Inescapable Tail Shock Stress Research Articles

Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 11659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats

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.

P.3.c.003 - N-Acetyl cysteine as therapeutic intervention in a “two-hit” model of maternal inflammation and post-natal methamphetamine exposure

Both early-life stress and immune system activation in adulthood have been linked independently to depression in a number of studies. However, the relationship between early-life infection, which may be considered a “stressor”, and later-life depression has not been explored. We have reported that neonatal bacterial infection in rats leads to exaggerated brain cytokine production, as well as memory impairments, to a subsequent peripheral immune challenge in adulthood, and therefore predicted that stressor-induced depressive-like symptoms would be more severe in these rats as well. Rats treated on postnatal day 4 with PBS or Escherichia coli were as adults exposed to inescapable tailshock stress (IS), and then tested for sucrose preference, social exploration with a juvenile, and overall activity, 1, 3, 5, and 7 days following the stressor. Serum corticosterone and extracellular 5-HT within the basolateral amygdala were measured in a second group of rats in response to the IS. IS resulted in profound depressive-like behaviors in adult rats, but, surprisingly, rats that suffered a bacterial infection early in life had blunted corticosterone responses to the stressor and were remarkably protected from the depressive symptoms compared to controls. These data suggest that early-life infection should be considered within a cost/benefit perspective, in which outcomes in adulthood may be differentially protected or impaired. These data also suggest that the immune system likely plays a previously unsuspected role in “homeostatic” HPA programming and brain development, which may ultimately lend insight into the often-contradictory literature on cytokines, inflammation, and depression.

160. Prebiotic-associated changes in gut microbiota affect the stress-induced inflammatory response

Stressor exposure increases inflammatory (CINC-1. IL1β, IL6, IL10) and stress (Hsp72) proteins in the absence of a foreign antigen. Recent evidence suggests that compromise of intestinal barrier integrity and increased translocation of bacteria from the intestinal lumen may contribute to these effects. Bifidobacteria and Lactobacilli are commensal bacteria found in the gut and are associated with improved intestinal epithelial integrity and attenuated bacterial translocation following stress. Prebiotics, a form of non-digestible dietary fiber, can promote the growth of select beneficial bacteria in the gut. We therefore hypothesized that two prebiotics, Galactooligosaccharides (GOS) and Polydextrose (PDX), would increase Bifidobacterium spp. and Lactobacillus spp. and reduce the impact of stress on inflammatory and stress proteins. Juvenile Fisher rats (PND 24, 8/grp) were fed a diet containing GOS/PDX for 4 weeks and exposed to inescapable tail shock stress (100 1.5 mA tail shocks) or remained in their home cages. Mesenteric lymph nodes, spleen, adipose and plasma were immediately collected after IS. Fecal samples were collected on the fourth week prior to stress, and plated on media selective for Bifidobacterium spp. and Lactobacillus spp. Diets containing GOS/PDX increased Bifidobacterium spp. and Lactobacillus spp., reduced stress-induced Hsp72 responses, and had little effect on the inflammatory protein response. These data suggest that GOS/PDX alters aspects of the stress response perhaps by modulating the gut microbiota. Supported by Mead Johnson Pediatric Nutrition Institute.

155. Dietary prebiotics increase Bifidobacterium spp. and Lactobacillus spp. in the gut and promote stress resistance

Prebiotics, a form of non-digestible dietary fiber, can selectively promote the expansion of beneficial microbial species in the mammalian gut. Two common prebiotics, Galactooligosaccharides (GOS) and Polydextrose (PDX), can increase Bifidobacterium spp. and Lactobacillus spp., implicated in attenuating depressive and anxiety-like behavior and generating adaptive changes in brain circuits implicated in mood. We therefore tested whether GOS and PDX would attenuate anxiety and depressive-like behavior produced by stressor exposure. Juvenile Fisher rats (PND 24, n = 9/grp) were fed a diet containing GOS/PDX (7.0 g/kg each) for 9 and 4 weeks. Fecal samples were collected after four weeks on the diet and plated on Lactobacillus spp. and Bifidobacterium spp. specific media. Rats were then either exposed to inescapable tail shock stress (IS; 100 1.5 mA tail shocks; a stressor that reliably produces anxiety-like behavior) or remained undisturbed in their home cages (HCC). Subsequently, rats were tested for anxiety/depressive-like behavior using shock-elicited freezing and shuttle-box escape tests 24 h later. Fecal cultures confirmed that GOS/PDX increased Lactobacillus spp. and Bifidobacterium spp. within the gut and both 9 and 4 weeks of diet containing GOS/PDX protected against the behavioral consequences of IS. In conclusion, GOS/PDX increase Lactobacillus spp. and Bifidobacterium spp. within the gut and promote stress resistance in Juvenile Fisher rats. Supported by Mead Johnson Pediatric Nutrition Institute.

Differential effects of inescapable stress on locus coeruleus GRK3, alpha 2-adrenoceptor and CRF 1 receptor levels in learned helpless and non-helpless rats: A potential link to stress resilience

Exposure of rats to unpredictable, inescapable stress results in two distinct behaviors during subsequent escape testing. One behavior, suggestive of lack of stress resilience, is prolonged escape latency compared to non-stressed rats and is labeled learned helplessness (LH). The other behavior suggestive of stress resilience is normal escape latency and is labeled non-helpless (NH). This study examines the effects of unpredictable, inescapable tail-shock stress (TSS) on alpha 2-adrenoceptor (α 2A-AR) and corticotropin-releasing factor 1 receptor (CRF 1-R) regulation as well as protein levels of G protein-coupled receptor kinase 3 (GRK3), GRK2, tyrosine hydroxylase (TH) plus carbonylated protein levels in locus coeruleus (LC), amygdala (AMG), cortex (COR) and striatum (STR). In NH rats, α 2A-AR and CRF 1-R were significantly down-regulated in LC after TSS. No changes in these receptor levels were observed in the LC of LH rats. GRK3, which phosphorylates receptors and thereby contributes to α 2A-AR and CRF 1-R down-regulation, was reduced in the LC of LH but not NH rats. GRK2 levels were unchanged. In AMG, GRK3 but not GRK2 levels were reduced in LH but not NH rats, and receptor regulation was impaired in LH rats. In STR, no changes in GRK3 or GRK2 levels were observed. Finally, protein carbonylation, an index of oxidative stress, was increased in the LC and AMG of LH but not NH rats. We suggest that reduced stress resilience after TSS may be related to oxidative stress, depletion of GRK3 and impaired regulation of α 2A-AR and CRF 1-R in LC.

Bacterial infection early in life protects against stressor-induced depressive-like symptoms in adult rats

Both early-life stress and immune system activation in adulthood have been linked independently to depression in a number of studies. However, the relationship between early-life infection, which may be considered a "stressor", and later-life depression has not been explored. We have reported that neonatal bacterial infection in rats leads to exaggerated brain cytokine production, as well as memory impairments, to a subsequent peripheral immune challenge in adulthood, and therefore predicted that stressor-induced depressive-like symptoms would be more severe in these rats as well. Rats treated on postnatal day 4 with PBS or Escherichia coli were as adults exposed to inescapable tailshock stress (IS), and then tested for sucrose preference, social exploration with a juvenile, and overall activity, 1, 3, 5, and 7 days following the stressor. Serum corticosterone and extracellular 5-HT within the basolateral amygdala were measured in a second group of rats in response to the IS. IS resulted in profound depressive-like behaviors in adult rats, but, surprisingly, rats that suffered a bacterial infection early in life had blunted corticosterone responses to the stressor and were remarkably protected from the depressive symptoms compared to controls. These data suggest that early-life infection should be considered within a cost/benefit perspective, in which outcomes in adulthood may be differentially protected or impaired. These data also suggest that the immune system likely plays a previously unsuspected role in "homeostatic" HPA programming and brain development, which may ultimately lend insight into the often-contradictory literature on cytokines, inflammation, and depression.

Habitual physical activity facilitates stress-induced HSP72 induction in brain, peripheral, and immune tissues.

The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.

Role of extracellular HSP72 in acute stress-induced potentiation of innate immunity in active rats.

Acute stress can compromise acquired, and potentiate innate, immunity. Recent evidence suggests that the impact of stress on measures of immunity can be modulated by the physical activity status of the organism and that extracellular heat shock protein 72 (eHSP72) contributes to the activation of innate immunity produced by stress. Therefore, this study investigated whether physical activity status would impact the immunologically enhancing effects of stressor exposure [inescapable tail-shock stress (IS)] on innate immunity and whether changes in eHSP72 responses could play a role. Adult, male Fischer 344 rats lived with mobile (physically active) or immobile (sedentary) running wheels. After 6 wk, rats were exposed to IS or to no stress. Immediately after IS, all rats were injected subcutaneously with live Escherichia coli. Inflammation was assessed daily, and plasma eHSP72 was measured at various time points. Rats exposed to IS resolved their inflammation faster than nonstressed rats, but the beneficial impact of stress on recovery was greater in physically active rats. All rats had equal increases in circulating eHSP72 after IS. Splenocytes harvested from a separate cohort of nonstressed rats were cultured with eHSP72, and nitric oxide and cytokines were measured. Physically active rats responded to eHSP72 stimulation in vitro with a greater nitric oxide and cytokine response than sedentary rats. Thus physically active rats both recover faster than sedentary rats after bacterial challenge + IS exposure and demonstrate potentiated cellular responses to eHSP72 activation that could be important for bacterial recovery.

The influence of gender and the estrous cycle on learned helplessness in the rat

Although the etiology of clinical depression is unknown, women are more likely to suffer from major depressive disorder than men. In addition, in some women, there is a clear association between depression and specific phases of the menstrual cycle. Surprisingly little research has examined gender differences and the influences of the estrous cycle in this and other animal behavioral models of clinical depression. Learned helplessness is a valid animal model of stress-induced behavioral depression in which prior exposure to inescapable stress produces deficits in escape testing. Learned helplessness was studied in rats using an inescapable tail shock stress followed by a shuttle box test to determine escape latencies. Animals with mean escape latencies of ≥20 s after shuttle-box testing are defined as learned helpless. Males and normal cycling female rats in the estrus and diestrus II phases were studied. Female rats in the diestrus II phase had significantly higher escape latencies and exhibited a more helpless behavior than female rats in the estrus phase. Male rat escape latencies were intermediate between the two female phases. These results suggest a role for gonadal hormones in the development of stress-induced behavioral depression or ‘learned helplessness.’

249. The effects of gender and the estrous cycle on learned helplessness in the rat

Women are more likely to suffer from major depressive disorder than men. Moreover, in depressed women, mood often fluctuates during the menstrual cycle. Learned helplessness is a valid animal model of stress-induced behavioral depression. Few studies have examined gender differences and influences of the estrous cycle in this and other animal behavioral models of clinical depression. We therefore tested the hypothesis that estrogen protects against depression. Learned helplessness was studied in rats using a inescapable tail shock stress followed by a shuttle box test to determine escape latencies. Rats studied were normal cycling female rats in the estrus and diestrus II phases, male and ovariectomized female rats. β-estradiol (10 μg/ml, sc) and progesterone (0.5 mg/kg) were administered to OVX females 12 or 72 hours prior to inescapable tail shock. Diazepam (2 mg/kg) and allopregnanolone (0.5 mg/kg) were administered to diestrus II females thirty minutes prior to inescapable tail shock. Pentylenetetrazole (20 mg/kg, PTZ) was administered to estrus females 10 minutes prior to shuttle box testing. Female rats in the diestrus II phase were significantly more helpless than female rats in the estrus phase. Estradiol decreased the escape latencies of OVX females. Progesterone produced a time-dependent effect such that after 12 hours escape latencies were increased but after 72 hours escape latencies were decreased. Diazepam and allopregnanolone prevented learned helplessness in diestrus II females. PTZ induced learned helplessness in estrus females. These results indicate an influence of the estrous cycle on the development of learned helplessness.

Acute stressor exposure both suppresses acquired immunity and potentiates innate immunity.

Acute stressor exposure alters immune function. Rats exposed to inescapable tail shock stress (IS) generate less antibody to a benign, antigenic protein, keyhole limpet hemocyanin (KLH). The following studies examined the effect of IS on peritoneal cavity, spleen, and mesenteric lymph node cell number, interferon-gamma (IFN-gamma) production, and nitrite production. Rats were injected intraperitoneally with KLH (200 microg) or saline immediately before IS exposure and killed 0, 48, and 96 h after IS termination. KLH immunization resulted in elevated cell numbers and IFN-gamma levels 2-4 days later in nonstressed control rats. In contrast, rats exposed to IS failed to increase cell number and IFN-gamma levels in response to KLH. The T cell subpopulations affected were CD4 T cells, specifically the Th1-like subset. In addition, in rats exposed to IS + KLH, nitrite production was potentiated 2-4 days after stressor termination. IS had little effect on these measures in saline-injected rats. These data support the conclusion that exposure to IS suppresses the expansion of anti-KLH lymphocytes, possibly anti-KLH Th1 cells. In addition, stressor exposure potentiates the production of nitrite. Importantly, this potentiated response occurred only in KLH-immunized animals, suggesting that macrophages may be primed by stressor exposure and thus respond more vigorously to antigen. The potential links between these changes are discussed.

Learned helplessness: In vivo biogenic amine release in hypothalamus

Learned helplessness (LH) is a behavioral depression caused by inescapable stress that models some aspects of human depression. We are developing a neuronal map of learned helplessness, which involves multiple neurotransmitters in several limbic regions. In frontal cortex, dopamine (DA), serotonin (5-HT), and GABA modulate the development and maintenance of LH, while in hippocampus interactions between GABA and norepinephrine (NE) play the major role. In hypothalamus, other investigators have reported decreased 5-HT uptake into synaptosomes and decreased basal and K+-stimulated 5-HT release from tissue slices of LH rats, as well as decreased paroxetine and unchanged [3-receptor binding. We have now applied in vivo microdialysis to measuring DA, NE, and 5-HT simultaneously in hypothalamus or caudate of conscious, freely moving rats in a LH paradigm. Rats received 100 trials of inescapable tailshock stress, and after testing for LH, microdialysis probes were implanted into either caudate or lateral hypothalamus. One day later, perfusion was maintained with Ringer's solution until a stable baseline was obtained, then switched to high K +. In caudate, no differences were found in DA or 5-HT among any of the three experimental groups--LH, non-helpless (NH), or unstressed tested controls (TC). In hypothalamus, no differences were observed among experimental groups for DA or 5-HT; however, NE showed significantly higher levels of basal release in LH rats, compared to NH or TC. Interestingly, NH rats had significantly higher K+-stimulated NE release compared to the other two groups. For all the stressed rats, there was a significant positive correlation between shuttlebox escape latencies and basal NE release. These data suggest that the caudate may not be involved in the biogenic amine neurochemistry of LH, and that in the lateral hypothalamus, NE mechanisms predominate in this animal model of depression.

Learned helplessness sensitizes hippocampal norepinephrine to mild restress

A proportion of rats exposed to inescapable tailshock stress displayed a performance deficit, termed learned helplessness, in a subsequent shuttlebox avoidance task. The technique of in vivo microdialysis was used to determine hippocampal norepinephrine levels in learned helpless, nonhelpless and nonprestressed control rats. Similar basal norepinephrine levels were detected in samples between rat groups. Following an exposure to a milder form of inescapable shock, an increase in norepinephrine output was detected in learned helpless rats, which was significantly greater than nonhelpless, nonprestressed, or control animals. Thus, inescapable stress appears to sensitize the hippocampus to increase norepinephrine release in response to a subsequent smaller stressor. This hypersensitivity might underlie the avoidance impairment of learned helplessness. Therefore, the possibility exists that similar neurochemical changes may also be responsible for some of the symptoms of human posttraumatic stress disorder (PTSD), such as the poor coping associated with seemingly mild stress.

Learned helplessness and in vivo hippocampal norepinephrine release

Hippocampal norepinephrine release was measured using in vivo microdialysis in rats before and after exposure to inescapable tail shock stress and after testing for learned helplessness. Rats that did not develop learned helplessness after stress had higher basal norepinephrine release after stress than rats developing learned helplessness or than control rats. After the shuttlebox test for learned helplessness, K +-stimulated norepinephrine release was lower in learned helpless than in nonhelpless or control rats. These results confirm an important role for the hippocampal noradrenergic system in differential behavioral responses to stress.