Evidence that preimmunization with a heat-killed preparation of Mycobacterium vaccae reduces corticotropin-releasing hormone mRNA expression in the extended amygdala in a fear-potentiated startle paradigm.

Posttraumatic stress disorder (PTSD) is a trauma and stressor-related disorder that is characterized by dysregulation of glucocorticoid signaling, chronic low-grade inflammation, and impairment in the ability to extinguish learned fear. Corticotropin-releasing hormone (Crh) is a stress- and immune-responsive neuropeptide secreted from the paraventricular nucleus of the hypothalamus (PVN) to stimulate the hypothalamic-pituitary-adrenal (HPA) axis; however, extra-hypothalamic sources of Crh from the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) govern specific fear- and anxiety-related defensive behavioral responses. We previously reported that preimmunization with a heat-killed preparation of the immunoregulatory environmental bacterium Mycobacterium vaccae NCTC 11659 enhances fear extinction in a fear-potentiated startle (FPS) paradigm. In this follow-up study, we utilized an in situ hybridization histochemistry technique to investigate Crh, Crhr1, and Crhr2 mRNA expression in the CeA, BNST, and PVN of the same rats from the original study [Fox et al., 2017, Brain, Behavior, and Immunity, 66: 70-84]. Here, we demonstrate that preimmunization with M. vaccae NCTC 11659 decreases Crh mRNA expression in the CeA and BNST of rats exposed to the FPS paradigm, and, further, that Crh mRNA expression in these regions is correlated with fear behavior during extinction training. These data are consistent with the hypothesis that M. vaccae promotes stress-resilience by attenuating Crh production in fear- and anxiety-related circuits. These data suggest that immunization with M. vaccae may be an effective strategy for prevention of fear- and anxiety-related disorders.

Treatment with a heat-killed preparation of Mycobacterium vaccae after fear conditioning enhances fear extinction in the fear-potentiated startle paradigm.

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 anxiety disorders, affective disorders, and trauma-and stressor-related 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 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 is a soil-derived bacterium with immunoregulatory and anti-inflammatory properties that has been demonstrated to enhance fear extinction in the fear-potentiated startle paradigm when given prior to fear conditioning. To determine if immunization with M. vaccae after fear conditioning also has protective effects, adult male Sprague Dawley rats underwent fear conditioning on days -37 and -36 followed by immunizations (3x), once per week beginning 24 h following fear conditioning, with a heat-killed preparation of M. vaccae NCTC 11659 (0.1 mg, s.c., in 100 µl borate-buffered saline) or vehicle, and, then, 3 weeks following the final immunization, were tested in the fear-potentiated startle paradigm (n = 12 per group). Rats underwent fear extinction training on days 1 through 6 followed by spontaneous recovery 14 days later (day 20). Rats were euthanized on day 21 and brain tissue was sectioned for analysis of Tph2, Htr1a, Slc6a4, Slc22a3, and Crhr2 mRNA expression throughout the brainstem dorsal and median raphe nuclei. Immunization with M. vaccae did not affect fear expression on day 1. However, M. vaccae-immunized rats showed enhanced enhanced within-session fear extinction on day 1 and enhanced between-session fear extinction beginning on day 2, relative to vehicle-immunized controls. Immunization with M. vaccae and fear-potentiated startle had minimal effects on serotonergic gene expression when assessed 42 days after the final immunization. Together with previous studies, these data are consistent with the hypothesis that immunoregulatory strategies, such as immunization with M. vaccae, have potential for both prevention and treatment of trauma- and stressor-related psychiatric disorders.

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice.

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

Preimmunization with a heat-killed preparation of Mycobacterium vaccae enhances fear extinction in the fear-potentiated startle paradigm.

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.

Exercise reduces the anxiogenic effects of meta-chlorophenylpiperazine: The role of 5-HT2C receptors in the bed nucleus of the stria terminalis.

Introduction: Two weeks of voluntary exercise in group-housed mice produces a reduction in anxiety-like behaviors across a number of different measures, including a reduction in the anxiety levels typically produced by the anxiogenic serotonergic drug m-chlorophenylpiperazine (mCPP), an agonist at 5-HT2C/2b receptors. We have previously demonstrated that 2-weeks of voluntary exercise blunted the anxiogenic effects of systemic mCPP, and we have also shown that mCPP infused into the bed nucleus of the stria terminalis (BNST) is anxiogenic. Here we follow up on these reports. Methods: In Experiment 1 we infused several doses of mCPP into the BNST with or without the 5-HT2C antagonist SB242084. In Experiment 2, we administered mCPP into amygdala subregions and the dorsal hippocampus to investigate site specificity. In Experiment 4 we lesioned the BNST and subsequently infused mCPP systemically, and in Experiment 4 we used RNAscope® to assess BNST 5-HT2C transcripts following wheel running. Results: BNST mCPP infusion increased acoustic startle responding, which was by 5-HT2C antagonism, while neither mCPP infused into the amygdala nor hippocampus was anxiogenic. Lesions of the BNST prevented the anxiogenic effect of systemically administered mCPP. Lastly, exercise reduced 5-HT2C transcripts in the BNST. Discussion: These results suggest that the BNST is a critical site of action for the effects of exercise on mCPP. Together these data suggest that exercise may reduce 5-HT2C receptor function in the BNST, which may, in part, explain some of the anxiolytic effects associated with wheel running.

Exercise is associated with reduction in the anxiogenic effect of mCPP on acoustic startle.

Voluntary exercise has been associated with reduced anxiety across several animal models. Manipulation of central 5-HT can alter anxiety-like behaviors and administration of the 5-HT agonist metachlorophenylpiperazine (mCPP) increases anxiety in rodents and humans. To examine whether the anxiolytic effect of exercise is associated with an alteration in 5-HT systems, we examined the anxiogenic effect of mCPP in exercising and nonexercising mice. C57BL/6J mice were given 2 weeks of free access to either a functioning or nonfunctioning running wheel. Mice were then tested for acoustic startle following systemic injection of either 0, 0.1, 0.3, or 1 mg/kg of mCPP. Consistent with its anxiogenic properties, mCPP produced a dose-dependent increase in acoustic startle in nonexercising mice. However, this anxiogenic effect was blunted in exercising mice. These findings suggest that exercise may help to reduce anxiety by altering 5-HT systems, perhaps by down-regulating postsynaptic 5HT 2B/2C receptors.

The Microbiota, Immunoregulation, and Mental Health: Implications for Public Health.

The hygiene or "Old Friends" hypothesis proposes that the epidemic of inflammatory disease in modern urban societies stems at least in part from reduced exposure to microbes that normally prime mammalian immunoregulatory circuits and suppress inappropriate inflammation. Such diseases include but are not limited to allergies and asthma; we and others have proposed that the markedly reduced exposure to these Old Friends in modern urban societies may also increase vulnerability to neurodevelopmental disorders and stress-related psychiatric disorders, such as anxiety and affective disorders, where data are emerging in support of inflammation as a risk factor. Here, we review recent advances in our understanding of the potential for Old Friends, including environmental microbial inputs, to modify risk for inflammatory disease, with a focus on neurodevelopmental and psychiatric conditions. We highlight potential mechanisms, involving bacterially derived metabolites, bacterial antigens, and helminthic antigens, through which these inputs promote immunoregulation. Though findings are encouraging, significant human subjects' research is required to evaluate the potential impact of Old Friends, including environmental microbial inputs, on biological signatures and clinically meaningful mental health prevention and intervention outcomes.

Corticotropin-releasing factor-related peptides, serotonergic systems, and emotional behavior.

Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide that is involved in stress-related physiology and behavior, including control of the hypothalamic-pituitary-adrenal (HPA) axis. Members of the CRF family of neuropeptides, including urocortin 1 (UCN 1), UCN 2, and UCN 3, bind to the G protein-coupled receptors, CRF type 1 (CRF1) and CRF2 receptors. In addition, CRF binding protein (CRFBP) binds both CRF and UCN 1 and can modulate their activities. There are multiple mechanisms through which CRF-related peptides may influence emotional behavior, one of which is through altering the activity of brainstem neuromodulatory systems, including serotonergic systems. CRF and CRF-related peptides act within the dorsal raphe nucleus (DR), the major source for serotonin (5-HT) in the brain, to alter the neuronal activity of specific subsets of serotonergic neurons and to influence stress-related behavior. CRF-containing axonal fibers innervate the DR in a topographically organized manner, which may contribute to the ability of CRF to alter the activity of specific subsets of serotonergic neurons. CRF and CRF-related peptides can either increase or decrease serotonergic neuronal firing rates and serotonin release, depending on their concentrations and on the specific CRF receptor subtype(s) involved. This review aims to describe the interactions between CRF-related peptides and serotonergic systems, the consequences for stress-related behavior, and implications for vulnerability to anxiety and affective disorders.

Voluntary exercise improves both learning and consolidation of cued conditioned fear in C57 mice.

Exercise is associated with improved cognitive function in humans as well as improved learning across a range of tasks in rodents. Although these studies provide a strong link between exercise and learning, to date studies have largely focused on tasks that principally involve the hippocampus. However, exercise has been shown to produce alterations in other brain areas suggesting that the cognitive enhancing effects of exercise may be more general. Therefore we set out to examine the effects of voluntary exercise on cued Pavlovian fear conditioning, a form of learning that is critically dependent on the amygdala. In Experiment 1 we showed that mice given 2 weeks of access to a running wheel prior to tone and foot shock fear conditioning showed enhanced conditioned fear as measured by fear-potentiated startle. This effect was not the result of altered shock reactivity nor was it to due to reduced baseline startle amplitude in exercising mice. In subsequent experiments we sought to examine whether the enhanced cued conditioned fear was the result of an improvement in learning, consolidation or retrieval of conditioned fear. In separate groups of mice, two weeks of access to a running wheel was begun either prior to fear conditioning, immediately after fear conditioning (consolidation period) or 2 weeks after fear conditioning. Compared to sedentary mice, mice that exercised either prior to fear conditioning, or immediately after fear conditioning, showed enhanced cued conditioned fear. Fear conditioning was not enhanced in mice that began exercising 2 weeks after fear conditioning. Taken together these results suggest that voluntary exercise improves the learning and consolidation of cued conditioned fear but does not improve the retrieval or performance of conditioned fear. Because a great deal is known about the neural circuit for cued conditioned fear, it is now possible to examine the cellular, molecular and pharmacological changes associated with exercise in this well-understood neural circuit.

Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety.

Voluntary wheel running in rodents is associated with a number of adaptive behavioral and physiological effects including improved learning, reduction in stress-associated behaviors, neurogenesis, angiogenesis, increases in neurotrophic factors, and changes in several signaling molecules. Exercise has also been reported to reduce anxiety-like behaviors. However, other studies have failed to find an anxiolytic effect of exercise. The inconsistencies in the literature may contribute to the scarcity of data examining the physiological correlates of the anxiolytic effect of exercise. Here we show that 2 weeks of voluntary exercise in male C57 mice is associated with reduced anxiety as measured with acoustic startle, stress-induced hyperthermia, social interaction, light-enhanced startle, and some, but not all, measures in the open field. A great deal is known about the neural circuits underlying anxiety. Given the consistency of the anxiolytic effect of voluntary exercise across several measures, it is now possible to begin a systematic analysis of the physiological basis of the anxiolytic effect of exercise.