Changes in Gene Expression in the Locus Coeruleus-Amygdala Circuitry in Inhibitory Avoidance PTSD Model.

The locus coeruleus (LC)-amygdala circuit is implicated in playing a key role in responses to emotionally arousing stimuli and in the manifestation of post-traumatic stress disorder (PTSD). Here, we examined changes in gene expression of a number of important mediators of the LC-amygdala circuitry in the inhibition avoidance model of PTSD. After testing for basal acoustic startle response (ASR), rats were exposed to a severe footshock (1.5 mA for 10 s) in the inhibitory avoidance apparatus. They were given contextual situational reminders every 5 day for 25 days. Controls were treated identically but with the footshock inactivated. Animals were re-tested on second ASR and decapitated 1 h later. The shock group had enhanced hyperarousal and several changes in gene expression compared to controls. In the LC, mRNA levels of norepinephrine (NE) biosynthetic enzymes (TH, DBH), NE transporter (NET), NPY receptors (Y1R, Y2R), and CB1 receptor of endocannabinoid system were elevated. In the basolateral amygdala (BLA), there were marked reductions in gene expression for CB1, and especially Y1R, with rise for corticotropin-releasing hormone (CRH) system (CRH, CRH receptor 1), and no significant changes in the central amygdala. Our results suggest a fast forward mechanism in the LC-amygdala circuitry in the shock group.

Locus coeruleus response to single-prolonged stress and early intervention with intranasal neuropeptide Y.

Dysregulation of the central noradrenergic system is a core feature of post-traumatic stress disorder (PTSD). Here, we examined molecular changes in locus coeruleus (LC) triggered by single-prolonged stress (SPS) PTSD model at a time when behavioral symptoms are manifested, and the effect of early intervention with intranasal neuropeptide Y (NPY). Immediately following SPS stressors, male SD rats were administered intranasal NPY (SPS/NPY) or vehicle (SPS/V). Seven days later, TH protein, but not mRNA, was elevated in LC only of the SPS/V group. Although 90% of TH positive cells expressed GR, its levels were unaltered. Compared to unstressed controls, LC of SPS/V, but not SPS/NPY, expressed less Y2 receptor mRNA with more CRHR1 mRNA in subset of animals, and elevated corticotropin-releasing hormone (CRH) in central nucleus of amygdala. Following testing for anxiety on elevated plus maze (EPM), there were significantly increased TH, DBH and NPY mRNAs in LC of SPS-treated, but not previously unstressed animals. Their levels highly correlated with each other but not with behavioral features on EPM. Thus, SPS triggers long-term noradrenergic activation and higher sensitivity to mild stressors, perhaps mediated by the up-regulation influence of amygdalar CRH input and down-regulation of Y2R presynaptic inhibition in LC. Results also demonstrate the therapeutic potential of early intervention with intranasal NPY for traumatic stress-elicited noradrenergic impairments. Single-prolonged stress (SPS)-triggered long-term changes in the locus coeruleus/norepinephrine (LC/NE) system with increased tyrosine hydroxylase (TH) protein and CRH receptor 1(CRHR1) mRNA and lower neuropeptide Y receptor 2 (Y2R) mRNA levels as well as elevated corticotropin-releasing hormone (CRH) in the central nucleus of amygdala (CeA) that were prevented by early intervention with intranasal neuropeptide Y (NPY). SPS treatment led to increased sensitivity of LC to mild stress of elevated plus maze (EPM), with elevated mRNA for NE biosynthetic enzymes in subset of animals.

Regulation of nonclassical renin-angiotensin system receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.

The involvement of the nonclassical renin-angiotensin system (RAS) in the adrenomedullary response to stress is unclear. Therefore, we examined basal and immobilization stress (IMO)-triggered changes in gene expression of the classical and nonclassical RAS receptors in the rat adrenal medulla, specifically the angiotensin II type 2 (AT2) and type 4 (AT4) receptors, (pro)renin receptor [(P)RR], and Mas receptor (MasR). All RAS receptors were identified, with AT2 receptor mRNA levels being the most abundant, followed by the (P)RR, AT1A receptor, AT4 receptor, and MasR. Following a single IMO, AT2 and AT4 receptor mRNA levels decreased by 90 and 50%, respectively. Their mRNA levels were also transiently decreased by repeated IMO. MasR mRNA levels displayed a 75% transient decrease as well. Conversely, (P)RR mRNA levels were increased by 50% following single or repeated IMO. Because of its abundance, the function of the (P)RR was explored in PC-12 cells. Prorenin activation of the (P)RR increased phosphorylation of extracellular signal-regulated kinase 1/2 and tyrosine hydroxylase at Ser(31), likely increasing its enzymatic activity and catecholamine biosynthesis. Together, the broad and dynamic changes in gene expression of the nonclassical RAS receptors implicate their role in the intricate response of the adrenomedullary catecholaminergic system to stress.

Blockage of melanocortin-4 receptors by intranasal HS014 attenuates single prolonged stress-triggered changes in several brain regions.

Melanocortin receptor four (MC4R) is implicated in regulation of stress-related functions. We previously demonstrated that intranasal infusion of MC4R antagonist HS014, shortly before single prolonged stress (SPS) animal model of post-traumatic stress disorder, lessened the development of anxiety- and depression-like behavior depending on the dose. Here, we evaluated effects of HS014 on SPS-elicited changes in hypothalamic-pituitary-adrenal axis and expression of several genes of interest in mediobasal hypothalamus, hippocampus, and locus coeruleus. Rats were given intranasal infusion of HS014 (3.5 ng or 100 µg) and 30 min later subjected to SPS stressors. Short-term responses of HS014 rats in comparison with vehicle-treated, evident 30 min following SPS stressors, included smaller rise in plasma corticosterone (100 µg HS014), absence of induction of corticotrophin-releasing hormone mRNA in mediobasal hypothalamus and of mRNA for tyrosine hydroxylase and dopamine-ß hydroxylase in locus coeruleus. Long-term responses found 7 days after SPS stressors, included lower induction corticotrophin-releasing hormone mRNA levels in the mediobasal hypothalamus without effect on mRNAs for the glucocorticoid receptor (GR) and FK506-binding protein 51 (FKBP5), a component of GR co-chaperone complex; and no induction of GR protein in ventral hippocampus. Thus, antagonism of MC4R prior to SPS attenuates development of several abnormalities in gene expression in regions implicated in post-traumatic stress disorder. Blockade of brain melanocortine receptor 4 (MC4R) with intranasal infusion of the MC4R antagonist HS014 to rats prior to single prolonged stress (SPS) leads to faster termination of stress responses (30 min later) and prevents or attenuates SPS-triggered abnormal gene expression related to post-traumatic stress disorder (7 days later). Targeting of brain MC4R is a promising strategy to protect HPA axis, LC-NE (locus coeruleus-norepinephrine) systems and hippocampus from overstimulation.

Microbial composition, functionality, and stress resilience or susceptibility: unraveling sex-specific patterns.

BACKGROUND: Following exposure to traumatic stress, women are twice as likely as men to develop mood disorders. Yet, individual responses to such stress vary, with some people developing stress-induced psychopathologies while others exhibit resilience. The factors influencing sex-related disparities in affective disorders as well as variations in resilience remain unclear; however, emerging evidence suggests differences in the gut microbiota play a role. In this study, using the single prolonged stress (SPS) model of post-traumatic stress disorder, we investigated pre- and post-existing differences in microbial composition, functionality, and metabolites that affect stress susceptibility or resilience in each sex. METHODS: Male and female Sprague-Dawley rats were randomly assigned to control or SPS groups. Two weeks following SPS, the animals were exposed to a battery of behavioral tests and decapitated a day later. Based on their anxiety index, they were further categorized as SPS-resilient (SPS-R) or SPS-susceptible (SPS-S). On the day of dissection, cecum, and selected brain tissues were isolated. Stool samples were collected before and after SPS, whereas urine samples were taken before and 30 min into the SPS. RESULTS: Before SPS exposure, the sympathoadrenal axis exhibited alterations within male subgroups only. Expression of tight junction protein claudin-5 was lower in brain of SPS-S males, but higher in SPS-R females following SPS. Across the study, alpha diversity remained consistently lower in males compared to females. Beta diversity revealed distinct separations between male and female susceptible groups before SPS, with this separation becoming evident in the resilient groups following SPS. At the genus level, Lactobacillus, Lachnospiraceae_Incertae_Sedis, and Barnesiella exhibited sex-specific alterations, displaying opposing abundances in each sex. Additionally, sex-specific changes were observed in microbial predictive functionality and targeted functional modules both before and after SPS. Alterations in the microbial short-chain fatty acids (SCFAs), were also observed, with major and minor SCFAs being lower in SPS-susceptible males whereas branched-chain SCFAs being higher in SPS-susceptible females. CONCLUSION: This study highlights distinct pre- and post-trauma differences in microbial composition, functionality, and metabolites, associated with stress resilience in male and female rats. The findings underscore the importance of developing sex-specific therapeutic strategies to effectively address stress-related disorders. Highlights SPS model induces divergent anxiety and social behavioral responses to traumatic stress in both male and female rodents. SPS-resilient females displayed less anxiety-like behavior and initiated more interactions towards a juvenile rat than SPS-resilient males. Sex-specific pre-existing and SPS-induced differences in the gut microbial composition and predictive functionality were observed in susceptible and resilient rats. SPS-resilient males displayed elevated cecal acetate levels, whereas SPS-susceptible females exhibited heightened branched-chain SCFAs.

After experiencing traumatic stress, women are more likely than men to develop mood disorders like anxiety and depression. However, people's responses to trauma vary­some develop mental health issues while others remain resilient. Recent research suggests that the bacteria in the gut might play a role in these differences. In this study, using a rat model of post-traumatic stress disorder (PTSD), we investigated whether there are differences in gut bacteria between male and female rats before and after stress exposure. The study involved two groups of rats­one not exposed to stress (control) and the other exposed to a traumatic event (stressed). The rats' behavior was evaluated using different tests to determine who among the males and females were vulnerable to stress and who were resilient. We found that even before the stress, there were differences in the types of bacteria and their functions in the guts of male and female rats. These differences were also linked to how they responded to stress. Interestingly, the bacteria that were more abundant in resilient males were found to be more abundant in vulnerable females. Additionally, the traumatic stress affected these bacteria and the substances they produce differently in males and females. In essence, our study demonstrates that the types of gut bacteria, their functions, and their products contribute to stress resilience in different ways for male and female rats. This insight suggests that tailored treatments specifically targeting these differences could be specially effective in treating stress-related issues.

Stress-induced changes in gene expression of urocortin 2 and other CRH peptides in rat adrenal medulla: involvement of glucocorticoids.

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Effect of acetate supplementation on traumatic stress-induced behavioral impairments in male rats.

Gut microbiota and their metabolites have emerged as key players in the pathogenesis of neuropsychiatric disorders. Recently, we demonstrated that animals susceptible to Single Prolonged Stress (SPS) have an overall pro-inflammatory gut microbiota and significantly lower cecal acetate levels than SPS-resilient rats, which correlated inversely with the anxiety index. Here, we investigated whether the microbial metabolite, acetate, could ameliorate SPS-triggered impairments. Male rats were randomly divided into unstressed controls or groups exposed to SPS. The groups received continued oral supplementation of either 150 mM of sodium acetate or 150 mM of sodium chloride-matched water. Two weeks after SPS, a battery of behavioral tests was performed, and the animals were euthanized the following day. While not affecting the unstressed controls, acetate supplementation reduced the impact of SPS on body weight gain and ameliorated SPS-induced anxiety-like behavior and the impairments in social interaction, but not depressive-like behavior. These changes were accompanied by several beneficial effects of acetate supplementation. Acetate alleviated the stress response by reducing urinary epinephrine levels, induced epigenetic modification by decreasing histone deacetylase (HDAC2) gene expression, inhibited neuroinflammation by reducing the density of Iba1+ cells and the gene expression of IL-1ß in the hippocampus, and increased serum ß-hydroxybutyrate levels. The findings reveal a causal relationship between oral acetate treatment and mitigation of several SPS-induced behavioral impairments. Mechanistically, it impacted neuronal and metabolic pathways including changes in stress response, epigenetic modifications, neuroinflammation and showed novel link to ketone body production. The study demonstrates the preventive-therapeutic potential of acetate supplementation to alleviate adverse responses to traumatic stress.

Potential benefits of intranasal neuropeptide Y include sustained extinction of fear memory.

Compelling evidence in animals and humans from a variety of approaches demonstrate that neuropeptide Y (NPY) in the brain can provide resilience to development of many stress-elicited symptoms. Preclinical experiments demonstrated that delivery of NPY by intranasal infusion to rats shortly after single exposure to traumatic stress in the single prolonged stress (SPS) rodent model of post-traumatic stress disorder (PTSD) can prevent development of many relevant behavioral alterations weeks later, including heightened anxiety and depressive-like behavior. Here, we examined responses to intranasal NPY in the absence of stress to evaluate the safety profile. Rats were administered intranasal NPY (150 µg/rat) or equal volume of vehicle (distilled water), and 7 days later they were tested on the elevated plus maze (EPM) and forced swim test (FST). There was no significant difference in the number of entries or duration in the open or closed arms, or in their anxiety index. Defecation on the EPM and immobility on the FST, measures of anxiety and depressive-like behavior respectively, were similar in both groups. To further characterize potential benefits of intranasal NPY, its effect on fear memory and extinction, important features of PTSD, were examined. Intranasal administration of NPY at the time of the traumatic stress had a profound effect on fear conditioning a week later. It prevented the SPS-triggered impairment in the retention of extinguished behavior, both contextual and cued. The findings support the translation of non-invasive intranasal NPY delivery to the brain for PTSD-behaviors including impairments in sustained extinction of fear memories.

Differences in gut microbiota associated with stress resilience and susceptibility to single prolonged stress in female rodents.

Exposure to traumatic stress is a major risk factor for the development of neuropsychiatric disorders in a subpopulation of individuals, whereas others remain resilient. The determinants of resilience and susceptibility remain unclear. Here, we aimed to characterize the microbial, immunological, and molecular differences between stress-susceptible and stress-resilient female rats before and after exposure to a traumatic experience. Animals were randomly divided into unstressed controls (n = 10) and experimental groups (n = 16) exposed to Single Prolonged Stress (SPS), an animal model of PTSD. Fourteen days later, all rats underwent a battery of behavioral tests and were sacrificed the following day to collect different organs. Stool samples were collected before and after SPS. Behavioral analyses revealed divergent responses to SPS. The SPS treated animals were further subdivided into SPS-resilient (SPS-R) and SPS-susceptible (SPS-S) subgroups. Comparative analysis of fecal 16S sequencing before and after SPS exposure indicated significant differences in the gut microbial composition, functionality, and metabolites of the SPS-R and SPS-S subgroups. In line with the observed distinct behavioral phenotypes, the SPS-S subgroup displayed higher blood-brain barrier permeability and neuroinflammation relative to the SPS-R and/or controls. These results indicate, for the first time, pre-existing and trauma-induced differences in the gut microbial composition and functionality of female rats that relate to their ability to cope with traumatic stress. Further characterization of these factors will be crucial for understanding susceptibility and fostering resilience, especially in females, who are more likely than males to develop mood disorders.

Transcriptome profiles associated with resilience and susceptibility to single prolonged stress in the locus coeruleus and nucleus accumbens in male sprague-dawley rats.

Although most people are subjected to traumatic stress at least once in their lifetime, only a subset develop long-lasting, stress-triggered neuropsychiatric disorders, such as PTSD. Here we examined different transcriptome profiles within the locus coeruleus (LC) and nucleus accumbens (NAc) that may contribute to stress susceptibility. Sprague Dawley male rats were exposed to the single prolonged stress (SPS) model for PTSD. Two weeks later they were tested for their anxiety/avoidance behavior on the Elevated Plus Maze (EPM) and were divided into high and low anxiety-like subgroups. RNA (n = 5 per group) was subsequently isolated from LC and NAc and subjected to RNAseq. Transcriptome analysis was used to identify differentially-expressed genes (DEGs) which differed by at least 50 % with significance of 0.01. The LC had more than six times the number of DEGs than the NAc. Only one DEG was regulated similarly in both locations. Many of the DEGs in the LC were associated with morphological changes, including regulation of actin cytoskeleton, growth factor activity, regulation of cell size, brain development and memory, with KEGG pathway of regulation of actin cytoskeleton. The DEGs in the NAc were primarily related to DNA repair and synthesis, and differential regulation of cytokine production. The analysis identified MTPN (myotrophin) and NR3C1 (glucocorticoid receptor) as important upstream regulators of stress susceptibility in the LC. Overall the study provides new insight into molecular pathways in the LC and NAc that are associated with anxiety-like behavior triggered by stress susceptibility or resilience.

Resilience or susceptibility to traumatic stress: Potential influence of the microbiome.

Exposure to traumatic stress is a major risk factor for development of neuropsychiatric disorders in a sub-population of individuals, while others remain resilient. The mechanisms and contributing factors differentiating between these phenotypes are still unclear. We hypothesize that inter-individual differences in the microbial composition and function contribute to host resilience or susceptibility to stress-induced psychopathologies. The current study aimed to characterize gut microbial community before and after exposure to traumatic stress in an animal model of PTSD. Sprague-Dawley male rats were randomly divided into unstressed controls and experimental group subjected to Single Prolonged Stress (SPS). After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into resilient (SPS-R) and susceptible (SPS-S) cohorts. The animals were sacrificed after the last behavioral test and cecum, colon, hippocampus, and medial prefrontal cortex were dissected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3-V4 ribosomal DNA sequencing, whereas urine samples were collected before SPS, 90 min into immobilization and on the day of sacrifice to measure epinephrine and norepinephrine levels. Analyses of the fecal microbiota revealed significant differences in microbial communities and in their predictive functionality among the groups before and after SPS stressors. Before SPS, the SPS-S subgroup harbored microbiota with an overall pro-inflammatory phenotype, whereas SPS-R subgroup had microbiota with an overall anti-inflammatory phenotype, with predictive functional pathways enriched in carbohydrate and lipid metabolism and decreased in amino acid metabolism and neurodegenerative diseases. After SPS, the gut microbial communities and their predictive functionality shifted especially in SPS cohorts, with volatility at the genus level correlating inversely with Anxiety Index. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS-S subgroup having significantly lower levels of acetate, valerate and caproate. The levels of acetate inversely correlated with Anxiety Index. Interestingly, urinary epinephrine and norepinephrine levels were also higher in the SPS-S subgroup at baseline and during stress, indicative of an altered sympathoadrenal stress axis. Finally, shorter colon (marker of intestinal inflammation) and a lower claudin-5 protein expression (marker for increased blood brain barrier permeability) were observed in the SPS-S subgroup. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiogenic symptoms following traumatic stress.

Intranasal Neuropeptide Y as a Potential Therapeutic for Depressive Behavior in the Rodent Single Prolonged Stress Model in Females.

The susceptibility to stress-elicited disorders is markedly influenced by sex. Women are twice as likely as men to develop posttraumatic stress disorder (PTSD), depression, anxiety disorders, and social impairments following exposure to traumatic stress. However, most of the studies in animal models examining putative therapeutics for stress-triggered impairments, including single prolonged stress (SPS), were performed predominantly with males. Previous studies in males demonstrated that intranasal neuropeptide Y (NPY) can provide therapeutic relief of many SPS-triggered behaviors, but is ineffective in females at the same dose. Thus, females may need a higher dose of exogenous NPY to attain a therapeutically significant concentration since the overwhelming majority of studies found that NPY levels in females in many brain regions are lower than in male rodents. Here, we examined SPS as an appropriate model to elicit many PTSD-associated symptoms in females and whether intranasal NPY at higher doses than with males is able to alter the development of SPS-triggered behavioral impairments. Sprague-Dawley female rats were exposed to SPS only, or in a separate cohort after SPS stressors were immediately infused intranasally with one of several doses of NPY, starting with 600 µg/rat-four times the dose effective in males. In the third cohort of animals, females were infused intranasally with either 600 µg NPY, omarigliptin [a dipeptidyl peptidase IV (DPP4) inhibitor], or both right after the SPS stressors. After 19 days they were tested on several behavioral tests. SPS elicited significant depressive/despair like behavior on the forced swim test (FST), anxiety behavior on the elevated plus maze (EPM), as well as impaired social interaction. On the FST, there was a dose-response effect of intranasal NPY, with 1,200 µg, but not 600 µg, preventing the development of the SPS-elicited depressive-like behavior. The omarigliptin and 600 µg NPY combined treatment, but neither alone, was also sufficient at preventing depressive-like behavior on the FST. The results demonstrate that: (1) SPS elicits several behavioral manifestations of PTSD in females; (2) early intervention with a high dose of intranasal NPY has therapeutic potential also for females; and (3) NPY cleavage by DPP4 may play a role in the higher dose requirement for females.

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Systematic Review and Methodological Considerations for the Use of Single Prolonged Stress and Fear Extinction Retention in Rodents.

Posttraumatic stress disorder (PTSD) is a mental health condition triggered by experiencing or witnessing a terrifying event that can lead to lifelong burden that increases mortality and adverse health outcomes. Yet, no new treatments have reached the market in two decades. Thus, screening potential interventions for PTSD is of high priority. Animal models often serve as a critical translational tool to bring new therapeutics from bench to bedside. However, the lack of concordance of some human clinical trial outcomes with preclinical animal efficacy findings has led to a questioning of the methods of how animal studies are conducted and translational validity established. Thus, we conducted a systematic review to determine methodological variability in studies that applied a prominent animal model of trauma-like stress, single prolonged stress (SPS). The SPS model has been utilized to evaluate a myriad of PTSD-relevant outcomes including extinction retention. Rodents exposed to SPS express an extinction retention deficit, a phenotype identified in humans with PTSD, in which fear memory is aberrantly retained after fear memory extinction. The current systematic review examines methodological variation across all phases of the SPS paradigm, as well as strategies for behavioral coding, data processing, statistical approach, and the depiction of data. Solutions for key challenges and sources of variation within these domains are discussed. In response to methodological variation in SPS studies, an expert panel was convened to generate methodological considerations to guide researchers in the application of SPS and the evaluation of extinction retention as a test for a PTSD-like phenotype. Many of these guidelines are applicable to all rodent paradigms developed to model trauma effects or learned fear processes relevant to PTSD, and not limited to SPS. Efforts toward optimizing preclinical model application are essential for enhancing the reproducibility and translational validity of preclinical findings, and should be conducted for all preclinical psychiatric research models.

Membrane-initiated estradiol signaling increases tyrosine hydroxylase promoter activity with ER alpha in PC12 cells.

Tyrosine hydroxylase (TH) promoter activity is induced by 17beta-estradiol (E(2)) in PC12 cells expressing estradiol receptor-alpha (ERalpha) requiring a cAMP/calcium response element (CRE/CaRE) at -45. To examine whether membrane-initiated estradiol signaling is underlying this induction, cells co-transfected with TH reporter construct and ERalpha expression vector were exposed to membrane-impermeant estradiol conjugate (beta-estradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin, E(2)BSA). TH promoter activity was elevated by E(2)BSA in dose- and time-dependent manner. E(2)BSA also elicited rapid phosphorylation of CRE binding protein (CREB) and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E(2)BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E(2) dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E(2) or E(2)BSA. Blocking S-palmitoylation of ERalpha with C451A mutation and/or pre-treatment with 2-Bromopalmitate did not prevent but instead enhanced E(2) or E(2)BSA-elicited induction of TH promoter activity. These findings reveal, for the first time, that estradiol induction of TH gene transcription with ERalpha in PC12 cells involves membrane-initiated estradiol signaling, rapid activation of dual PKA/MEK signaling pathways, leading to CREB phosphorylation, acting at CRE/CaRE. The data demonstrate possible mechanism whereby estradiol affects catecholaminergic systems in vivo.

Vesicular monoamine transporters (VMATs) in adrenal chromaffin cells: stress-triggered induction of VMAT2 and expression in epinephrine synthesizing cells.

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.

Sex Differences in the Neuropeptide Y System and Implications for Stress Related Disorders.

The neuropeptide Y (NPY) system is emerging as a promising therapeutic target for neuropsychiatric disorders by intranasal delivery to the brain. However, the vast majority of underlying research has been performed with males despite females being twice as susceptible to many stress-triggered disorders such as posttraumatic stress disorder, depression, anorexia nervosa, and anxiety disorders. Here, we review sex differences in the NPY system in basal and stressed conditions and how it relates to varied susceptibility to stress-related disorders. The majority of studies demonstrate that NPY expression in many brain areas under basal, unstressed conditions is lower in females than in males. This could put them at a disadvantage in dealing with stress. Knock out animals and Flinders genetic models show that NPY is important for attenuating depression in both sexes, while its effects on anxiety appear more pronounced in males. In females, NPY expression after exposure to stress may depend on age, timing, and nature and duration of the stressors and may be especially pronounced in the catecholaminergic systems. Furthermore, alterations in NPY receptor expression and affinity may contribute to the sex differences in the NPY system. Overall, the review highlights the important role of NPY and sex differences in manifestation of neuropsychiatric disorders.

Effect of intranasal administration of neuropeptide Y and single prolonged stress on food consumption and body weight in male rats.

Emerging evidence indicates that intranasal delivery of neuropeptide Y (NPY) to the brain has therapeutic potential for management of stress-triggered neuropsychiatric disorders. Here we aimed to determine how intranasal administration of NPY, either before or immediately after, traumatic stress in single prolonged stress (SPS) rodent model of Post-traumatic stress disorder (PTSD) impacts food consumption and body weight. SPS stressors suppressed food consumption for at least two days in the vehicle-treated animals. When given prior to SPS stressors, intranasal NPY prevented the SPS-elicited reduction in food intake only for several hours afterwards. When given after the SPS stressors, under conditions shown to prevent behavioral and biochemical impairments, intranasal NPY had no effect on food intake. Although all groups showed circadian variation, the SPS-exposed rats ate less than unstressed animals during the dark (active) phase. Seven days after exposure to SPS stressors, there were no differences in food intake, although body weight was still lower than unstressed controls in all the experimental groups. Thus, traumatic stress has pronounced effect on food consumption during the rodent's active phase, and a prolonged effect on body weight. Single intranasal infusion of NPY, which was previously shown to prevent development of several PTSD associated behavioral and neuroendocrine impairments, did not elicit prolonged changes in stress triggered food consumption nor regulation of body weight.

Activation of NPY receptor subtype 1 by [D-His26]NPY is sufficient to prevent development of anxiety and depressive like effects in the single prolonged stress rodent model of PTSD.

The neuropeptide Y (NPY) system plays an important role in mediating resilience to the harmful effect of stress in post-traumatic stress disorder (PTSD). It can mediate its effects via several G-protein coupled receptors: Y1R, Y2R, Y4R and Y5R. To investigate the role of individual NPY receptors in the resilience effects of NPY to traumatic stress, intranasal infusion of either Y1R agonists [D-His26]NPY, [Leu31Pro34]NPY, Y2R agonist NPY (3-36) or NPY were administered to male Sprague-Dawley rats immediately following the last stressor of the single prolonged stress (SPS) protocol, a widely used PTSD animal model. After 7 or 14 days, effects of the treatments were measured on the elevated plus maze (EPM) for anxiety, in forced swim test (FST) for development of depressive-like or re-experiencing behavior, in social interaction (SI) test for impaired social behavior, and acoustic startle response (ASR) for hyperarousal. [D-His26]NPY, but not [Leu31Pro34]NPY nor NPY (3-36) Y2R, was effective in preventing the SPS-elicited development of anxiety. Y1R, but not Y2R agonists prevented development of depressive- feature on FST, with [D-His26]NPY superior to NPY. The results demonstrate that [D-His26]NPY was sufficient to prevent development of anxiety, social impairment and depressive symptoms, and has promise as an early intervention therapy following traumatic stress.

Neuropeptide Y and cannabinoids interaction in the amygdala after exposure to shock and reminders model of PTSD.

Modulation of cannabinoid and neuropeptide Y (NPY) receptors may offer therapeutic benefits for post-traumatic stress disorder (PTSD). In this study, we aimed to investigate the functional interaction between these systems in the basolateral amygdala (BLA) in a rat model of PTSD. Rats were exposed to the shock and reminders model of PTSD and tested for hyper arousal/PTSD- and depression-like behaviors 3 weeks later. Immediately after shock exposure rats were microinjected into the BLA with URB597, a selective inhibitor of fatty acid amide hydrolase (FAAH) that increases the levels of the endocannabinoid anandamide or with the NPY1 receptor agonist Leu31,Pro34-NPY (Leu). Intra-BLA URB597 prevented the shock/reminders-induced PTSD- behaviors (extinction, startle) and depression-behaviors (despair, social impairments). These preventing effects of URB597 on PTSD- and depression-like behaviors were shown to be mostly mediated by cannabinoid CB1 and NPY1 receptors, as they were blocked when URB597 was co-administered with a low dose of a CB1 or NPY1 receptor antagonist. Similarly, intra-BLA Leu prevented development of all the behaviors. Interestingly, a CB1 antagonist prevented the effects of Leu on despair and social behavior, but not the effects on extinction and startle. Moreover, exposure to shock and reminders upregulated CB1 and NPY1 receptors in the BLA and infralimbic prefrontal cortex and this upregulation was restored to normal with intra-BLA URB597 or Leu. The findings suggest that the functional interaction between the eCB and NPY1 systems is complex and provide a rationale for exploring novel therapeutic strategies that target the cannabinoid and NPY systems for stress-related diseases.