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.

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.

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.

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.

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.

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.

Preclinical findings on the potential of intranasal neuropeptide Y for treating hyperarousal features of PTSD.

Acoustic startle response (ASR) assesses hyperarousal, a core symptom of posttraumatic stress disorder (PTSD). Intranasal neuropeptide Y (NPY) administration was shown to prevent hyperarousal in single prolonged stress (SPS) rodent PTSD model. However, it is unclear how ASR itself alters responses to stress. Rats (A-S-A) were exposed to acoustic startle (AS) 1 day before SPS (ASR1) and 2 weeks afterward (ASR2). Other groups were exposed in parallel to either AS (A-A) or SPS or neither. SPS enhanced ASR2. In relevant brain areas, mRNA levels were determined by qRT-PCR. In mediobasal hypothalamus, AS or SPS each increased CRH mRNA levels without an additive effect. Exposure to AS appeared to dampen some responses to SPS. The SPS-triggered reduction of GR and FKBP5 gene expression was not observed in A-S-A group. In locus coeruleus, SPS increased CRHR1 and reduced Y2R mRNAs, but not in A-S-A group. In both regions, AS altered NPY receptor gene expression, which may mediate dampening responses to SPS. In second experiment, intranasal NPY administered 2 weeks after SPS reversed hyperarousal symptoms for at least 7 days. This study reveals important effects of AS on the NPY system and demonstrates that intranasal NPY elicits long-lasting reversal of traumatic stress-triggered hyperarousal.

Single prolonged stress PTSD model triggers progressive severity of anxiety, altered gene expression in locus coeruleus and hypothalamus and effected sensitivity to NPY.

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 µg/rat, but not 150 µg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder.

Single Prolonged Stress as a Prospective Model for Posttraumatic Stress Disorder in Females.

Sex plays an important role in susceptibility to stress triggered disorders. Posttraumatic Stress disorder (PTSD), a debilitating psychiatric disorder developed after exposure to a traumatic event, is two times more prevalent in women than men. However, the vast majority of animal models of PTSD, including single prolonged stress (SPS), were performed mostly with males. Here, we evaluated SPS as an appropriate PTSD model for females in terms of anxiety, depressive symptoms and changes in gene expression in the noradrenergic system in the brain. In addition, we examined intranasal neuropeptide Y (NPY) as a possible treatment in females. Female rats were subjected to SPS and given either intranasal NPY or vehicle in two separate experiments. In the first experiment, stressed females were compared to unstressed controls on forced swim test (FST) and for levels of expression of several genes in the locus coeruleus (LC) 12 days after SPS exposure. Using a separate cohort of animals, experiment two examined stressed females and unstressed controls on the elevated plus maze (EPM) and LC gene expression 7 days after SPS stressors. SPS led to increased anxiety-like behavior on EPM and depressive-like behavior on FST. Following FST, the rats displayed elevated tyrosine hydroxylase (TH), CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system. The expression level of these mRNAs was unchanged following EPM, except Y1R. Intranasal NPY at the doses shown to be effective in males, did not prevent development of depressive or anxiety-like behavior or molecular changes in the LC. The results indicate that while SPS could be an appropriate PTSD model for females, sex differences, such as response to NPY, are important to consider.

Potential of Intranasal Neuropeptide Y (NPY) and/or Melanocortin 4 Receptor (MC4R) Antagonists for Preventing or Treating PTSD.

There is a great need for effective treatment options for post-traumatic stress disorder (PTSD). Neuropeptide Y (NPY) is associated with resilience to traumatic stress. MC4R antagonists, such as HS014, also reduce response to stress. Both regulate stress-responsive systems - the hypothalamic-pituitary-axis (HPA) and the noradrenergic nervous system and their associated behaviors. Therefore, we examined if their intranasal delivery to brain could attenuate development of PTSD-related symptoms in single prolonged stress (SPS) rodent PTSD model. Three regimens were used: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, (3) therapeutic treatment when PTSD behaviors are manifested 1 wk or more after the traumatic stress. NPY delivered by regimen 1 or 2 prevented SPS-triggered elevation in anxiety, depressive-like behavior, and hyperarousal and reduced dysregulation of HPA axis. Hypothalamic CRH mRNA and GR in ventral hippocampus were significantly induced in vehicle- but not NPY-treated group. NPY also prevented hypersensitivity of LC/NE system to novel mild stressor and induction of CRH in amygdala. Some of these impairments were also reduced with HS014, alone or together with NPY. When given after symptoms were manifested (regiment 3), NPY attenuated anxiety and depressive behaviors. This demonstrates strong preclinical proof of concept for intranasal NPY, and perhaps MC4R antagonists, for non-invasive early pharmacological interventions for PTSD and comorbid disorders and possibly also as therapeutic strategy.

Glucocorticoid withdrawal affects stress-induced changes of urocortin 2 gene expression in rat adrenal medulla and brain.

Corticotropin-releasing factor is well known activator of the hypothalamic-pituitary-adrenocortical axis, that represents crucial system participating on stress response of the organism. Urocortins are members of the corticotropin-releasing factor family of peptides with proposed effects on neuroendocrine and behavioral stress response mechanisms. Urocortin 2, one of three known urocortins, is present in central and peripheral stress response system and its expression can be augmented by glucocorticoids. In the present study we have examined how glucocorticoid withdrawal affects urocortin 2 gene expression after acute immobilization in the adrenal medulla and selected brain areas in rats. We used pharmacological adrenalectomy to block synthesis of corticosterone. Our results show that the immobilization-induced rise in urocortin 2 mRNA levels in rat adrenal medulla was not inhibited by glucocorticoid withdrawal. On the other hand, observed changes in the brain indicate that the effect of stress and pharmacological adrenalectomy on urocortin 2 gene expression is site-specific. While in the paraventricular nucleus and locus coeruleus the immobilization induced rise of urocortin 2 was not inhibited by pharmacological adrenalectomy in the arcuate nucleus and central amygdala it was. Moreover, we have seen a significant depletion of urocortin 2 plasma levels after immobilization. The immobilization induced rise of urocortin 2 gene expression in rat adrenal medulla and brain areas regulating stress response pathways and preservation of its induction after adrenalectomy suggests a role of urocortin 2 in the neuroendocrine stress response of an organism. This article is protected by copyright. All rights reserved.

Cardiovascular responses to intranasal neuropeptide Y in single prolonged stress rodent model of post-traumatic stress disorder.

Delivery of neuropeptide Y (NPY) to the brain by intranasal administration shows promise as non-invasive means for preventing or treating PTSD symptoms. Here, radiotelemetry and echocardiography were used to determine effects of intranasal NPY on cardiovascular functions in absence and presence of stress. Male adult Sprague Dawley rats were implanted with radiotelemetric probes, and subjected to single prolonged stress (SPS), followed by intranasal vehicle (V) or NPY (150µg) under conditions shown to prevent development of many of the behavioral neuroendocrine and biochemical impairments. In both groups, mean arterial pressure (MAP) rose rapidly peaking at about 125mmHg, remaining near maximal levels for 1h. SPS also elicited robust rise in heart rate (HR) which was mitigated by intranasal NPY, and significantly lower than V-treated rats 12-50min after exposure to SPS stressors. In the first hr. after SPS, locomotor activity was elevated but only in the V-treated group. By 3h, MAP returned to pre-stress levels in both groups with no further change when monitored for 6days. HR remained elevated during the 6h remaining light phase after SPS. Subsequently HR was at pre-SPS levels during the remaining days. However dark phase HR was low following SPS, gradually recovered over 6days and was associated with reduced activity. When administered in the absence of further stress, intranasal NPY or V elicited similar much smaller, short-lived rises in MAP and HR. Echocardiography revealed no change in HR, stroke volume (SV) or cardiac output (Q) with intranasal NPY in the absence of stress. SPS led to reduced SV and Q but was not affected by NPY. Overall the results demonstrate no major cardiovascular effects of intranasal NPY and indicate possible benefit from transient amelioration of HR response in line with its translational potential to combat PTSD and comorbid impairments.