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Stress-related psychiatric disorders including anxiety disorders, mood disorders, and trauma and stressor-related disorders, such as posttraumatic stress disorder (PTSD), affect millions of people world-wide each year. Individuals with stress-related psychiatric disorders have been found to have poor immunoregulation, increased proinflammatory markers, and dysregulation of fear memory. The "Old Friends" hypothesis proposes that a lack of immunoregulatory inputs has led to a higher prevalence of inflammatory disorders and stress-related psychiatric disorders, in which inappropriate inflammation is thought to be a risk factor. Immunization with a soil-derived saprophytic bacterium with anti-inflammatory and immunoregulatory properties, Mycobacterium vaccae NCTC 11659, can lower proinflammatory biomarkers, increase stress resilience, and, when given prior to or after fear conditioning in a rat model of fear-potentiated startle, enhance fear extinction. In this study, we investigated whether immunization with heat-killed M. vaccae NCTC 11659 would enhance fear extinction in contextual or auditory-cued fear conditioning paradigms and whether M. vaccae NCTC 11659 would prevent stress-induced exaggeration of fear expression or stress-induced resistance to extinction learning. Adult male Sprague Dawley rats were immunized with M. vaccae NCTC 11659 (subcutaneous injections once a week for three weeks), and underwent either: Experiment 1) one-trial contextual fear conditioning; Experiment 2) two-trial contextual fear conditioning; Experiment 3) stress-induced enhancement of contextual fear conditioning; Experiment 4) stress-induced enhancement of auditory-cued fear conditioning; or Experiment 5) stress-induced enhancement of auditory-cued fear conditioning exploring short-term memory. Immunizations with M. vaccae NCTC 11659 had no effect on one- or two-trial contextual fear conditioning or contextual fear extinction, with or without exposure to inescapable stress. However, inescapable stress increased resistance to auditory-cued fear extinction. Immunization with M. vaccae NCTC 11659 prevented the stress-induced increase in resistance to auditory-cued fear extinction learning. Finally, in an auditory-cued fear conditioning paradigm exploring short-term memory and fear acquisition, immunization with M. vaccae did not prevent fear acquisition, either with or without exposure to inescapable stress, consistent with the hypothesis that M. vaccae NCTC 11659 has no effect on fear acquisition but enhances fear extinction. These data are consistent with the hypothesis that increased immunoregulation following immunization with M. vaccae NCTC 11659 promotes stress resilience, in particular by preventing stress-induced resistance to fear extinction, and may be a potential therapeutic intervention for trauma- and stressor-related disorders such as PTSD.

Posttraumatic stress disorder (PTSD) imposes a significant burden on patients and communities. Although the microbiome-gut-brain axis has been proposed as a mediator or moderator of PTSD risk and persistence of symptoms, clinical data directly delineating the gut microbiome's relationship to PTSD are sparse. This study investigated associations between the gut microbiome and mental health outcomes in participants with PTSD (n = 79) and trauma-exposed controls (TECs) (n = 58). Diagnoses of PTSD, major depressive disorder (MDD), and childhood trauma were made using the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5), MINI International Neuropsychiatric Interview (MINI), and Childhood Trauma Questionnaire (CTQ), respectively. Microbial communities from stool samples were profiled using 16S ribosomal RNA gene V4 amplicon sequencing and tested for associations with PTSD-related variables of interest. Random forest models identified a consortium of four genera, i.e., a combination of Mitsuokella, Odoribacter, Catenibacterium, and Olsenella, previously associated with periodontal disease, that could distinguish PTSD status with 66.4% accuracy. The relative abundance of this consortium was higher in the PTSD group and correlated positively with CAPS-5 and CTQ scores. MDD diagnosis was also associated with increased relative abundance of the Bacteroidetes phylum. Current use of psychotropics significantly impacted community composition and the relative abundances of several taxa. Early life trauma may prime the microbiome for changes in composition that facilitate a pro-inflammatory cascade and increase the risk of development of PTSD. Future studies should rigorously stratify participants into healthy controls, TECs, and PTSD (stratified by psychotropic drug use) to explore the role of the oral-gut-microbiome-brain axis in trauma-related disorders. ispartof: EUROPEAN NEUROPSYCHOPHARMACOLOGY vol:56 pages:24-38 ispartof: location:Netherlands status: published

Chronic disruption of rhythms (CDR) impacts sleep and can result in circadian misalignment of physiological systems which, in turn, is associated with increased disease risk. Exposure to repeated or severe stressors also disturbs sleep and diurnal rhythms. Prebiotic nutrients produce favorable changes in gut microbial ecology, the gut metabolome, and reduce several negative impacts of acute severe stressor exposure, including disturbed sleep, core body temperature rhythmicity, and gut microbial dysbiosis. In light of previous compelling evidence that prebiotic diet broadly reduces negative impacts of acute, severe stressors, we hypothesize that prebiotic diet will also effectively mitigate the negative impacts of chronic disruption of circadian rhythms on physiology and sleep/wake behavior. Male, Sprague Dawley rats were fed diets enriched in prebiotic substrates or calorically matched control chow. After 5 weeks on diet, rats were exposed to CDR (12 h light/dark reversal, weekly for 8 weeks) or remained on undisturbed normal light/dark cycles (NLD). Sleep EEG, core body temperature, and locomotor activity were recorded via biotelemetry in freely moving rats. Fecal samples were collected on experimental days –33, 0 (day of onset of CDR), and 42. Taxonomic identification and relative abundances of gut microbes were measured in fecal samples using 16S rRNA gene sequencing and shotgun metagenomics. Fecal primary, bacterially modified secondary, and conjugated bile acids were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prebiotic diet produced rapid and stable increases in the relative abundances of Parabacteroides distasonis and Ruminiclostridium 5. Shotgun metagenomics analyses confirmed reliable increases in relative abundances of Parabacteroides distasonis and Clostridium leptum, a member of the Ruminiclostridium genus. Prebiotic diet also modified fecal bile acid profiles; and based on correlational and step-wise regression analyses, Parabacteroides distasonis and Ruminiclostridium 5 were positively associated with each other and negatively associated with secondary and conjugated bile acids. Prebiotic diet, but not CDR, impacted beta diversity. Measures of alpha diversity evenness were decreased by CDR and prebiotic diet prevented that effect. Rats exposed to CDR while eating prebiotic, compared to control diet, more quickly realigned NREM sleep and core body temperature (ClockLab) diurnal rhythms to the altered light/dark cycle. Finally, both cholic acid and Ruminiclostridium 5 prior to CDR were associated with time to realign CBT rhythms to the new light/dark cycle after CDR; whereas both Ruminiclostridium 5 and taurocholic acid prior to CDR were associated with NREM sleep recovery after CDR. These results support our hypothesis and suggest that ingestion of prebiotic substrates is an effective strategy to increase the relative abundance of health promoting microbes, alter the fecal bile acid profile, and facilitate the recovery and realignment of sleep and diurnal rhythms after circadian disruption.

Stress-related disorders, such as posttraumatic stress disorder (PTSD), are highly prevalent and often difficult to treat. In rodents, stress-related, anxiety-like defensive behavioral responses may be characterized by social avoidance, exacerbated inflammation, and altered metabolic states. We have previously shown that, in rodents, subcutaneous injections of a heat-killed preparation of the soil-derived bacterium Mycobacterium vaccae NCTC 11659 promotes stress resilience effects that are associated with immunoregulatory signaling in the periphery and the brain. In the current study, we sought to determine whether treatment with a heat-killed preparation of the closely related M. vaccae type strain, M. vaccae ATCC 15483, would also promote stress-resilience in adult male rats, likely due to biologically similar characteristics of the two strains. Here we show that immunization with either M. vaccae NCTC 11659 or M. vaccae ATCC 15483 prevents stress-induced increases in hippocampal interleukin 6 mRNA expression, consistent with previous studies showing that M. vaccae NCTC 11659 prevents stress-induced increases in peripheral IL-6 secretion, and prevents exaggeration of anxiety-like defensive behavioral responses assessed 24 h after exposure to inescapable tail shock stress (IS) in adult male rats. Analysis of mRNA expression, protein abundance, and flow cytometry data demonstrate overlapping but also unique effects of treatment with the two M. vaccae strains on immunological and metabolic signaling in the host. These data support the hypothesis that treatment with different M. vaccae strains may immunize the host against stress-induced dysregulation of physiology and behavior.

Chronic psychosocial stress is a risk factor for many mental disorders, including affective disorders, anxiety disorders, and trauma- and stressor-related disorders (i.e., posttraumatic stress disorder, PTSD). As these disorders are associated with an overreactive immune system and chronic low-grade inflammation, immunoregulatory approaches counterbalancing basal and/or stress-induced immune activation should be protective in this context. In support of this hypothesis, we recently demonstrated that repeated subcutaneous (s.c.) preimmunization with a heat-killed preparation of the immunoregulatory bacterium Mycobacterium vaccae (M. vaccae; National Collection of Type Culture (NCTC) 11659) promoted proactive stress coping and protected against stress-induced anxiety and intestinal pathology in a mouse model of chronic psychosocial stress. To induce development of a chronic anxiety-like state, the chronic subordinate colony housing (CSC) paradigm was used. Here we employed the CSC paradigm (start day 1) to confirm the stress-protective effects of repeated s.c. M. vaccae administrations prior to CSC exposure (days -21, -14, and -7) and to extend these findings to the stress-protective role of M. vaccae when administered repeatedly during CSC exposure (days 2, 8 and 15). As readouts we assessed the stress coping behavior on days 1, 8, and 15 and general and/or social anxiety-related behavior on days 19 (elevated plus-maze), 20 (open-field/novel object test), and day 21 (social preference/avoidance test) of CSC exposure. In line with our previous study, M. vaccae administered prior to CSC strongly promoted active stress coping and moderately reduced CSC-induced general and social anxiety. Although M. vaccae administered during CSC did not affect stress coping, this treatment protocol profoundly protected against CSC-induced general, and to a lesser extent social, anxiety. Taken together, these data broaden the framework for developing bioimmunoregulatory approaches, based on the administration of immunoregulatory microorganisms, for the prevention and/or treatment of affective disorders, anxiety disorders, and trauma- and stressor-related psychiatric disorders like PTSD.

An increasing body of evidence indicates that immunodysregulation and subsequent chronic low-grade inflammation can promote the development of stress-related somatic and psychiatric pathologies, including inflammatory bowel disease (IBD) and posttraumatic stress disorder (PTSD). Thus, immunoregulatory approaches counterbalancing basal and/or stress-induced immune activation should have stress-protective potential. In support of this hypothesis, we recently demonstrated that repeated s.c. preimmunization with a heat-killed preparation of the immunoregulatory bacterium Mycobacterium vaccae ( M. vaccae ; National Collection of Type Culture (NCTC) 11659), protects mice against stress-induced general anxiety, spontaneous colitis, and aggravation of dextran sulfate sodium (DSS)-induced colitis in the chronic subordinate colony housing (CSC) paradigm, a validated model for PTSD in male mice. In the current study, we repeatedly administered M. vaccae via the non-invasive intranasal (i.n.; 0.1 mg/mouse/administration) route, prior to or during CSC exposure or single housed control (SHC) conditions, and assessed the effects on general and social anxiety, and on parameters related to the severity of DSS-induced colitis. While administration of M. vaccae prior to the onset of CSC exposure only had minor stress-protective effects, administration of M. vaccae during CSC completely prevented CSC-induced aggravation of DSS colitis. As CSC in the current experimental setting did not reliably increase general anxiety-related behavior, potential stress-protective effects of M.vaccae are difficult to interpret. Taken together, these data broaden the framework for developing bioimmunoregulatory approaches, based on the administration of microorganisms with anti-inflammatory and immunoregulatory properties, for the prevention of stress-related disorders.

Most people spend the majority of their lives indoors. Research over the last thirty years has focused on investigating the mechanisms through which specific elements of the built environment, such as indoor air quality, influence the physical health of occupants. However, similar effort has not been expended in regard to mental health, a significant public health concern. One in five Americans has been diagnosed with a mental health disorder in the past year, and, in the United States, the number of suicide deaths are similar to the number of deaths due to breast cancer. Increases in mental health disorders in Western societies may be due, in part, to increased systemic inflammation, secondary to decreased exposures to a diverse microbial environment (i.e., the hygiene hypothesis, “Old Friends” hypothesis, “missing microbes” hypothesis, or biodiversity hypothesis), as well as increased environmental exposures that lead to chronic low-grade inflammation. In this review, we provide an assessment that integrates historical research across disciplines. We offer ten questions that highlight the importance of current lessons learned regarding the built environment and mental health, including a potential role for the microbiome of the built environment to influence mental health. Suggested areas for future investigation are also highlighted.

Organic cation transporter 3 (OCT3) is a corticosterone-sensitive, low-affinity, high-capacity transporter. This transporter functions, in part, to clear monoamines, including serotonin (5-HT), from the extracellular space. The central nucleus of the amygdala (CeA) is an important structure controlling fear- and anxiety-related behaviors. The CeA has reciprocal connections with brainstem nuclei containing monoaminergic systems, including serotonergic systems arising from the dorsal raphe nucleus, which are thought to play an important role in modulation of CeA-mediated behavioral responses. Organic cation transporter 3 (OCT3) is expressed in the CeA, but little is known about the role of OCT3 within the CeA in modulating serotonergic signaling. We hypothesized that inhibition of OCT3-mediated transport in the CeA during restraint stress would increase extracellular 5-HT. In Experiment 1, rats received unilateral reverse dialysis of either corticosterone or normetanephrine, which interfere with OCT3-mediated transport, into the CeA under home cage control conditions. In Experiment 2, rats received unilateral reverse dialysis of corticosterone, normetanephrine, or vehicle into the CeA, while undergoing a 40-min period of restraint stress. Infusion of these drugs had no effect on extracellular concentrations of 5-HT during home cage control conditions, but, in contrast, markedly increased extracellular concentrations of 5-HT during restraint stress, relative to vehicle-treated controls. These findings suggest a role for OCT3 in the CeA in control of serotonergic signaling during stressful conditions.

Autism spectrum disorder (ASD) is a heterogeneous and highly heritable condition with multiple aetiologies. Although the biological mechanisms underlying ASD are not fully understood, evidence suggests that dysregulation of serotonergic systems play an important role in ASD psychopathology. Preclinical models using mice with altered serotonergic neurotransmission may provide insight into the role of serotonin in behaviours relevant to clinical features of ASD. For example, BALB/c mice carry a loss-of-function single nucleotide polymorphism (SNP; C1473 G) in tryptophan hydroxylase 2 (Tph2), which encodes the brain-specific isoform of the rate-limiting enzyme for serotonin synthesis, and these mice frequently have been used to model symptoms of ASD. In this study, juvenile male BALB/c (G/G; loss-of-function variant) and C57BL/6 J (C/C; wild type variant) mice, were exposed to the three-chamber sociability test, and one week later to the elevated plus-maze (EPM). Tryptophan hydroxylase 2 (TPH2) activity was measured following injection of the aromatic amino acid decarboxylase (AADC)-inhibitor, NSD-1015, and subsequent HPLC detection of 5-hydroxytryptophan (5-HTP) within subregions of the dorsal raphe nucleus (DR) and median raphe nucleus (MnR). The BALB/c mice showed reduced social behaviour and increased anxious behaviour, as well as decreased 5-HTP accumulation in the rostral and mid-rostrocaudal DR. In the full cohort of mice, TPH2 activity in the mid-rostrocaudal DR was correlated with anxious behaviour in the EPM, however these correlations were not statistically significant within each strain, suggesting that TPH2 activity was not directly associated with either anxiety or sociability. Further research is therefore required to more fully understand how serotonergic systems are involved in mouse behaviours that resemble some of the clinical features of ASD.

Although the neurobiological mechanisms underlying autism spectrum disorder (ASD) are still unknown, dysregulation of serotonergic systems has been implicated in the etiology of ASD, and serotonergic antidepressant drugs are often prescribed to treat some symptoms of ASD. The BALB/c strain of mice express a dysregulated serotonergic system and a phenotype that is relevant to ASD. In this study, juvenile male BALB/c mice were exposed to the selective serotonin reuptake inhibitor fluoxetine either chronically (18 mg/kg/day in drinking water, post-natal day (PND) 28–39) or acutely (18 mg/kg, i.p.; PND40), or to vehicle control conditions (0.9% sterile saline, i.p.; PND40), prior to being exposed to the three-chambered sociability test (SAT; PND40). One cohort of mice then received an injection of the aromatic amino acid decarboxylase inhibitor, NSD-1015, and one hour later brain tissue was collected for quantification of 5-hydroxytryptophan accumulation in the dorsal raphe nucleus (DR) as a measure of TPH2 activity. For the second cohort, brain tissue was collected ninety minutes after the onset of the social phase of the SAT and prepared for immunohistochemical staining for c-Fos and TPH2 to measure the activation of serotonergic neurons within subregions of the DR. Acute fluoxetine decreased social behavior, while chronic fluoxetine increased social behavior compared with vehicle-treated controls. Furthermore, acute and chronic fluoxetine treatments were without effect on TPH2 activity but differentially affected populations of serotonergic neurons in the DR. These data are consistent with the hypothesis that serotonergic systems are implicated in social behavior that is relevant for ASD.

Flooding is the most common natural hazard, leading to property damage, injuries, and death. Despite the potential for major consequences, urban flooding remains difficult to forecast, largely due to a lack of data availability at fine spatial scales and associated predictive capabilities. Crowdsourcing of public webcams, social media, and citizen science represent potentially important data sources for obtaining fine-scale hydrological data, but also raise novel challenges related to data reliability and consistency. We provide a review of literature and analysis of existing databases regarding the availability and quality of these unconventional sources that then drives a discussion of their potential to support fine-grained urban flood modelling and prediction. Our review and analysis suggest that crowdsourced data are increasingly available in urban contexts and have considerable potential. Integration of crowdsourced data could help ameliorate quality and completeness issues in any one source. Yet, substantial weaknesses and challenges remain to be addressed.

The hygiene hypothesis or "Old Friends" hypothesis proposes that inflammatory diseases are increasing in modern urban societies, due in part to reduced exposure to microorganisms that drive immunoregulatory circuits, and a failure to terminate inappropriate inflammatory responses. Inappropriate inflammation is also emerging as a risk factor for trauma-related, anxiety, and affective disorders, including posttraumatic stress disorder (PTSD), which is characterized as persistent re-experiencing of the trauma after a traumatic experience. Traumatic experiences can lead to long-lasting fear memories and exaggerated fear potentiation of the acoustic startle reflex. The acoustic startle reflex is an ethologically relevant reflex and can be potentiated in both humans and rats through Pavlovian conditioning. Mycobacterium vaccae NCTC 11659 is a soil-derived bacterium with immunoregulatory and anti-inflammatory properties that has been demonstrated to confer stress resilience in mice. Here we immunized adult male Sprague Dawley rats 3×, once per week, with a heat-killed preparation of M. vaccae NCTC 11659 (0.1mg, s.c., in 100µl borate-buffered saline) or vehicle, and, then, 3weeks following the final immunization, tested them in the fear-potentiated startle paradigm; controls were maintained under home cage control conditions throughout the experiment (n=11-12 per group). Rats were tested on days 1 and 2 for baseline acoustic startle, received fear conditioning on days 3 and 4, and underwent fear extinction training on days 5-10. Rats were euthanized on day 11 and brain tissue was sectioned for analysis of mRNA expression for genes important in control of brain serotonergic signaling, including tph2, htr1a, slc6a4, and slc22a3, throughout the brainstem dorsal and median raphe nuclei. Immunization with M. vaccae had no effect on baseline acoustic startle or fear expression on day 5. However, M. vaccae-immunized rats showed enhanced between-session and within-session extinction on day 6, relative to vehicle-immunized controls. Immunization with M. vaccae and fear-potentiated startle altered serotonergic gene expression in a gene- and subregion-specific manner. These data are consistent with the hypothesis that immunoregulatory strategies, such as preimmunization with M. vaccae, have potential for prevention of stress- and trauma-related psychiatric disorders.

Chronic subordinate colony housing (CSC), an established mouse model for chronic psychosocial stress, promotes a microbial signature of gut inflammation, characterized by expansion of Proteobacteria, specifically Helicobacter spp., in association with colitis development. However, whether the presence of Helicobacter spp. during CSC is critically required for colitis development is unknown. Notably, during previous CSC studies performed at Regensburg University (University 1), male specific-pathogen-free (SPF) CSC mice lived in continuous subordination to a physically present and Helicobacter spp.-positive resident. Therefore, it is likely that CSC mice were colonized, during the CSC procedure, with Helicobacter spp. originating from the dominant resident. In the present study we show that employing SPF CSC mice and Helicobacter spp.-free SPF residents at Ulm University (University 2), results in physiological responses that are typical of chronic psychosocial stress, including increased adrenal and decreased thymus weights, decreased adrenal in vitro adrenocorticotropic hormone (ACTH) responsiveness, and increased anxiety-related behavior. However, in contrast to previous studies that used Helicobacter spp.-positive resident mice, use of Helicobacter spp.-negative resident mice failed to induce spontaneous colitis in SPF CSC mice. Consistent with the hypothesis that the latter is due to a lack of Helicobacter spp. transmission from dominant residents to subordinate mice during the CSC procedure, colonization of SPF residents with Helicobacter typhlonius at University 2, prior to the start of the CSC model, rescued the colitis-inducing potential of CSC exposure. Furthermore, using SPF CSC mice and H. typhlonius-free SPF residents at University 1 prevented CSC-induced colitis. In summary, our data support the hypothesis that the presence or absence of exposure to certain pathobionts contributes to individual variability in susceptibility to stress-/trauma-associated pathologies and to reproducibility of stress-related outcomes between laboratories.

Fluxes across the groundwater-surface water interface are spatially and temporally variable, difficult to observe and measure, and play a major role in regulating ecological habitat distributions, water quality, and water quantity. The long-term quantification of these fluxes is difficult as field conditions dictate when data can be collected. Ultimately there is a pressing necessity to quantify these long-term flow regimes, as impacts from a changing climate are altering the timing and extent of key groundwater-surface water interactions. The use of periodic temperature time series data is one method that can be utilized to capture these fluxes over long periods of time. Long term deployment of temperature sensors, continuously logging temperature time series data, can be leveraged into time-varying Darcy fluxes via time series analysis and the advection–dispersion equation. However, as hydrologic boundary conditions change, fluxes transition in both magnitude and direction and these temperature time series-based methods are less capable of accurately quantifying fluxes. Real world data taken from five existing United States Geological Survey paired stream gages and riparian groundwater wells sites were used as boundary conditions to inform a one-dimensional heat and mass transport model, with the simulated temperatures used to quantify Darcy fluxes through time. Comparing the Darcy fluxes found using Darcy's Law and the estimated Darcy fluxes from the use of heat as an environmental tracer, periodic temperature time series derived fluxes accurately matched those derived from Darcy's law at three of the sites. For the remaining two sites, hydrologic conditions resulted in erroneous flux estimates, allowing for the identification of specific conditions where temperature time series methods relying on a periodic signal cannot be applied.

A 7-year-old boy attended the emergency department after falling from a climbing frame onto his outstretched left wrist. On examination, there was mild swelling to the left elbow and tenderness to the antecubital fossa. There was also tenderness diffusely to the distal ulnar and radius. There was no neurovascular deficit. Radiographs revealed a plastic deformation fracture of the left radius and ulna, with dislocations of the ipsilateral radiocapitellar joint and distal radioulnar joint. A diagnosis of combined Monteggia and Galeazzi-type fractures of the left forearm was made. It is rare to find cases of combined Monteggia and Galeazzi fractures to the same forearm. Furthermore, to our knowledge, ipsilateral plastic deformation Monteggia and Galeazzi-type fractures in children have not been reported in the literature.

Anxiety and affective disorders are often associated with hypercortisolism and dysfunctional serotonergic systems, including increased expression of TPH2, the gene encoding the rate-limiting enzyme of neuronal serotonin synthesis. We previously reported that chronic glucocorticoid exposure is anxiogenic and increases rat Tph2 mRNA expression, but it was still unclear if this also translates to increased TPH2 protein levels and in vivo activity of the enzyme. Here, we found that adult male rats treated with corticosterone (CORT, 100 μg/ml) via the drinking water for 21 days indeed show increased TPH2 protein expression in the dorsal and ventral part of the dorsal raphe nucleus (DRD, DRV) during the light phase, abolishing the enzyme's diurnal rhythm. In a second study, we systemically blocked the conversion of 5-hydroxytryptophan (5-HTP) to serotonin immediately before rats treated with CORT or vehicle were either exposed to 30 min acoustic startle stress or home cage control conditions. This allowed us to measure 5-HTP accumulation as a direct readout of basal versus stress-induced in vivo TPH2 activity. As expected, basal TPH2 activity was elevated in the DRD, DRV and MnR of CORT-treated rats. In response to stress, a multitude of serotonergic systems reacted with increased TPH2 activity, but the stress-, anxiety-, and learned helplessness-related dorsal and caudal DR (DRD/DRC) displayed stress-induced increases in TPH2 activity only after chronic CORT-treatment. To address the mechanisms underlying this region-specific CORT-dependent sensitization, we stereotaxically implanted CORT-treated rats with cannulae targeting the DR, and pharmacologically blocked either corticotropin-releasing hormone receptor type 1 (CRHR1) or type 2 (CRHR2) 10 min prior to acoustic startle stress. CRHR2 blockade prevented stress-induced increases of TPH2 activity within the DRD/DRC, while blockade of CRHR1 potentiated stress-induced TPH2 activity in the entire DR. Stress-induced TPH2 activity in the DRD/DRC furthermore predicted TPH2 activity in the amygdala and in the caudal pontine reticular nucleus (PnC), while serotonin synthesis in the PnC was strongly correlated with the maximum startle response. Our data demonstrate that chronically elevated glucocorticoids sensitize stress- and anxiety-related serotonergic systems, and for the first time reveal competing roles of CRHR1 and CRHR2 on stress-induced in vivo serotonin synthesis.

The basolateral and lateral amygdala nuclei complex (BLC) is implicated in a number of emotional responses including conditioned fear and social anxiety. Based on previous studies demonstrating that enhanced serotonin release in the BLC leads to increased anxiety and fear responses, we hypothesized that pharmacologically depleting serotonin in the BLC using 5,7-dihydroxytryptamine (5,7-DHT) injections would lead to diminished anxiety and disrupted fear conditioning. To test this hypothesis, 5,7-DHT (a serotonin-depleting agent) was bilaterally injected into the BLC. Desipramine (a norepinephrine reuptake inhibitor) was systemically administered to prevent non-selective effects on norepinephrine. After 5 days, 5-7-DHT-treated rats showed increases in the duration of social interaction (SI) time, suggestive of reduced anxiety-like behavior. We then used a cue-induced fear conditioning protocol with shock as the unconditioned stimulus and tone as the conditioned stimulus for rats pretreated with bilateral 5,7-DHT, or vehicle, injections into the BLC. Compared to vehicle-treated rats, 5,7-DHT rats had reduced acquisition of fear during conditioning (measured by freezing time during tone), also had reduced fear retrieval/recall on subsequent testing days. Ex vivo analyses revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by ∼40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior and fear-associated memories through its actions within the BLC.

The bed nucleus of the stria terminalis (BNST) is a nodal structure in neural circuits controlling anxiety-related defensive behavioral responses. It contains neurons expressing the stress- and anxiety-related neuropeptide corticotropin-releasing hormone (Crh) as well as Crh receptors. Repeated daily subthreshold activation of Crh receptors in the BNST is known to induce a chronic anxiety-like state, but how this affects neurotransmitter-relevant gene expression in target regions of the BNST is still unclear. Since the BNST projects heavily to the dorsal raphe nucleus (DR), the main source of brain serotonin, we here tested the hypothesis that such repeated, anxiety-inducing activation of Crh receptors in the BNST alters the expression of serotonergic genes in the DR, including tph2, the gene encoding the rate-limiting enzyme for brain serotonin synthesis, and slc6a4, the gene encoding the serotonin transporter (SERT). For 5 days, adult male Wistar rats received daily, bilateral, intra-BNST microinjections of vehicle (1% bovine serum albumin in 0.9% saline, n = 11) or behaviorally subthreshold doses of urocortin 1 (Ucn1, n = 11), a potent Crh receptor agonist. Priming with Ucn1 increased tph2 and slc6a4 mRNA expression selectively within the anxiety-related dorsal part of the DR (DRD) and decreased social interaction (SI) time, a measure of anxiety-related defensive behavioral responses in rodents. Decreased social interaction was strongly correlated with increased tph2 mRNA expression in the DRD. Together with previous studies, our data are consistent with the hypothesis that Crh-mediated control of the BNST/DRD-serotonergic system plays a key role in the development of chronic anxiety states, possibly also contributing to stress-induced relapses in drug abuse and addiction behavior.

The Deakin/Graeff hypothesis proposes that different subpopulations of serotonergic neurons through topographically organized projections to forebrain and brainstem structures modulate the response to acute and chronic stressors, and that dysfunction of these neurons increases vulnerability to affective and anxiety disorders, including panic disorder. We outline evidence supporting the existence of a serotonergic system originally discussed by Deakin/Graeff that is implicated in the inhibition of panic-like behavioral and physiological responses. Evidence supporting this panic inhibition system comes from the following observations: (1) serotonergic neurons located in the ‘ventrolateral dorsal raphe nucleus’ (DRVL) as well as the ventrolateral periaqueductal gray (VLPAG) inhibit dorsal periaqueductal gray-elicited panic-like responses; (2) chronic, but not acute, antidepressant treatment potentiates serotonin's panicolytic effect; (3) contextual fear activates a central nucleus of the amygdala-DRVL/VLPAG circuit implicated in mediating freezing and inhibiting panic-like escape behaviors; (4) DRVL/VLPAG serotonergic neurons are central chemoreceptors and modulate the behavioral and cardiorespiratory response to panicogenic agents such as sodium lactate and CO2. Implications of the panic inhibition system are discussed.

Serotonergic neurons in the dorsal raphe nucleus (DR) are organized in anatomically distinct subregions that form connections with specific brain structures to modulate diverse behaviors, including anxiety-like behavior. It is unclear if the functional heterogeneity of these neurons is coupled to their developmental heterogeneity, and if abnormal development of specific DR serotonergic subregions can permanently impact anxiety circuits and behavior. The goal of this study was to examine if deficiencies in different components of fibroblast growth factor (Fgf) signaling could preferentially impact the development of specific populations of DR serotonergic neurons to alter anxiety-like behavior in adulthood. Wild-type and heterozygous male mice globally hypomorphic for Fgf8, Fgfr1, or both (Fgfr1/Fgf8) were tested in an anxiety-related behavioral battery. Both Fgf8- and Fgfr1/Fgf8-deficient mice display increased anxiety-like behavior as measured in the elevated plus-maze and the open-field tests. Immunohistochemical staining of a serotonergic marker, tryptophan hydroxylase (Tph), revealed reductions in specific populations of serotonergic neurons in the ventral, interfascicular, and ventrolateral/ventrolateral periaqueductal gray subregions of the DR in all Fgf-deficient mice, suggesting a neuroanatomical basis for increased anxiety-like behavior. Overall, this study suggests Fgf signaling selectively modulates the development of different serotonergic neuron subpopulations. Further, it suggests anxiety-like behavior may stem from developmental disruption of these neurons, and individuals with inactivating mutations in Fgf signaling genes may be predisposed to anxiety disorders.

Previous studies have highlighted interactions between serotonergic systems and adverse early life experience as important gene x environment determinants of risk of stress-related psychiatric disorders. Evidence suggests that mice deficient in Tph2, the rate-limiting enzyme for brain serotonin synthesis, display disruptions in behavioral phenotypes relevant to stress-related psychiatric disorders. The aim of this study was to determine how maternal separation in wild-type, heterozygous, and Tph2 knockout mice affects mRNA expression of serotonin-related genes. Serotonergic genes studied included Tph2, the high-affinity, low-capacity, sodium-dependent serotonin transporter (Slc6a4), the serotonin type 1a receptor (Htr1a), and the corticosterone-sensitive, low-affinity, high-capacity sodium-independent serotonin transporter, organic cation transporter 3 (Slc22a3). Furthermore, we studied corticotropin-releasing hormone receptors 1 (Crhr1) and 2 (Crhr2), which play important roles in controlling serotonergic neuronal activity. For this study, offspring of Tph2 heterozygous dams were exposed to daily maternal separation for the first two weeks of life. Adult, male wild-type, heterozygous, and homozygous offspring were subsequently used for molecular analysis. Maternal separation differentially altered serotonergic gene expression in a genotype- and topographically-specific manner. For example, maternal separation increased Slc6a4 mRNA expression in the dorsal part of the dorsal raphe nucleus in Tph2 heterozygous mice, but not in wild-type or knockout mice. Overall, these data are consistent with the hypothesis that gene x environment interactions, including serotonergic genes and adverse early life experience, play an important role in vulnerability to stress-related psychiatric disorders.

Advanced age is a major risk factor for developing postoperative cognitive dysfunction (POCD). Age-related neuroinflammatory “priming” may contribute to POCD: peripheral immune stimuli (e.g., infection or surgery) cause exaggerated pro-inflammatory responses in the aged brain that can elicit pathology. Exposure to micro-organisms with immunoregulatory and anti-inflammatory properties may be a promising strategy to quell neuroinflammatory priming. We hypothesized that immunization with Mycobacterium vaccae (M. vaccae; NCTC11659) would reduce neuroinflammation and cognitive impairments in aged rats post-surgery. Aged (24mos) and adult (3mos) male F344XBN rats received subcutaneous injections of heat-killed M. vaccae (3 injections, once per week) and then 5 days following the final M. vaccae injection rats underwent a laparotomy or sham (anesthesia control) procedure. Three days post-surgery, rats were trained in a fear conditioning paradigm or tissue was collected. Aged (but not young) rats showed post-operative memory deficits. Prophylactic treatment with M. vaccae protected aged rats from surgery induced-cognitive impairments. Furthermore, M. vaccae treatment shifted the aged pro-inflammatory hippocampal microenvironment (increased IL-1beta and NFKBIA) towards an anti-inflammatory phenotype (increased IL-4 and Arg1). Microglia may mediate the anti-inflammatory effects of M. vaccae in the brain: prior in vivo treatment with M. vaccae reduced hyperinflammatory responses to ex vivo lipopolysaccharide in microglia isolated from aged rats. Overall, our novel data suggest that M. vaccae can re-direct a primed neuroimmune environment in aged rats and prevent POCD.

Prior adverse experience alters behavioral responses to subsequent stressors. For example, exposure to a brief swim increases immobility in a subsequent swim test 24 h later. In order to determine if qualitative differences (e.g. 19 °C versus 25 °C) in an initial stressor (15 min swim) impact behavioral, physiological, and associated neural responses in a 5 min, 25 °C swim test 24 h later, rats were surgically implanted with biotelemetry devices one week prior to experimentation then randomly assigned to one of 6 conditions (Day 1 (15 min)/Day 2 (5 min)): 1) home cage (HC)/HC, 2) HC/25 °C swim, 3) 19 °C swim/HC, 4) 19 °C swim/25 °C swim, 5) 25 °C swim/HC, 6) 25 °C swim/25 °C swim. Core body temperature (Tb) was measured on Days 1 and 2 using biotelemetry; behavior was measured on Day 2. Rats were transcardially perfused with fixative 2 h following the onset of the swim on Day 2 for analysis of c-Fos expression in midbrain serotonergic neurons. Cold water (19 °C) swim on Day 1 reduced Tb, compared to both 25 °C swim and HC groups on Day 1, and, relative to rats exposed to HC conditions on Day 1, reduced the hypothermic response to the 25 °C swim on Day 2. The 19 °C swim on Day 1, relative to HC exposure on Day 1, increased immobility during the 5 min swim on Day 2. Also, 19 °C swim, relative to HC conditions, on Day 1 reduced swim (25 °C)-induced increases in c-Fos expression in serotonergic neurons within the dorsal (DRD) and interfascicular (DRI) parts of the dorsal raphe nucleus (DR). These results suggest that exposure to a 5 min 19 °C cold water swim, but not exposure to a 5 min 25 °C swim alters physiological, behavioral and serotonergic responses to a subsequent stressor.

Rats treated with three daily urocortin 1 (UCN) injections into the basolateral amygdala (BLA; i.e., UCN/BLA-primed rats) develop prolonged anxiety-associated behavior and vulnerability to panic-like physiological responses (i.e., tachycardia, hypertension and tachypnea) following intravenous infusions of 0.5 M sodium lactate (NaLac, an ordinarily mild interoceptive stressor). In these UCN-primed rats, the osmosensitive subfornical organ (SFO) may be a potential site that detects increases in plasma NaLac and mobilizes panic pathways since inhibiting the SFO blocks panic following NaLac in this model. Furthermore, since SFO neurons synthesize angiotensin II (A-II), we hypothesized that the SFO projects to the BLA and releases A-II to mobilizing panic responses in UCN/BLA-primed rats following NaLac infusions. To test this hypothesis, rats received daily bilateral injections of UCN or vehicle into the BLA daily for 3 days. Five to seven days following the intra-BLA injections, we microinjected either the nonspecific A-II type 1 (AT1r) and 2 (AT2r) receptor antagonist saralasin, or the AT2r-selective antagonist PD123319 into the BLA prior to the NaLac challenge. The UCN/BLA-primed rats pre-injected with saralasin, but not PD123319 or vehicle, had reduced NaLac-induced anxiety-associated behavior and panic-associated tachycardia and tachypnea responses. We then confirmed the presence of AT1rs in the BLA using immunohistochemistry which, combined with the previous data, suggest that A-II's panicogenic effects in the BLA is AT1r dependent. Surprisingly, the SFO had almost no neurons that directly innervate the BLA, which suggests an indirect pathway for relaying the NaLac signal. Overall these results are the first to implicate A-II and AT1rs as putative neurotransmitter-receptors in NaLac induced panic-like responses in UCN/BLA-primed rats.

Adverse early life experience is thought to increase an individual's susceptibility to mental health disorders, including anxiety and affective disorders, later in life. Our previous studies have shown that post-weaning social isolation of female rats during a critical period of development sensitizes an anxiety-related serotonergic dorsal raphe nucleus (DR) system in adulthood. Therefore, we investigated how post-weaning social isolation, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the GABAA receptor), affects home cage behavior and serotonergic gene expression in the DR of female rats using in situ hybridization histochemistry. Juvenile female rats were reared in isolation or groups of three for a 3-week period from weaning (postnatal day (PD) 21 to mid-adolescence (PD42)), after which all rats were group-reared for an additional 16 days until adulthood. Among vehicle-treated rats, isolation-reared rats had decreased rodent tryptophan hydroxylase 2 (tph2) mRNA expression in ventral and ventrolateral subdivisions of the DR, a pattern observed previously in a rat model of panic disorder. Isolation-reared rats, but not group-reared rats, responded to FG-7142 with increased duration of vigilance and arousal behaviors. In addition, FG-7142 decreased tph2 expression, measured 4h following treatment, in multiple subregions of the DR of group-reared rats but had no effect in isolation-reared rats. No treatment effects were observed on 5-HT1A receptor or serotonin transporter gene expression. These data suggest that adolescent social isolation alters tph2 expression in specific subregions of the DR and alters the effects of stress-related stimuli on behavior and serotonergic systems.

Previous studies have found that adolescent social isolation of rats can lead to an increased anxiety state during adulthood, while chronic anxiety states are associated with dysregulated local GABAergic inhibition within the basolateral amygdala (BL). Therefore, we investigated the effects of post-weaning social isolation of female rats, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142), on a subset of GABAergic interneurons in the BL in adulthood using dual immunohistochemical staining for c-Fos and parvalbumin. Juvenile female rats were reared in isolation or in groups of three for a 3-week period from weaning to mid-adolescence, after which all rats were group-housed for an additional 2 weeks. Group-reared rats and isolation-reared rats injected with FG-7142 had increased c-Fos expression in GABAergic interneurons in the anterior part of the BL compared to group-reared rats and isolation-reared rats, respectively, injected with vehicle. Isolation rearing had a main effect to decrease c-Fos expression in GABAergic interneurons in the anterior part of the BL compared to group-reared rats. These data suggest that post-weaning social isolation of female rats leads to dysregulation of a parvalbumin-containing subset of local GABAergic interneurons in the anterior part of the BL, which have previously been implicated in the pathophysiology of chronic anxiety states. These cellular changes may lead to an increased vulnerability to stress- and anxiety-related responses in adulthood.

Background Although the hypothalamic orexin system is known to regulate appetitive behaviors and promote wakefulness and arousal (Sakurai, 2007 [56] ), this system may also be important in adaptive and pathological anxiety/stress responses (Suzuki et al., 2005 [4]). In a recent study, we demonstrated that CSF orexin levels were significantly higher in patients experiencing panic attacks compared to non-panicking depressed subjects (Johnson et al., 2010 [9]). Furthermore, genetically silencing orexin synthesis or blocking orexin 1 receptors attenuated lactate-induced panic in an animal model of panic disorder. Therefore, in the present study, we tested if orexin (ORX) modulates panic responses and brain pathways activated by two different panicogenic drugs. Methods We conducted a series of pharmacological, behavioral, physiological and immunohistochemical experiments to study the modulation by the orexinergic inputs of anxiety behaviors, autonomic responses, and activation of brain pathways elicited by systemic injections of anxiogenic/panicogenic drugs in rats. Results We show that systemic injections of two different anxiogenic/panicogenic drugs (FG-7142, an inverse agonist at the benzodiazepine site of the GABA A receptor, and caffeine, a nonselective competitive adenosine receptor antagonist) increased c-Fos induction in a specific subset of orexin neurons located in the dorsomedial/perifornical (DMH/PeF) but not the lateral hypothalamus. Pretreating rats with an orexin 1 receptor antagonist attenuated the FG-7142-induced anxiety-like behaviors, increased heart rate, and neuronal activation in key panic pathways, including subregions of the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray and in the rostroventrolateral medulla. Conclusion Overall, the data here suggest that the ORX neurons in the DMH/PeF region are critical to eliciting coordinated panic responses and that ORX1 receptor antagonists constitute a potential novel treatment strategy for panic and related anxiety disorders. The neural pathways through which ORX1 receptor antagonists attenuate panic responses involve the extended amygdala, periaqueductal gray, and medullary autonomic centers.

Remote telemetry has a long history of use for collection of environmental measurements. With the rise of mobile phones and SMS text-messaging capacity, many members of the general pubic carry communications equipment in their pockets at all times. Enabling the general public to provide environmental data through text messages has the potential both to provide additional data to scientific projects and also to raise awareness of the projects through participation. Hydrologic measurements - some of which can be made without training, involve a single measurement, and are often made in rural areas - are well-suited to text-message conveyance. Many other environmental measurements are similarly well-suited for this technology. Social.Water is a software package, written in Python, that collects, parses, and categorizes text messages sent to a dedicated phone number, updates a simple database, and posts both graphical results and the database on the Web. Social.Water was designed as the backend to the Crowdhydrology project and is written in an object-oriented design that makes customization and modification straightforward.

HPA axis; Stress; ACTH; Adrenocorticotropin; Depression; Anxiety; Raphe; tph2; Tryptophan hydroxylase; Serotonin; 5-HT; Glucose Summary Both hypothalamic—pituitary—adrenal (HPA) axis activity and serotonergic systems are commonly dysregulated in stress-related psychiatric disorders. We describe here a non-invasive rat model for hypercortisolism, as observed in major depression, and its effects on physiology, behavior, and the expression of tph2, the gene encoding tryptophan hydroxylase 2, the rate-limiting enzyme for brain serotonin (5-hydroxytryptamine; 5-HT) synthesis. We delivered corticosterone (40 mg/ml, 100 mg/ml or 400 mg/ml) or vehicle to adrenal-intact adult, male rats via the drinking water for 3 weeks. On days 15, 16, 17 and 18, respectively, the rats’ emotionality was assessed in the open-field (OF), social interaction (SI), elevated plus-maze (EPM), and forced swim tests (FST). On day 21, half of the rats in each group were killed 2 h into the dark phase of a 12/12 h reversed light/ dark cycle; the other half were killed 2 h into the light phase. We then measured indices of HPA axis activity, plasma glucose and interleukin-6 (IL-6) availability, and neuronal tph2 expression at each time point. Chronic corticosterone intake was sufficient to cause increased anxiety- and depressivelike behavior in a dose-dependent manner. It also disrupted the diurnal pattern of plasma adrenocorticotropin (ACTH), corticosterone, and glucose concentrations, caused adrenal atrophy, and prevented regular weight gain. No diurnal or treatment-dependent changes were found for plasma concentrations of IL-6. Remarkably, all doses of corticosterone treatment abolished the diurnal variation of tph2 mRNA expression in the brainstem dorsal raphe nucleus (DR) by elevating the gene’s expression during the animals’ inactive (light) phase. Our data demonstrate that chronic elevation of corticosterone creates a vulnerability to a depression-like syndrome that is associated with increased tph2 expression, similar to that observed in depressed patients.

Our previous studies have shown that post-weaning social isolation of male rats leads to sensitization of serotonergic systems and increases in anxiety-like behavior in adulthood. Although studies in humans suggest that females have an increased sensitivity to stress and risk for the development of neuropsychiatric illnesses, most studies involving laboratory rats have focused on males while females have been insufficiently studied. The objective of this study was to investigate the effects of post-weaning social isolation on subsequent responses of an anxiety-related dorsal raphe nucleus (DR)-basolateral amygdala system to pharmacological challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the γ-aminobutyric acid (GABA)(A) receptor). Juvenile female rats were reared in isolation or in groups of three for a 3-week period from weaning to mid-adolescence, after which all rats were group-reared for an additional 2 weeks. We then used dual immunohistochemical staining for c-Fos and tryptophan hydroxylase in the DR or single immunohistochemical staining for c-Fos in the basolateral amygdala. Isolation-reared rats, but not group-reared rats, injected with FG-7142 had increased c-Fos expression within the basolateral amygdala and in serotonergic neurons in the dorsal, ventrolateral, caudal and interfascicular parts of the DR relative to appropriate vehicle-injected control groups. These data suggest that post-weaning social isolation of female rats sensitizes a DR-basolateral amygdala system to stress-related stimuli, which may lead to an increased sensitivity to stress- and anxiety-related responses in adulthood.

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24 h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.

Tryptophan hydroxylase is the rate-limiting enzyme in the biosynthesis of serotonin (5-hydroxytryptamine; 5-HT). Two isoforms of tryptophan hydroxylase, derived from different genes, tph1 and tph2, have been identified. The tph1 isoform is expressed in peripheral tissues, whereas tph2 is brain and neuron-specific. Recent studies suggest that tph2 expression and brain serotonin turnover are upregulated in depressed suicide patients, and drug-free depressed patients, respectively. Increased tph2 expression could result from genetic influences, early life developmental influences, adverse experience during adulthood, or interactions among these factors. Studies in rodents support the hypothesis that interactions between early life developmental influences and adverse experience during adulthood play an important role in determining tph2 expression. In this review, we highlight the evidence for the effects of adverse early life experience and stressful experience during adulthood on both tph1 and tph2 expression.