdala and hippocampus at baseline and different time points after CF learning. We observed an overall decrease in transcriptional changes in DKO mice across all time points, but most strikingly, at periods when memory consolidation and retention should be occurring. Further, we identified a shared set of transcription factor binding sites in genes upregulated in wild-type mice that were associated with downregulated genes in DKO mice. To prove the temporal and regional importance of AC activity on different stages of memory processing, the tetracycline-off system was used to produce mice with forebrain-specific inducible expression of AC8 on a DKO background. CF behavioral results reveal that adult restoration of AC8 activity in the forebrain is sufficient for intact learning, while cessation of this expression at any time point across learning causes memory deficits. CONCLUSIONS/SIGNIFICANCE: Overall, these studies demonstrate that the Ca2+-stimulated ACs contribute to the formation and maintenance of fear memory by a network of long-term transcriptional changes.

Transient early-life forebrain corticotropin-releasing hormone elevation causes long-lasting anxiogenic and despair-like changes in mice.

During development, early-life stress, such as abuse or trauma, induces long-lasting changes that are linked to adult anxiety and depressive behavior. It has been postulated that altered expression of corticotropin-releasing hormone (CRH) can at least partially account for the various effects of stress on behavior. In accord with this hypothesis, evidence from pharmacological and genetic studies has indicated the capacity of differing levels of CRH activity in different brain areas to produce behavioral changes. Furthermore, stress during early life or adulthood causes an increase in CRH release in a variety of neural sites. To evaluate the temporal and spatial specificity of the effect of early-life CRH exposure on adult behavior, the tetracycline-off system was used to produce mice with forebrain-restricted inducible expression of CRH. After transient elevation of CRH during development only, behavioral testing in adult mice revealed a persistent anxiogenic and despair-like phenotype. These behavioral changes were not associated with alterations in adult circadian or stress-induced corticosterone release but were associated with changes in CRH receptor type 1 expression. Furthermore, the despair-like changes were normalized with antidepressant treatment. Overall, these studies suggest that forebrain-restricted CRH signaling during development can permanently alter stress adaptation leading to increases in maladaptive behavior in adulthood.

TAK1 targeting by glucocorticoids determines JNK and IkappaB regulation in Toll-like receptor-stimulated macrophages.

Glucocorticoids potently attenuate the production of inflammatory mediators by macrophages, a primary effector of innate immunity. Activation of different macrophage Toll-like receptors (TLRs) by their respective ligands presents a powerful system by which to evaluate stimulus-dependent glucocorticoid effects in the same cell type. Here, we test the hypothesis that glucocorticoids, acting through the glucocorticoid receptor, modulate macrophage activation preferentially depending upon the TLR-selective ligand and TLR adapters. We established that 2 adapters, Trif, MyD88, or both, determine the ability of glucocorticoids to suppress inhibitor of kappaB (IkappaB) degradation or Janus kinase (JNK) activation. Moreover, the sensitivity of transforming growth factor beta-activated kinase 1 (TAK1) activation to glucocorticoids determines these effects. These findings identify TAK1 as a novel target for glucocorticoids that integrates their anti-inflammatory action in innate immunity signaling pathways.

Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase.

To explore the role of glucocorticoids in regulation of kinase pathways during innate immune responses, we generated mice with conditional deletion of glucocorticoid receptor (GR) in macrophages (MGRKO). Activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) caused greater mortality and cytokine production in MGRKO mice than in controls. Ex vivo, treatment with dexamethasone (Dex) markedly inhibited LPS-mediated induction of inflammatory genes in control but not GR-deficient macrophages. We show that Dex inhibits p38 MAPK, but not PI3K/Akt, ERK, or JNK, in control macrophages. Associated with p38 inhibition, Dex induced MAP kinase phosphatase-1 (MKP-1) in control, but not MGRKO, macrophages. Consistent with the ex vivo studies, treatment with a p38 MAPK-specific inhibitor resulted in rescue of MGRKO mice from LPS-induced lethality. Taken together, we identify p38 MAPK and its downstream targets as essential for GR-mediated immunosuppression in macrophages.

Cortisol and its action on the glucocorticoid receptor in malnutrition and acute infection.

Severe malnutrition alone is believed to cause hypercortisolemia. Cortisol's effects are mediated through the glucocorticoid receptor, which binds the hormone in the cytosol, translocates to the nucleus, and promotes gene transcription. This observational study in marasmic children with and without acute infection tested the hypothesis that marasmus is associated with hypercortisolemia, less glucocorticoid receptor, and less receptor translocation to the nucleus. Twenty-eight Malawian children participated; 14 with marasmus and infection, 6 with marasmus without infection, and 8 well nourished with infection. Free serum cortisol, interleukin 6 and tumor necrosis factor alpha, leucine derived from whole-body proteolysis, and the amount of whole-cell and nuclear leukocyte glucocorticoid receptor were measured upon admission. Free serum cortisol concentration was increased in marasmic and well-nourished children with infection compared with uninfected children with marasmus (14.2 [8.5, 16.3], 24.4 [15.0, 39.2], 5.1 [3.5, 7.0] microg/L, median [25th, 75th percentiles]; P < .05 by Kruskal-Wallis test). The amount of whole-cell leukocyte glucocorticoid receptor was similar in all children (0.48 +/- 0.33 signal units), but the amount in the nucleus was greatest in marasmic children with infection, followed by the amount in uninfected marasmic children, and then in well-nourished infected children (0.54 +/- 0.58, 0.19 +/- 0.13, 0.02 +/- 0.5 signal units [mean +/- SD]; P < .05 for all comparisons by analysis of variance). These findings suggest that hypercortisolemia is not associated with malnutrition alone, but does occur appropriately with acute infection. The increased nuclear glucocorticoid receptor abundance in marasmus demonstrates that nutritional status modulates glucocorticoid receptor action by mechanisms in addition to circulating glucocorticoid concentrations.

Forebrain glucocorticoid receptors modulate anxiety-associated locomotor activation and adrenal responsiveness.

Stress potently modulates anxiety- and depression-related behaviors. In response to stressors, the hypothalamic-pituitary-adrenal (HPA) axis is activated, resulting in the release of glucocorticoids from the adrenal cortex. These hormones act peripherally to restore homeostasis but also feed back to the CNS to control the intensity and duration of the stress response. Glucocorticoids act in limbic areas of the CNS to mediate the psychological and behavioral effects of stress. In this study, we investigate the effect of forebrain-specific disruption of the glucocorticoid receptor (GR) on stress- and anxiety-related behaviors. We demonstrate that mice with disruption of forebrain GR show alterations in stress-induced locomotor activation in a number of anxiety-related behavioral paradigms. These changes are associated with alterations in stress-induced HPA axis activation and, importantly, are not attenuated by chronic treatment with the tricyclic antidepressant imipramine. These data demonstrate the importance of forebrain GR in regulation of physiological and behavioral stress reactivity and suggest that distinct pathways regulate despair- and anxiety-related behaviors.