Stress-induced Neuronal Activation Research Articles

Characterization of transcriptional profiles associated with stress-induced neuronal activation in Arc-GFP mice.

Chronic stress has become a predominant factor associated with a variety of psychiatric disorders, such as depression and anxiety, in both human and animal models. Although multiple studies have looked at transcriptional changes after social defeat stress, these studies primarily focus on bulk tissues, which might dilute important molecular signatures of social interaction in activated cells. In this study, we employed the Arc-GFP mouse model in conjunction with chronic social defeat (CSD) to selectively isolate activated nuclei (AN) populations in the ventral hippocampus (vHIP) and prefrontal cortex (PFC) of resilient and susceptible animals. Nuclear RNA-seq of susceptible vs. resilient populations revealed distinct transcriptional profiles linked predominantly with neuronal and synaptic regulation mechanisms. In the vHIP, susceptible AN exhibited increased expression of genes related to the cytoskeleton and synaptic organization. At the same time, resilient AN showed upregulation of cell adhesion genes and differential expression of major glutamatergic subunits. In the PFC, susceptible mice exhibited upregulation of synaptotagmins and immediate early genes (IEGs), suggesting a potentially over-amplified neuronal activity state. Our findings provide a novel view of stress-exposed neuronal activation and the molecular response mechanisms in stress-susceptible vs. resilient animals, which may have important implications for understanding mental resilience.

Comparisons of stress-related neuronal activation induced by restraint in adult male rat offspring with prenatal exposure to buprenorphine, methadone, or morphine.

Prenatal opioid exposure may impede the development of adaptive responses to environmental stimuli by altering the stress-sensitive brain circuitry located at the paraventricular nucleus of the hypothalamus (PVH) and locus coeruleus (LC). Corticotropin-releasing factor (CRF) released from neurons in the PVH has emerged as a key molecule to initiate and integrate the stress response. Methadone (Meth) and buprenorphine (Bu) are two major types of synthetic opioid agonists for first-line medication-assisted treatment of opioid (e.g., morphine, Mor) use disorder in pregnant women. No studies have compared the detrimental effects of prenatal exposure to Meth versus Bu on the stress response of their offspring upon reaching adulthood. In this study, we aimed to compare stress-related neuronal activation in the PVH and LC induced by restraint (RST) stress in adult male rat offspring with prenatal exposure to the vehicle (Veh), Bu, Meth, or Mor. CFos-immunoreactive cells were used as an indicator for neuronal activation. We found that RST induced less neuronal activation in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. RST-induced neuronal activation was completely prevented by central administration of a CRF receptor antagonist (α-helical CRF9-41, 10 μg/3 μL) in all exposure groups, suggesting the crucial role of CRF in this stress response. In offspring without RST, central administration of CRF (0.5 μg/3 μL)-induced neuronal activation in the PVH and LC. CRF-induced neuronal activation was lessened in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. Moreover, RST- or CRF-induced neuronal activation in the Meth exposure group was comparable with that in the Mor exposure group. Further immunohistochemical analysis revealed that the Meth and Mor exposure groups displayed less CRF neurons in the PVH of offspring with or without RST compared with the Bu or Veh groups. Thus, stress-induced neuronal activation in the PVH and LC was well preserved in adult male rat offspring with prenatal exposure to Bu, but it was substantially lessened in those with prenatal exposure to Meth or Mor. Lowered neuronal activation found in the Meth or Mor exposure groups may be, at least in part, due to the reduction in the density of CRF neurons in the PVH.

High fat diet blunts stress-induced hypophagia and activation of Glp1r dorsal lateral septum neurons in male but not in female mice

ObjectiveWhile stress typically reduces caloric intake (hypophagia) in chow-fed rodents, presentation of palatable, high calorie substances during stress can increase caloric consumption (i.e. “comfort feeding”) and promote obesity. However, little is known about how obesity itself affects feeding behavior in response to stress and the mechanisms that can influence stress-associated feeding in the context of obesity. MethodsWe assessed food intake and other metabolic parameters in lean and obese male and female mice following acute restraint stress. We also measured real-time activity of glucagon-like peptide-1 (Glp1) receptor (Glp1r)-expressing neurons in the dorsal lateral septum (dLS) during stress in lean and obese mice using fiber photometry. Glp1r activation in various brain regions, including the dLS, promotes hypophagia in response to stress. Finally, we used inhibitory Designer Receptors Activated Exclusively by Designer Drugs (DREADDs) to test whether activation of Glp1r-expressing neurons in the LS is required for stress-induced hypophagia. ResultsLean male mice display the expected hypophagic response following acute restraint stress, but obese male mice are resistant to this acute stress-induced hypophagia. Glp1r-positive neurons in the dLS are robustly activated during acute restraint stress in lean but not in obese male mice. This raises the possibility that activation of dLS Glp1r neurons during restraint stress contributes to subsequent hypophagia. Supporting this, we show that chemogenetic inhibition of LS Glp1r neurons attenuates acute restraint stress hypophagia in male mice. Surprisingly, we show that both lean and obese female mice are resistant to acute restraint stress-induced hypophagia and activation of dLS Glp1r neurons. ConclusionsThese results suggest that dLS Glp1r neurons contribute to the hypophagic response to acute restraint stress in male mice, but not in female mice, and that obesity disrupts this response in male mice. Broadly, these findings show sexually dimorphic mechanisms and feeding behaviors in lean vs. obese mice in response to acute stress.

Androgen Regulation of Corticotropin Releasing Factor Receptor 1 in the Mouse Brain

Stress-related mood disorders like anxiety and depression are more prevalent in women than men and are often associated with hypothalamic–pituitary-adrenal (HPA) axis dysregulation. Androgen actions through androgen receptors (ARs) decrease HPA axis responses and stress-associated behaviors. Corticotropin releasing factor (CRF) and its binding to CRF receptor 1 (CRFR1) is also critical for regulation of the HPA axis, anxiety, and depression. We first determined CRFR1/AR co-localization patterns in male and female CRFR1-GFP mice. High co-localization was found within the paraventricular nucleus (PVN), dorsolateral and anteroventral subdivisions of the bed nucleus of the stria terminalis (BSTdl and BSTav), medial preoptic area (MPOA), and posterodorsal medial amygdala (MePD). We next determined whether the non-aromatizable androgen dihydrotestosterone (DHT) regulates CRFR1 expression and stress-induced activation of CRFR1-expressing cells. In the PVN, CRFR1-GFP cell number decreased following gonadectomy (GDX), but DHT treatment reversed this effect. GDX-DHT treated mice also had a decreased CRFR1-GFP cell number within the BSTdl compared to gonad intact and GDX-untreated groups. Following restraint stress GDX-blank mice showed fewer c-Fos/CRFR1 co-localized neurons in the MePD compared to gonad intact and GDX-DHT groups indicating decreased stress-induced activation of CRFR1 neurons following GDX. Higher plasma corticosterone (CORT) was found in GDX males compared to GDX-DHT and sham males following restraint stress, with a negative correlation between PVN CRFR1+ neurons and corticosterone levels 30- and 90-min following restraint. Together these findings show androgens can directly alter CRFR1 levels in the brain which may have implications for sex differences in regulation of the HPA axis and stress-related behaviors.

Effects of fluoxetine on stress-induced neuronal activity in the ventral hippocampus

Effects of fluoxetine on stress-induced neuronal activity in the ventral hippocampus

Low Fos expression in newly generated neurons of the main and accessory olfactory bulb following single maternal separation

The main and accessory olfactory bulbs (MOB and AOB) are unique in that they produce new neurons throughout adulthood. Despite the recent knowledge about the involvement of postnatally generated cells in several aspects of olfaction, the functional role of these neurons is still not sufficiently understood. The function of newly generated olfactory bulb neurons is primarily investigated in relation to activities related to smell. Stress-induced activation of new olfactory neurons has not yet been studied. Thus, our work was aimed to investigate whether a stressful event, such as maternal separation (MS) can induce Fos expression in postnatally-born neurons in the MOB and AOB. Rat pups were exposed to single maternal separation (SMS) for 2 h at the postnatal days: P7, P14, and P21. Quantification of immunohistochemically labeled Fos + cells revealed that exposure to SMS in different age stages during the first postnatal month stimulates activity in cells of individual MOB/AOB layers in an age-dependent manner. In order to find out whether newly generated cells in the MOB/AOB could express Fos protein as a response to SMS, newborn rats were administrated with the marker of proliferation, bromodeoxyuridine (BrdU) at P0, and three weeks later (at P21) colocalization of Fos and BrdU in the neurons of the MOB and AOB was assessed. Quantitative analysis of BrdU/Fos double-labeled cells showed that Fos is expressed only in a small number of postnatally generated cells within the MOB/AOB. Our results indicate that postnatally generated MOB/AOB neurons are less sensitive to stress caused by MS than preexisting ones. LAY SUMMARY Our results showed that single maternal separation (SMS) is a stressful event that in age-dependent manner stimulates cellular activity in the main and accessory olfactory bulb (AOB) – the structures dedicated to odor information processing. The low level of Fos expression in newborn neurons of the main and accessory bulb indicates that postnatally generated cells are less sensitive to neonatal stress than preexisting neurons.

Long non-coding RNA Neat1 regulates adaptive behavioural response to stress in mice

NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1−/− mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1−/− mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1−/− mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1−/− neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1−/− mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress.

Diazepam limits microglia-mediated neuronal remodeling in the prefrontal cortex and associated behavioral consequences following chronic unpredictable stress.

Chronic stress induces neuronal atrophy and synaptic loss in the medial prefrontal cortex (PFC), and this leads to behavioral and cognitive impairments. Our recent findings indicate that microglia contribute to structural remodeling of neurons via increased colony-stimulating factor (CSF)-1 in the medial PFC. Other work shows that chronic stress induces aberrant neuronal activity in the medial PFC, and that neuronal hyperactivity increases CSF1 signaling and alters microglia function. Thus, the present studies were designed to examine the role of neuronal activity in stress-induced CSF1 signaling and microglia-mediated neuronal remodeling in the medial PFC. Additional analyses probed stress effects on the dorsal hippocampus (HPC), basolateral amygdala (BLA), and somatosensory cortex (SSCTX). Mice were exposed to chronic unpredictable stress (CUS) or handled intermittently as controls, and received daily injection of vehicle or diazepam (1 mg/kg). As anticipated, diazepam attenuated CUS-induced behavioral despair and cognitive impairments. Further studies showed that diazepam normalized Csf1 and C3 mRNA in the PFC, and prevented increases in Csf1r and Cd11b in frontal cortex microglia following CUS. Stress had no effect on neuroimmune gene expression in the HPC. Confocal imaging in Thy1-GFP(M) mice demonstrated that diazepam limited microglial engulfment of neuronal elements and blocked CUS-induced dendritic spine loss in the medial PFC. Altogether, these findings indicate that modulation of chronic stress-induced neuronal activity limits microglia-mediated neuronal remodeling in the medial PFC, and subsequent behavioral and cognitive consequences.

Neonatal shaking brain injury changes psychological stress-induced neuronal activity in adult male rats

Neonatal shaking brain injury (SBI) leads to increases in anxiety-like behavior and altered hormonal responses to psychological stressors as adults. These abnormalities are hypothesized to be due to a change in sensitization in neuronal circuits as a consequence of neonatal SBI. We examined the effects of neonatal SBI on neuronal activity in the anxiety- and/or stress-related areas of adult rats using Fos immunohistochemistry. Exposure to a novel elevated plus maze (EPM) resulted in a marked increase in Fos expression in the parvocellular (PVNp) and magnocellular parts of the paraventricular nucleus and the ventral part of the bed nucleus of the stria terminalis (vBNST) of shaken rats (S group) compared to non-shaken control rats (C group). On the contrary, Fos expression was significantly lower in the medial nucleus of the amygdala and the ventral subiculum (vS) of S group rats than C group rats exposed to EPM. Although we found no significant correlation in the number of Fos-expressing cells in the vBNST and PVNp in the C group rats, these numbers were significantly correlated in the S group rats. Furthermore, in the S group rats, but not in the C group rats, the number of Fos-expressing cells in the vBNST was inversely correlated with that in the vS. Interestingly, previous neuronal tracing studies have demonstrated direct projections from the vS to the vBNST and from the vBNST to the PVNp. The present data suggest that neonatal SBI can alter neuronal activity in anxiety- and/or stress-related neuronal circuits.

Corticotropin-Releasing Factor-Producing Cells in the Paraventricular Nucleus of the Hypothalamus and Extended Amygdala Show Age-Dependent FOS and FOSB/DeltaFOSB Immunoreactivity in Acute and Chronic Stress Models in the Rat.

Corticotropin-releasing factor (CRF) immunoreactive (ir) neurons of the paraventricular nucleus of the hypothalamus (PVN) play pivotal role in the coordination of stress response. CRF-producing cells in the central nucleus of amygdala (CeA) and oval division of the bed nucleus of stria terminalis (BNSTov) are also involved in stress adaptation and mood control. Immediate early gene products, subunits of the transcription factor activator protein 1 (AP1) are commonly used as acute (FOS) and/or chronic (FOSB/deltaFOSB) markers for the neuronal activity in stress research. It is well known that the course of aging affects stress adaptation, but little is known about the aging-related stress sensitivity of CRF neurons. To the best of our knowledge, the stress-induced neuronal activity of CRF neurons in the course of aging in acute and chronic stress models was not studied systematically yet. Therefore, the aim of the present study was to quantify the acute restraint stress (ARS) and chronic variable mild stress (CVMS) evoked neuronal activity in CRF cells of the PVN, CeA, and BNSTov using triple-label immunofluorescence throughout the whole lifespan in the rat. We hypothesized that the FOS and FOSB content of CRF cells upon ARS or CVMS decreases with age. Our results showed that the FOS and FOSB response to ARS declined with age in the PVN-CRF cells. BNSTov and CeA CRF cells did not show remarkable stress-induced elevation of these markers neither in ARS, nor in CVMS. Exposure to CVMS resulted in an age-independent significant increase of FOSB/delta FOSB immunosignal in PVN-CRF neurons. Unexpectedly, we detected a remarkable stress-independent FOSB/deltaFOSB signal in CeA- and BNSTov-CRF cells that declined with the course of aging. In summary, PVN-CRF cells show decreasing acute stress sensitivity (i.e., FOS and FOSB immunoreactivity) with the course of aging, while their (FOSB/deltaFOSB) responsivity to chronic challenge is maintained till senescence. Stress exposure does not affect the occurrence of the examined Fos gene products in CeA- and BNSTov-CRF cells remarkably suggesting that their contribution to stress adaptation response does not require AP1-controlled transcriptional changes.

Neuroligin-2 Determines Inhibitory Synaptic Transmission in the Lateral Septum to Optimize Stress-Induced Neuronal Activation and Avoidance Behavior

BackgroundInvestigations in the neocortex have revealed that the balance of excitatory and inhibitory synaptic transmission (E/I ratio) is important for proper information processing. The disturbance of this balance underlies many neuropsychiatric illnesses, including autism spectrum disorder and schizophrenia. However, little is known about the contribution of E/I balance to the functioning of subcortical brain regions, such as the lateral septum (LS), a structure that plays important roles in regulating anxiety-related behavior. MethodsWe manipulated E/I balance in the mouse LS by localized conditional deletion of neuroligin-2, a postsynaptic cell adhesion protein located at gamma-aminobutyric acidergic synapses and important for inhibitory synaptic transmission. We then performed analyses of synaptic transmission in the LS, stress-induced expression of immediate early gene c-fos, and anxiety-related and depression-related behavior. ResultsThe absence of neuroligin-2 in the LS in the mature mouse brain resulted in postsynaptic impairment of inhibitory synaptic transmission. Importantly, the reduced inhibition and resulting E/I imbalance decreased the responsiveness of LS neurons to stress. Furthermore, this E/I imbalance in the LS was associated with impaired stress-induced activation of downstream hypothalamic nuclei and reduced avoidance behavior of the animals in the elevated plus maze. ConclusionsOur results described the synaptic function of neuroligin-2 in the LS, uncovered a positive association between c-Fos–expressing neurons in the LS and downstream hypothalamic areas and avoidance behavior, and demonstrated that intact inhibitory synaptic transmission and proper E/I balance are required for the optimal functioning of this subcortical circuit.

Chronic stress produces enduring sex- and region-specific alterations in novel stress-induced c-Fos expression

Prolonged or repeated exposure to stress increases risk for a variety of psychological disorders, many of which are marked by dysfunction of corticolimbic brain regions. Notably, women are more likely than men to be diagnosed with these disorders, especially when onset of symptoms follows stressful life events. Using rodent models, investigators have recently begun to elucidate sex-specific changes in the brain and behavior that occur immediately following chronic stress. However, little is known regarding the lasting sequelae of chronic stress, as well as how potential changes may impact responsivity to future stressors. We recently demonstrated that male and female rats show different patterns of dendritic reorganization in medial prefrontal cortex in the days following chronic stress. Here, we examined the immediate and lasting effects of chronic restraint stress (CRS; 3 h/day, 10 days) on neuronal activation, across several corticolimbic brain regions, induced by novel acute stress exposure. Chronically stressed male and female rats were exposed to acute elevated platform stress (EPS) either 1 (CRS-EPS) or 7 (CRS-Rest-EPS) days after CRS. Compared to rats exposed to EPS only, significant reductions in acute stress-induced c-Fos expression were observed in the medial prefrontal cortex, hippocampus, and paraventricular nucleus of the hypothalamus (PVN) in CRS-EPS male rats, some of which persisted to 7 days post-stress. In contrast, we found little modulation of novel stress-induced c-Fos expression in CRS-EPS female rats. However, CRS-Rest-EPS female rats exhibited a significant enhancement of acute stress-induced neuronal activity in the PVN. Together, these data show that prior chronic stress produces sex- and region-specific alterations in novel stress-induced neuronal activation, which are dependent on the presence or absence of a rest period following chronic stress. These findings suggest that the post-stress rest period may give rise to sex-specific neuroadaptations to stress, which may underlie sex differences in stress susceptibility versus resilience.

Neuroinflammation Contributes to High Salt Intake-Augmented Neuronal Activation and Active Coping Responses to Acute Stress.

High dietary salt intake increases risk of stress-related neuropsychiatric disorders. Here, we explored the contribution of high dietary salt intake-induced neuroinflammation in key stress-responsive brain regions, the hypothalamic paraventricular nucleus and basolateral amygdala, in promoting exaggerated neuronal activation and coping behaviors in response to acute psychogenic stress. Mice that underwent high dietary salt intake exhibited increased active stress coping behaviors during and after an acute swim stress, and these were reduced by concurrent administration of minocycline, an inhibitor of microglial activation, without affecting body fluid hyperosmolality caused by high dietary salt intake. Moreover, minocycline attenuated high dietary salt intake-induced increases of paraventricular nucleus tumor necrosis factor-α, activated microglia (ionized calcium-binding adaptor molecule 1), and acute swim stress-induced neuronal activation (c-Fos). In the basolateral amygdala, similar effects were observed on ionized calcium-binding adaptor molecule 1+ and c-Fos+ counts, but not tumor necrosis factor-α levels. These data indicate that high dietary salt intake promotes neuroinflammation, increasing recruitment of neurons in key stress-associated brain regions and augmenting behavioral hyper-responsivity to acute psychological stress.

Both Basal and Acute Restraint Stress-Induced c-Fos Expression Is Influenced by Age in the Extended Amygdala and Brainstem Stress Centers in Male Rats.

The hypothalamus-pituitary-adrenal axis (HPA) is the main regulator of the stress response. The key of the HPA is the parvocellular paraventricular nucleus of the hypothalamus (pPVN) controlled by higher-order limbic stress centers. The reactivity of the HPA axis is considered to be a function of age, but to date, little is known about the background of this age-dependency. Sporadic literature data suggest that the stress sensitivity as assessed by semi-quantitation of the neuronal activity marker c-Fos may also be influenced by age. Here, we aimed at investigating the HPA activity and c-Fos immunoreactivity 2 h after the beginning of a single 60 min acute restraint stress in eight age groups of male Wistar rats. We hypothesized that the function of the HPA axis (i.e., pPVN c-Fos and blood corticosterone (CORT) level), the neuronal activity of nine stress-related limbic areas (i.e., magnocellular PVN (mPVN), medial (MeA), central (CeA), basolateral nuclei of the amygdala, the oval (ovBNST), dorsolateral (dlBNST), dorsomedial (dmBNST), ventral and fusiform (fuBNST) divisions of the bed nucleus of the stria terminalis (BNST)), and two brainstem stress centers such as the centrally projecting Edinger-Westphal nucleus (cpEW) and dorsal raphe nucleus (DR) show age dependency in their c-Fos response. The somatosensory barrel cortex area (S1) was evaluated to test whether the age dependency is specific for stress-centers. Our results indicate that the stress-induced rise in blood CORT titer was lower in young age reflecting relatively low HPA activity. All 12 stress-related brain areas showed c-Fos response that peaked at 2 months of age. The magnitude of c-Fos immunoreactivity correlated negatively with age in seven regions (MeA, CeA, ovBNST, dlBNST, dmBNST, fuBNST and pPVN). Unexpectedly, the CeA, ovBNST and cpEW showed a considerable basal c-Fos expression in 1-month-old rats which decreased with age. The S1 showed a U-shaped age-related dynamics in contrast to the decline observed in stress centers. We conclude that the age- and brain area dependent dynamics in stress-induced neuronal activity pattern may contribute to the age dependance of the stress reactivity. Further studies are in progress to determine the neurochemical identity of neurons showing age-dependent basal and/or stress-induced c-Fos expression.

C-Fos mapping of brain regions activated by multi-modal and electric foot shock stress

Real-world stressors are complex and multimodal, involving physical, psychological, and social dimensions. However, the brain networks that mediate stress responses to these stimuli need to be further studied. We used c-Fos mapping in mice to characterize brain circuits activated by exposure to a single episode of multimodal stress (MMS), and compared these to circuits activated by electric foot shocks (EFS). We focused on characterizing c-Fos activity in stress-relevant brain regions including the paraventricular nucleus (PVN) of the hypothalamus and the bed nucleus of the stria terminalis (BNST). We also assessed stress-induced activation of CRH-positive neurons in each of these structures. MMS and EFS activated an overlapping network of brain regions with a similar time course. c-Fos expression within the PVN and the BNST peaked 30–60 min after exposure to both MMS and EFS, and returned to baseline levels within 24 h. Quantification of c-Fos expression within BNST subregions revealed that while c-Fos expression peaked in all subregions 30–60 min after MMS and EFS exposure, the neuronal density of c-Fos expression was significantly higher in the dorsomedial and ventral BNST relative to the dorsolateral BNST. Our preliminary assessment indicated that a great majority of MMS or EFS-activated neurons in the PVN were CRH-positive (>87%); in contrast, about 6–35% of activated neurons in the BNST were CRH-positive. Our findings indicate that both MMS and EFS are effective at activating stress-relevant brain areas and support the use of MMS as an effective approach for studying multidimensional stress in animal models. The results also reveal that the PVN and BNST are part of a common neural circuit substrate involved in neural processing related to stress.

Regional Specific Modulation of Stress-Induced Neuronal Activation Associated with the PSD95/NOS Interaction Inhibitor ZL006 in the Wistar Kyoto Rat.

BackgroundTo determine brain areas involved in the antidepressant-related behavioral effects of the selective neuronal nitric oxide synthase inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) and experimental test compound 4-((3,5-dichloro-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid (ZL006), an inhibitor of the PSD of 95 kDa/neuronal nitric oxide synthase interaction in the N-methyl-D-aspartic acid receptor signalling pathway, regional specific expression of the neuronal activation marker c-FOS was assessed following exposure to the forced swimming test in the Wistar Kyoto rat.MethodsWistar Kyoto rats were subjected to a 15-minute swim pretest (pre-forced swimming test) period on day 1. At 24, 5, and 1 hour prior to the 5-minute test, which took place 24 hours following the pre-forced swimming test, animals were treated with TRIM (50 mg/kg; i.p.), ZL006 (10 mg/kg; i.p.), or saline vehicle (1 mL/kg i.p). Behavior was recorded during both pretest and test periods.ResultsBoth TRIM and ZL006 decreased immobility time in Wistar Kyoto rats in the forced swimming test. Exposure to the forced swimming test increased c-FOS immunoreactivity in the lateral septum, paraventricular nucleus of the hypothalamus, periaqueductal grey, dentate gyrus, and ventral CA1 of the hippocampus compared with non-forced swimming test-exposed controls. Forced swimming test-induced c-FOS immunoreactivity was further increased in the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus following treatment with TRIM or ZL006. By contrast, forced swimming test-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus and ventral CA1 following treatment with TRIM or ZL006. Exposure to the forced swimming test resulted in an increase in NADPH diaphorase staining in the paraventricular nucleus of the hypothalamus. This forced swimming test-induced increase was attenuated following treatment with ZL006 and points to the paraventricular nucleus as a brain region where ZL006 acts to attenuate forced swimming test-induced neuronal nitric oxide synthase activity while concomitantly regulating region specific neuronal activation associated with an antidepressant-related response.ConclusionsThis study identified a pattern of enhanced and reduced forced swimming test-related c-FOS immunoreactivity indicative of a regulated network where inhibition of nitric oxide coupled to the N-methyl-D-aspartic acid receptor leads to activation of the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus with concomitant inhibition of the hippocampus.

Co-localization of the cannabinoid type 1 receptor with corticotropin-releasing factor-containing afferents in the noradrenergic nucleus locus coeruleus: implications for the cognitive limb of the stress response.

The noradrenergic system has been shown to play a key role in the regulation of stress responses, arousal, mood, and emotional states. Corticotropin-releasing factor (CRF) is a primary mediator of stress-induced activation of noradrenergic neurons in the nucleus locus coeruleus (LC). The endocannabinoid (eCB) system also plays a key role in modulating stress responses, acting as an "anti-stress" neuro-mediator. In the present study, we investigated the cellular sites for interactions between the cannabinoid receptor type 1 (CB1r) and CRF in the LC. Immunofluorescence and high-resolution immunoelectron microscopy showed co-localization of CB1r and CRF in both the core and peri-LC areas. Semi-quantitative analysis revealed that 44% (208/468) of CRF-containing axon terminals in the core and 35% (104/294) in the peri-LC expressed CB1r, while 18% (85/468) of CRF-containing axon terminals in the core and 6.5% (19/294) in the peri-LC were presynaptic to CB1r-containing dendrites. In the LC core, CB1r + CRF axon terminals were more frequently of the symmetric (inhibitory) type; while in the peri-LC, a majority were of the asymmetric (excitatory) type. Triple label immunofluorescence results supported the ultrastructural analysis indicating that CB1r + CRF axon terminals contained either gamma amino butyric acid or glutamate. Finally, anterograde transport from the central nucleus of the amygdala revealed that CRF-amygdalar afferents projecting to the LC contain CB1r. Taken together, these results indicate that the eCB system is poised to directly modulate stress-integrative heterogeneous CRF afferents in the LC, some of which arise from limbic sources.

A mixed glucocorticoid/mineralocorticoid receptor modulator dampens endocrine and hippocampal stress responsivity in male rats

Aberrant glucocorticoid secretion is implicated in the pathophysiology of stress-related disorders (i.e., depression, anxiety). Glucocorticoids exert biological effects via mineralocorticoid (MR) and glucocorticoid (GR) receptors. Previous data from our laboratory indicate that GR antagonism/modulation (i.e., mifepristone, CORT 108297) regulate endocrine, behavioral, and central stress responses. Because of the dynamic interplay between MR and GR on HPA axis regulation and emotionality, compounds targeting both receptors are of interest for stress-related pathology. We investigated the effects of CORT 118335 (a dual selective GR modulator/MR antagonist) on endocrine, behavioral, and central (c-Fos) stress responses in male rats. Rats were treated for five days with CORT 118335, imipramine (positive control), or vehicle and exposed to restraint or forced swim stress (FST). CORT 118335 dampened corticosterone responses to both stressors, without a concomitant antidepressant-like effect in the FST. Imipramine decreased corticosterone responses to restraint stress; however, the antidepressant-like effect of imipramine in the FST was independent of circulating glucocorticoids. These findings indicate dissociation between endocrine and behavioral stress responses in the FST. CORT 118335 decreased c-Fos expression only in the CA1 division of the hippocampus. Imipramine decreased c-Fos expression in the basolateral amygdala and CA1 and CA3 divisions of the hippocampus. Overall, the data indicate differential effects of CORT 118335 and imipramine on stress-induced neuronal activity in various brain regions. The data also highlight a complex relationship between neuronal activation in stress and mood regulatory brain regions and the ensuing impact on endocrine and behavioral stress responses.

Stress-induced mast cell activation contributes to atherosclerotic plaque destabilization

Abstract Mast cells accumulate in the perivascular tissue during plaque progression and upon activation release pro-inflammatory cytokines and extracellular matrix degrading enzymes which fuel the inflammatory process and decrease lesion stability. Interestingly, mast cells have been shown to co-localize with peripheral neurons and contain receptors for various hormones and neuropeptides which are released upon exposure to stress. Therefore, we hypothesize that stress-induced neuronal activation of mast cells contributes to plaque destabilization. In apoE−/− mice the acute stress induced by 120′ restraint caused a significant increase in mast cell activation in the heart (37.3 ± 1.8% vs. 50.1 ± 4.9%). In parallel with a rise in serum corticosterone levels we observed a transient increase mast cell-specific β-hexosaminidase levels and the pro-inflammatory cytokine, interleukin-6 in the stressed mice compared to controls (55.9 ± 11.0 pg/ml vs. 29.6 ± 5.49 pg/ml). Subsequent characterization of atherosclerotic lesions in the aortic root revealed a significant reduction in lesion collagen content and a higher incidence of intraplaque hemorrhages, a hallmark of the vulnerable plaque (38.5% vs. 7.7%). Importantly, both these effects were reduced in mice treated with the mast cell stabilizer cromolyn and completely abolished in mast cell deficient (apoE−/−/Kit(W-sh/W-sh)) mice, thus strongly indicating the involvement of a mast cell-dependent response to stress in atherosclerotic plaque destabilization. We demonstrate that acute stress activates mast cells near atherosclerotic lesions and contributes to plaque destabilization, identifying acute stress as a risk factor for acute cardiovascular syndromes.

Mice selected for extremes in stress reactivity reveal key endophenotypes of major depression: A translational approach

Summary Clear evidence has linked dysregulated hypothalamus–pituitary–adrenocortical (HPA) axis function to the aetiology and pathophysiology of major depression (MD), as observed in the majority of patients. Increased stress reactivity and hyperactivity of the HPA axis seem characteristic for psychotic/melancholic depression, while the atypical subtype of depression has been connected with the opposing phenotypes. However, the underlying molecular-genetic mechanisms are poorly understood. In the present study, mouse lines selectively bred for extremes in stress reactivity (SR), i.e. presenting high (HR) or low (LR) corticosterone secretion in response to stressors, were used to characterise the molecular alterations on all levels of the HPA axis. Results were contrasted with clinical phenotypes of MD patients from the Munich Antidepressant Response Signature project, stratified according to their cortisol response in the Dex/CRH test. Distinct differences between HR and LR mice were found in the expression of HPA axis-related genes in the adrenals, pituitary and selected brain areas. Moreover, HR animals presented an enhanced adrenal sensitivity, increased stress-induced neuronal activation in the PVN and an overshooting Dex/CRH test response, whereas LR animals showed a blunted response in these paradigms. Interestingly, analogous neuroendocrine, morphometric, psychopathological and behavioural differences were observed between the respective high and low HPA axis responder groups of MD patients. Our findings suggests that (i) the SR mouse model can serve as a valuable tool to elucidate HPA axis-related mechanisms underlying affective disorders and (ii) a stratification of MD patients according to their HPA axis-related neuroendocrine function should be considered for clinical research and treatment.