Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.

Effects of maternal separation on serotonergic systems in the dorsal and median raphe nuclei of adult male Tph2-deficient mice.

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.

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.