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.

Evidence for the Integration of Stress-Related Signals by the Rostral Posterior Hypothalamic Nucleus in the Regulation of Acute and Repeated Stress-Evoked Hypothalamo-Pituitary-Adrenal Response in Rat.

A likely adaptive process mitigating the effects of chronic stress is the phenomenon of stress habituation, which frequently reduces multiple stress-evoked responses to the same (homotypic) stressor experienced repeatedly. The current studies investigated putative brain circuits that may coordinate the reduction of stress-related responses associated with stress habituation, a process that is inadequately understood. Initially, two rat premotor regions that respectively regulate neuroendocrine (medial parvicellular region of the paraventricular hypothalamic nucleus [PaMP]) and autonomic (rostral medullary raphe pallidus [RPa]) responses were targeted with distinguishable retrograde tracers. Two to 3 weeks later, injected animals underwent loud noise stress, and their brains were processed for fluorescent immunohistochemical detection of the tracers and the immediate early gene Fos. A rostral region of the posterior hypothalamic nucleus (rPH), and to a lesser extent, the median preoptic nucleus, exhibited the highest numbers of retrogradely labeled cells from both the RPa and PaMP that were colocalized with loud noise-induced Fos expression. Injections of an anterograde tracer in the rPH confirmed these connections and suggested that this region may contribute to the coordination of multiple stress-related responses. This hypothesis was partially tested by posterior hypothalamic injections of small volumes of muscimol, which disrupts normal synaptic functions, before acute and repeated loud noise or restraint exposures. In addition to significantly reduced corticosterone release in response to these two distinct stressors, rPH muscimol disrupted habituation to each stressor modality, suggesting a novel and important contribution of the rostral posterior hypothalamic nucleus in this category of adaptive processes. Significance statement: Habituation to stress is a process that possibly diminishes the detrimental health consequences of chronic stress by reducing the amplitude of many responses when the same challenging conditions are experienced repeatedly. Stress elicits a highly coordinated set of neuroendocrine, autonomic, and behavioral responses that are independently and relatively well defined; however, how the brain achieves coordination of these responses and their habituation-related declines is not well understood. The current studies provide some of the first anatomical and functional results suggesting that a specific region of the hypothalamus, the rostral posterior hypothalamic nucleus, targets multiple premotor regions and contributes to the regulation of acute neuroendocrine responses and their habituation to repeated stress.

Early life diets with prebiotics and bioactive milk fractions attenuate the impact of stress on learned helplessness behaviours and alter gene expression within neural circuits important for stress resistance.

Manipulating gut microbes may improve mental health. Prebiotics are indigestible compounds that increase the growth and activity of health-promoting microorganisms, yet few studies have examined how prebiotics affect CNS function. Using an acute inescapable stressor known to produce learned helplessness behaviours such as failure to escape and exaggerated fear, we tested whether early life supplementation of a blend of two prebiotics, galactooligosaccharide (GOS) and polydextrose (PDX), and the glycoprotein lactoferrin (LAC) would attenuate behavioural and biological responses to stress later in life. Juvenile, male F344 rats were fed diets containing either GOS and PDX alone, LAC alone, or GOS, PDX and LAC. All diets altered gut bacteria, while diets containing GOS and PDX increased Lactobacillus spp. After 4 weeks, rats were exposed to inescapable stress, and either immediately killed for blood and tissues, or assessed for learned helplessness 24 h later. Diets did not attenuate stress effects on spleen weight, corticosterone and blood glucose; however, all diets differentially attenuated stress-induced learned helplessness. Notably, in situ hybridization revealed that all diets reduced stress-evoked cfos mRNA in the dorsal raphe nucleus (DRN), a structure important for learned helplessness behaviours. In addition, GOS, PDX and LAC diet attenuated stress-evoked decreases in mRNA for the 5-HT1A autoreceptor in the DRN and increased basal BDNF mRNA within the prefrontal cortex. These data suggest early life diets containing prebiotics and/or LAC promote behavioural stress resistance and uniquely modulate gene expression in corresponding circuits.

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.

Reversible inactivation of rostral nucleus raphe pallidus attenuates acute autonomic responses but not their habituation to repeated audiogenic stress in rats.

The medullary nucleus raphe pallidus (RPa) mediates several autonomic responses evoked by acute stress exposure, including tachycardia and hyperthermia. The present study assessed whether the RPa contributes to the decline/habituation of these responses observed during repeated audiogenic stress. Adult male rats were implanted with cannulae aimed at the RPa, and abdominal E-mitters that wirelessly acquire heart rate and core body temperature. After surgical recovery, animals were injected with muscimol or vehicle (aCSF) in the RPa region, followed by 30 min of 95-dBA loud noise or no noise control exposures on 3 consecutive days at 24-h intervals. Forty-eight hours after the third exposure, animals were exposed to an additional, but injection-free, loud noise or no noise test to assess habituation of hyperthermia and tachycardia. Three days later, rats were restrained for 30-min to evaluate their ability to display normal acute autonomic responses following the repeated muscimol injection regimen. The results indicated that the inhibition of cellular activity induced by the GABAA-receptor agonist muscimol centered in the RPa region reliably attenuated acute audiogenic stress-evoked tachycardia and hyperthermia, compared with vehicle-injected rats. Animals in the stress groups exhibited similar attenuated tachycardia and hyperthermia during the injection-free fourth audiogenic stress exposure, and displayed similar and robust increases in these responses to the subsequent restraint test. These results suggest that cellular activity in neurons of the RPa region is necessary for the expression of acute audiogenic stress-induced tachycardia and hyperthermia, but may not be necessary for the acquisition of habituated tachycardic responses to repeated stress.

Voluntary exercise during extinction of auditory fear conditioning reduces the relapse of fear associated with potentiated activity of striatal direct pathway neurons.

Relapse of previously extinguished fear presents a significant, pervasive obstacle to the successful long-term treatment of anxiety and trauma-related disorders. Thus, identification of a novel means to enhance fear extinction to stand the passage of time and generalize across contexts is of the utmost importance. Acute bouts of exercise can be used as inexpensive, noninvasive treatment strategies to reduce anxiety, and have been shown to enhance memory for extinction when performed in close temporal proximity to the extinction session. However, it is unclear whether acute exercise can be used to prevent relapse of fear, and the neural mechanisms underlying this potential effect are unknown. The current study therefore examined whether acute exercise during extinction of auditory fear can protect against the later relapse of fear. Male F344 rats lacking an extended history of wheel running were conditioned to fear a tone CS and subsequently extinguished within either a freely mobile running wheel, a locked wheel, or a control context lacking a wheel. Rats exposed to fear extinction within a freely mobile wheel ran during fear extinction, and demonstrated reduced fear as well as attenuated corticosterone levels during re-exposure to the extinguished CS during the relapse test in a novel context 1week later. Examination of cfos mRNA patterns elicited by re-exposure to the extinguished CS during the relapse test revealed that acute exercise during extinction decreased activation of brain circuits classically involved in driving fear expression and interestingly, increased activity within neurons of the direct striatal pathway involved in reward signaling. These data suggest that exercise during extinction reduces relapse through a mechanism involving the direct pathway of the striatum. It is suggested that a positive affective state could become associated with the CS during exercise during extinction, thus resulting in a relapse-resistant extinction memory.

Habituation of hypothalamic-pituitary-adrenocortical axis hormones to repeated homotypic stress and subsequent heterotypic stressor exposure in male and female rats.

Understanding potential sex differences in repeated stress-induced hypothalamic-pituitary-adrenocortical (HPA) axis habituation could provide insight into the sex-biased prevalence of certain affective disorders such as anxiety and depression. Therefore in these studies, male and female rats were exposed to 30 min of either audiogenic or restraint stress daily for 10 days in order to determine whether sex regulates the extent to which HPA axis hormone release is attenuated upon repeated homotypic stressor presentation. In response to the initial exposure, both stressors robustly increased plasma concentrations of both adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in both sexes. Acutely, females displayed higher ACTH and CORT concentrations following restraint stress, whereas males exhibited higher hormone concentrations following loud noise stress. HPA axis hormone responses to both stressors decreased incrementally over successive days of exposure to each respective stressor. Despite the differential effect of sex on acute hormone responses, the extent to which HPA axis hormone response was attenuated did not differ between male and female animals following either stressor. Furthermore, ACTH and CORT responses to a novel environment were not affected by prior exposure to stress of either modality in either male or female rats. These experiments demonstrate that despite the acute stress response, male and female rats exhibit similar habituation of HPA axis hormones upon repeated homotypic stressor presentations, and that exposure to repeated stress does not produce exaggerated HPA axis hormone responses to a novel environment in either female or male rats.

Stressor-specific effects of sex on HPA axis hormones and activation of stress-related neurocircuitry.

Experiencing stress can be physically and psychologically debilitating to an organism. Women have a higher prevalence of some stress-related mental illnesses, the reasons for which are unknown. These experiments explore differential HPA axis hormone release in male and female rats following acute stress. Female rats had a similar threshold of HPA axis hormone release following low intensity noise stress as male rats. Sex did not affect the acute release, or the return of HPA axis hormones to baseline following moderate intensity noise stress. Sensitive indices of auditory functioning obtained by modulation of the acoustic startle reflex by weak pre-pulses did not reveal any sexual dimorphism. Furthermore, male and female rats exhibited similar c-fos mRNA expression in the brain following noise stress, including several sex-influenced stress-related regions. The HPA axis response to noise stress was not affected by stage of estrous cycle, and ovariectomy significantly increased hormone release. Direct comparison of HPA axis hormone release to two different stressors in the same animals revealed that although female rats exhibit robustly higher HPA axis hormone release after restraint stress, the same effect was not observed following moderate and high intensity loud noise stress. Finally, the differential effect of sex on HPA axis responses to noise and restraint stress cannot readily be explained by differential social cues or general pain processing. These studies suggest the effect of sex on acute stress-induced HPA axis hormone activity is highly dependent on the type of stressor.

Little exercise, big effects: reversing aging and infection-induced memory deficits, and underlying processes.

We have previously found that healthy aged rats are more likely to suffer profound memory impairments following a severe bacterial infection than are younger adult rats. Such a peripheral challenge is capable of producing a neuroinflammatory response, and in the aged brain this response is exaggerated and prolonged. Normal aging primes, or sensitizes, microglia, and this appears to be the source of this amplified inflammatory response. Among the outcomes of this exaggerated neuroinflammatory response are impairments in synaptic plasticity and reductions of brain-derived neurotrophic factor (BDNF), both of which have been associated with cognitive impairments. Since it has been shown that physical exercise increases BDNF mRNA in the hippocampus, the present study examined voluntary exercise in 24-month-old F344×BN rats as a neuroprotective therapeutic in our bacterial infection model. Although aged rats ran only an average of 0.7 km per week, this small amount of exercise was sufficient to completely reverse infection-induced impairments in hippocampus-dependent long-term memory compared with sedentary animals. Strikingly, exercise prevented the infection-induced exaggerated neuroinflammatory response and the blunted BDNF mRNA induction seen in the hippocampus of sedentary rats. Moreover, voluntary exercise abrogated age-related microglial sensitization, suggesting a possible mechanism for exercise-induced neuroprotection in aging.

Effects of voluntary wheel running on heart rate, body temperature, and locomotor activity in response to acute and repeated stressor exposures in rats.

Stress often negatively impacts physical and mental health but it has been suggested that voluntary physical activity may benefit health by reducing some of the effects of stress. The present experiments tested whether voluntary exercise can reduce heart rate, core body temperature and locomotor activity responses to acute (novelty or loud noise) or repeated stress (loud noise). After 6 weeks of running-wheel access, rats exposed to a novel environment had reduced heart rate, core body temperature, and locomotor activity responses compared to rats housed under sedentary conditions. In contrast, none of these measures were different between exercised and sedentary rats following acute 30-min noise exposures, at either 85 or 98 dB. Following 10 weeks of running-wheel access, both groups displayed significant habituation of all these responses to 10 consecutive daily 30-min presentations of 98 dB noise stress. However, the extent of habituation of all three responses was significantly enhanced in exercised compared to sedentary animals on the last exposure to noise. These results suggest that in physically active animals, under some conditions, acute responses to stress exposure may be reduced, and response habituation to repeated stress may be enhanced, which ultimately may reduce the negative and cumulative impact of stress.

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.

Long-term habituation to repeated loud noise is impaired by relatively short interstressor intervals in rats.

The phenomenon of spaced (longer intertrial interval) compared with massed (shorter intertrial interval) training leading to better long-term habituation and associative learning is well documented. However, the effects of intertrial intervals on response habituation to repeated stress exposures have not been previously examined. The present experiments found that massed (six 30-min exposures of 95 dB white noise in 6 hr) and spaced (one 30-min exposure daily for 6 days) noise exposures led to similar habituation of plasma corticosterone and ACTH responses, heart rate, and core body temperature after the 6th exposure in male Sprague-Dawley rats. However, these habituated responses were not retained in the massed group on a similar noise re-exposure 48 hr later, compared with the spaced group. The habituated responses found in the massed group after the 6 noise exposures were not due to differential hearing threshold shifts, as examined with modifications of the acoustic startle reflex. These data indicate that relatively short interstressor intervals impair long-term stress adaptation. This series of studies supports the idea of distinct short- and long-term habituation processes to stress responsiveness.

Chronic voluntary wheel running facilitates corticosterone response habituation to repeated audiogenic stress exposure in male rats.

Voluntary exercise is associated with the prevention and treatment of numerous physical and psychological illnesses, yet the mechanisms by which it confers this protection remain unclear. In contrast, stress, particularly under conditions of prolonged or repeated exposure when glucocorticoid levels are consistently elevated, can have a devastating impact on health. It has been suggested that the benefits of physical exercise may lie in an ability to reduce some of the more deleterious health effects of stress and stress hormones. The present series of experiments provides evidence that voluntary exercise facilitates habituation of corticosterone but not adrenocorticotropin hormone responses to repeated stress presentations. After 6 weeks of running wheel access or sedentary housing conditions, rats were exposed to 11 consecutive daily 30 min presentations of 98 dB noise stress. Similar corticosterone responses in exercised rats and sedentary controls were observed following the first, acute stress presentation. While both groups demonstrated habituation of corticosterone secretory responses with repeated noise stress exposures, the rate of habituation was significantly facilitated in exercised animals. These results suggest that voluntary exercise may reduce the negative impact of prolonged or repeated stress on health by enhancing habituation of the corticosterone response ultimately reducing the amount of glucocorticoids the body and brain are exposed to.

Conditioned fear inhibits c-fos mRNA expression in the central extended amygdala.

We have shown previously that unconditioned stressors inhibit neurons of the lateral/capsular division of the central nucleus of the amygdala (CEAl/c) and oval division of the bed nucleus of the stria terminalis (BSTov), which form part of the central extended amygdala. The current study investigated whether conditioned fear inhibits c-fos mRNA expression in these regions. Male rats were trained either to associate a visual stimulus (light) with footshock or were exposed to the light alone. After training, animals were replaced in the apparatus, and 2 h later injected remotely, via a catheter, with amphetamine (2 mg/kg i.p.), to induce c-fos mRNA and allow inhibition of expression to be measured. The rats were then presented with 15 visual stimuli over a 30 minute period. As expected, fear conditioned animals that were not injected with amphetamine, had extremely low levels of c-fos mRNA in the central extended amygdala. In contrast, animals that were trained with the light alone (no fear conditioning) and were injected with amphetamine had high levels of c-fos mRNA in the CEAl/c and BSTov. Animals that underwent fear conditioning, and were re-exposed to the conditioned stimulus after amphetamine injection had significantly reduced levels of c-fos mRNA in both the BSTov and CEAl/c, compared to the non-conditioned animals. These data suggest that conditioned fear can inhibit neurons of the central extended amygdala. Because these neurons are GABAergic, and project to the medial CEA (an amygdaloid output region), this may be a novel mechanism whereby conditioned fear potentiates amygdaloid output.

Regulation of hippocampal alpha1d adrenergic receptor mRNA by corticosterone in adrenalectomized rats.

The hippocampal formation receives extensive noradrenergic projections and expresses high levels of mineralocorticoid (MR) and glucocorticoid (GR) receptors. Considerable evidence suggests that the noradrenergic system influences hippocampal corticosteroid receptors. However, there is relatively little data describing the influence of glucocorticoids on noradrenergic receptors in the hippocampal formation. alpha1d adrenergic receptor (ADR) mRNA is expressed at high levels in the hippocampal formation, within cells that express MR or GR. In order to determine whether expression of alpha1d ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7 or 14 days. Levels of alpha1d ADR mRNA were profoundly decreased in hippocampal subfields CA1, CA2 and CA3 and the medial and lateral blades of the dentate gyrus, as early as 1day after ADX, as determined by in situ hybridization. The effect was specific for the hippocampal formation, with levels of alpha1d mRNA unaltered by ADX in the lateral amygdala, reticular thalamic nucleus, retrosplenial cortex or primary somatosensory cortex. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50mg) or placebo for 7 days. Corticosterone replacement prevented the ADX-induced decrease in hippocampal alpha1d ADR mRNA, with the magnitude of effect depending on corticosterone dose and hippocampal subregion. These data indicate that alpha1d ADR mRNA expression in the hippocampal formation is highly sensitive to circulating levels of corticosterone, and provides further evidence for a close interaction between glucocorticoids and the noradrenergic system in the hippocampus.

Modulation of the hypothalamo-pituitary-adrenocortical axis by caffeine.

Although caffeine is the most consumed psychoactive substance in the world, the extents of many of its effects are unknown. High doses of caffeine have been shown to activate the HPA axis while the effects of low to moderate doses have usually not been described in detail. Moreover, although several lines of evidence suggest that low doses of caffeine may restrain some negative affective states, the possible modulatory role of caffeine on HPA axis activation induced by a stressful stimulus has not been described. Thus, the present studies investigated the possible modulatory effects of low to moderate doses of caffeine on moderate to high HPA axis activation induced by different intensities of loud noise. First, in order to test this modulation, time courses for adrenocorticotropic hormone (ACTH) and corticosterone responses to loud noise stress and to caffeine were defined, in rats. Plasma ACTH and corticosterone levels peaked 30 min from the onset of noise presentation, and rapidly declined after noise termination. A low caffeine dose of 2 mg/kg significantly increased plasma corticosterone and ACTH levels 30 min following injections, but levels returned to baseline 60 min following injections. Caffeine doses of 30 mg/kg and higher elevated plasma hormone levels for at least 2h. Doses of 2 or 10mg/kg, however, did not modulate endocrine responses to loud noise presentation. It is concluded that although caffeine activates the HPA axis, low to moderate doses do not modulate HPA axis responses to stressful stimuli.

Elevated central monoamine receptor mRNA in rats bred for high endurance capacity: implications for central fatigue.

Although alteration to peripheral systems at the skeletal muscle level can contribute to one's ability to sustain endurance capacity, neural circuits regulating fatigue may also play a critical role. Previous studies demonstrated that increasing brain serotonin (5-HT) release is sufficient to hasten the onset of exercise-induced fatigue, while manipulations that increase brain dopamine (DA) release can delay the onset of fatigue. These results suggest that individual differences in endurance capacity could be due to factors capable of influencing the activity of 5-HT and DA systems. We evaluated possible differences in central fatigue pathways between two contrasting rat groups selectively bred for high (HCR) or low (LCR) capacity running. Using quantitative in situ hybridization, we measured messenger RNA (mRNA) levels of tryptophan hydroxylase (TPH), 5-HT transporter (5-HTT), 5-HT1A and 5-HT1B autoreceptors, dopamine receptor-D2 (DR-D2) autoreceptors and postsynaptic receptors, and dopamine receptor-D1 (DR-D1) postsynaptic receptors, in discrete brain regions of HCR and LCR. HCR expressed higher levels of 5-HT1B autoreceptor mRNA in the raphe nuclei relative to LCR, but similar levels of TPH, 5-HTT, and 5-HT1A mRNA in these areas. Surprisingly, HCR expressed higher levels of DR-D2 autoreceptor mRNA in the midbrain, while simultaneously expressing greater DR-D2 postsynaptic mRNA in the striatum compared to LCR. There were no differences in DR-D1 mRNA levels in the striatum or cortex between groups. These data suggest that central serotonergic and dopaminergic systems may be involved in the mechanisms by which HCR have delayed onset of exercise-induced fatigue compared to LCR.

Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons.

Serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) are implicated in mediating learned helplessness (LH) behaviors, such as poor escape responding and expression of exaggerated conditioned fear, induced by acute exposure to uncontrollable stress. DRN 5-HT neurons are hyperactive during uncontrollable stress, resulting in desensitization of 5-HT type 1A (5-HT1A) inhibitory autoreceptors in the DRN. 5-HT1A autoreceptor downregulation is thought to induce transient sensitization of DRN 5-HT neurons, resulting in excessive 5-HT activity in brain areas that control the expression of learned helplessness behaviors. Habitual physical activity has antidepressant/anxiolytic properties and results in dramatic alterations in physiological stress responses, but the neurochemical mediators of these effects are unknown. The current study determined the effects of 6 weeks of voluntary freewheel running on LH behaviors, uncontrollable stress-induced activity of DRN 5-HT neurons, and basal expression of DRN 5-HT1A autoreceptor mRNA. Freewheel running prevented the shuttle box escape deficit and the exaggerated conditioned fear that is induced by uncontrollable tail shock in sedentary rats. Furthermore, double c-Fos/5-HT immunohistochemistry revealed that physical activity attenuated tail shock-induced activity of 5-HT neurons in the rostral-mid DRN. Six weeks of freewheel running also resulted in a basal increase in 5-HT1A inhibitory autoreceptor mRNA in the rostral-mid DRN. Results suggest that freewheel running prevents behavioral depression/LH and attenuates DRN 5-HT neural activity during uncontrollable stress. An increase in 5-HT1A inhibitory autoreceptor expression may contribute to the attenuation of DRN 5-HT activity and the prevention of LH in physically active rats.

Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei.

BACKGROUND: Altered serotonergic (5-HT) neurotransmission is implicated in the antidepressant and anxiolytic properties of physical activity. In the current study, we investigated whether physical activity alters factors involved in the regulation of central 5-HT neural activity. METHODS: In situ hybridization was used to quantify levels of 5-HT transporter (5-HTT), 5-HT(1A), 5-HT(1B), and alpha(1b)-adrenergic receptor (alpha(1b) ADR) messenger ribonucleic acids (mRNAs) in the dorsal (DRN) and median raphe (MR) nuclei of male Fischer rats after either sedentary housing or 3 days, 3 weeks, or 6 weeks of wheel running. RESULTS: Wheel running produced a rapid and lasting reduction of 5-HT(1B) mRNA in the ventral DRN. Three weeks of wheel running decreased 5-HTT mRNA in the DRN and MR and increased alpha(1b) ADR mRNA in the DRN. After 6 weeks of wheel running, 5-HTT mRNA remained reduced, but alpha(1b) ADR mRNA returned to sedentary levels. Serotonin(1A) mRNA was increased in the MR and certain DRN subregions after 6 weeks only. CONCLUSIONS: Data suggest that the central 5-HT system is sensitive to wheel running in a time-dependent manner. The observed changes in mRNA regulation in a subset of raphe nuclei might contribute to the stress resistance produced by wheel running and the antidepressant and anxiolytic effects of physical activity.

A detailed characterization of loud noise stress: Intensity analysis of hypothalamo-pituitary-adrenocortical axis and brain activation.

The present studies were undertaken to help determine the putative neural circuits mediating activation of the hypothalamo-pituitary-adrenocortical (HPA) axis and the release of adrenocorticotropin hormone (ACTH) and corticosterone in response to the perceived threat of loud noise. This experiment involved placing rats in acoustic chambers overnight to avoid any handling and context changes prior to noise exposure, which was done for 30 min (between 9:00 and 10:00 am) at intensities of 80, 85, 90, 95, 100, 105, and 110 dBA in different groups (n = 8), and included a background condition (60 dBA ambient noise). This manipulation produced a noise-intensity-related increase in plasma ACTH and corticosterone levels, with levels beginning to rise at approximately 85 dBA. c-fos mRNA induction was very low in the brains of the control and 80 dBA groups, but several brain regions displayed a noise-intensity-related induction. Of these, several forebrain regions displayed c-fos mRNA induction highly correlated (r > 0.70) with that observed in the paraventricular hypothalamic nucleus and plasma ACTH levels. These regions included the ventrolateral septum, the anteroventral subiculum, several preoptic nuclei, the anterior bed nucleus of the stria terminalis (BNST), the anterior paraventricular nucleus of the thalamus, and the medial subdivision of the medial geniculate body. Together with prior findings with audiogenic stress, the present results suggest that either or both the anterior BNST or the lateral septum is ideally situated to trigger HPA axis activation by stimuli that are potentially threatening.