George A. Saviolakis

Acute social defeat reduces neurotrophin expression in brain cortical and subcortical areas in mice.

Acute social defeat in mice activates the hypothalamic-pituitary-adrenal axis (HPA) and induces long-term behavioral changes, including exaggerated fear responses and inhibition of territorial behavior. Stress-induced hormonal and neurotransmitter release may contribute to disruption of expression of genes important for cell survival, neuronal plasticity, and neuronal remodeling. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor associated with structural cellular changes that occur during nervous system development and contributes to neural plasticity in the adult brain. In rats, acute (1-2 h) restraint stress transiently reduces BDNF mRNA expression in the hippocampus, a region important in the memory and in HPA regulation; restraint stress also decreases BDNF expression in the basolateral amygdala (BLA), a region important for fear consolidation and emotional memory. We hypothesized that a brief (10 min) exposure to intense social stress, a more naturalistic stressor than restraint stress, would also reduce BDNF mRNA in the hippocampus and BLA of mice. In the present study, we examined the time course of expression of BDNF mRNA expression in the hippocampus and amygdala, as well as other subcortical and cortical brain regions, following acute social stress. In situ hybridization analysis for BDNF mRNA expression showed that there was a significant decrease in BDNF mRNA expression in all regions studied in mice 24 h after social defeat when compared to control (naive) mice (P<0.05). These findings support our hypothesis that BDNF mRNA levels are reduced by social stress, and may have implications for brain plasticity and behavioral changes following social stress.

A CRH1 Antagonist into the Amygdala of Mice Prevents Defeat-Induced Defensive Behavior

Abstract: Corticotropin‐releasing hormone (CRH) is believed to play an important role in the regulation of behavioral responses to stress. CRH1 receptor antagonists may reduce stress responsivity. Stress increases CRH in the amygdala, important in memory consolidation. We hypothesized that infusion of a CRH1 antagonist into the amygdala following social defeat would prevent the development of generalized fear responses. Acute social defeat in mice increases defense towards intruders, even nonaggressive intruders, placed within their home cage. We infused the CRH1 antagonist antalarmin (0.25 μg/125 nl) bilaterally into the amygdala of mice immediately after defeat and measured their response to a nonaggressive intruder stimulus mouse placed within their home cage 24 h after defeat. Defeated mice that received vehicle displayed high levels of crouch defensive posture and numerous flights from intruders, relative to nondefeated mice that received vehicle. Defeated mice that received antalarmin into the amygdala exhibited significantly less defensive posture than did vehicle‐treated defeated mice. Display of defensive posture in antalarmin‐treated mice approached that of vehicle‐treated nondefeated mice. These findings support a role for CRH in the amygdala to promote consolidation of emotional memory and indicate that antagonism of CRH1 receptors in the amygdala may prevent the development of exaggerated fear responses in stressed mice.

The combined effects of pyridostigmine and chronic stress on brain cortical and blood acetylcholinesterase, corticosterone, prolactin and alternation performance in rats

Thousands of soldiers who served in the Gulf War have symptoms that have been collectively termed Gulf War Illness (GWI). It has been suggested that a combination of operational stress and pyridostigmine, a drug given as a pretreatment to protect soldiers against the effects of exposure to nerve agents, might have had unexpected adverse health effects causing these symptoms. Our laboratory has previously modeled operational stress in rats using a paradigm of around-the-clock intermittent signalled footshock. In the present studies, this model was used to investigate the potential synergistic effects of chronic stress and pyridostigmine on physiology and behavior. Seventy-two rats were trained to perform an alternation lever pressing task to earn their entire daily food intake. The rats were then implanted with osmotic minipumps containing vehicle, pyridostigmine (25 mg/ml pyridostigmine bromide) or physostigmine (20 mg/ml eserine hemisulfate). The pumps delivered 1 microl/h, which resulted in a cumulative dosing of approximately 1.5 mg/kg/day of pyridostigmine or 1.2 mg/kg/day of physostigmine, equimolar doses of the two drugs. The rats were then returned to their home cages where performance continued to be measured 24 h/day. After 4 days, 24 of the 72 rats were trained to escape signalled footshock (avoidance-escape group) and 24 other rats (yoked-stressed group) were each paired to a rat in the avoidance-escape group. The remaining 24 rats were not subjected to footshock (unstressed group). Shock trials were intermittently presented in the home cage 24 h/day for 3 days, while alternation performance continued to be measured. Since only 12 test cages were available, each condition was repeated to achieve a final n of six rats per group. Pyridostigmine and physostigmine each decreased blood acetylcholinesterase levels by approximately 50%. Physostigmine also decreased brain cortical acetylcholinesterase levels by approximately 50%, while pyridostigmine had no effect on cortical acetylcholinesterase activity. Alternation performance was impaired on the first day of stress and then recovered. Neither pyridostigmine nor physostigmine affected performance in the absence of stress or increased the effects of stress alone. Corticosterone was significantly increased in the yoked stress group compared to unstressed controls. These data suggest that pyridostigmine does not exacerbate the effects of stress on performance or levels of stress hormones. Furthermore, these data do not suggest that stress enables pyridostigmine to cross the blood brain barrier.

Sexually stimulated testosterone release in male mice (Mus musculus): Roles of genotype and sexual arousal

In virtually every mammalian species examined, some males exhibit reflexive testosterone release upon encountering a novel female (or female-related stimulus). At the same time, not every individual male (or every published study) provides evidence for reflexive testosterone release. Four experiments using house mice (Mus musculus) examined the hypothesis that both the males genotype and his degree of sexual arousal (as indexed by ultrasonic mating calls) are related to such variability. In Experiment 1, CF-1 males exhibited reflexive testosterone elevations 30 min after encountering female urine. CK males, on the other hand, did not exhibit testosterone elevations 20, 30, 50, 60, or 80 min after encountering female urine (Experiments 1 and 2) suggesting this strain incapable of reflexive release. In Experiment 3, we measured both mating calls and reflexive testosterone release in response to female urine in CF-1 and CK males. Most males of both strains called vigorously to female urine but not to water. But, only CF-1 males exhibited significant testosterone elevations to female urine. In Experiment 4, DBA/2J males called vigorously to females followed by testosterone elevations 30 min later. The first 3 experiments support the hypothesis that male genotype is an important variable underlying mammalian reflexive testosterone release. Statistically significant correlations between mating calls in the first minute after stimulus exposure and testosterone elevations 30 min later (Experiments 3 and 4) support the hypothesis that, in capable males, reflexive testosterone release is related to the males initial sexual arousal.

Evaluating performance of law enforcement personnel during a stressful Training scenario

Abstract: Police trainees who were ready to graduate from the Federal Law Enforcement Training Center (FLETC) volunteered to participate in an exercise designed to evaluate their survivability. In a highly stressful interactive scenario, which included a hostage situation, performance was evaluated for a range of responses, including: shooting judgment and accuracy, communications, and coping with a weapon malfunction. Nineteen percent of subjects shot the hostage, a failure rate that falls in the reported range of friendly fire casualties in military combat. The Spielberger Trait Anger Scale showed an association with shot placement and performance during the gunfight as well as with overall performance scores.

Chronic sustained stress increases levels of anterior pituitary prolactin mRNA.

Our laboratory is investigating the effects of chronic stress on physiological, endocrine and behavioral measures, in order to elucidate the neuronal substrates for the pathophysiological consequences of stress in humans. In these studies, we have employed a rodent model of sustained stress in which rats are exposed to around-the-clock intermittent footshock that can be avoided or escaped by rats in the controllable stress group, but not by rats in the uncontrollable stress group. Each rat in the uncontrollable stress group is paired (yoked) to a rat in the controllable stress group such that the controllable stress group rat avoids or escapes shock for both rats. A third group of rats receives no shock (controls). We have previously reported that in male rats, plasma prolactin levels were elevated after 3 days of stress in both stress groups. In the present experiments we determined whether the increases in plasma prolactin were correlated with increases in anterior pituitary prolactin mRNA. In addition, we measured hormones and mRNA at three time points and we examined these responses in female as well as male rats. Adult male and female Sprague-Dawley rats were exposed to chronic stress for 1, 3 or 14 days. In unstressed control rats, levels of anterior pituitary prolactin mRNA were fivefold higher in female as compared to male rats. However, stress increased levels of anterior pituitary prolactin mRNA over baseline in both genders. After 1 day of stress, anterior pituitary prolactin mRNA levels increased in male and female rats belonging to both stress groups, with no significant difference seen between the uncontrollable vs. controllable stress groups. After 3 days of stress, anterior pituitary prolactin mRNA levels were even more elevated, and rats in the uncontrollable stress group had higher anterior pituitary prolactin mRNA levels than those in the controllable stress group. After 14 days of stress, there were no significant differences in control and stressed groups with respect to anterior pituitary prolactin mRNA. These data suggest that chronic sustained stress increases the synthesis of anterior pituitary prolactin mRNA during the first days of stress, and that levels return to prestress values sometime between 3 and 14 days of stress.

Accelerated Barnes Maze Test in Mice for Assessment of Stress Effects on Memory

Abstract: Repeated restraint stress in rodents impairs spatial memory in a Y‐maze test and induces hippocampal neuronal changes that last up to 5 d after the stressor ends. Our goal was to implement a Barnes maze spatial memory test in mice that could be used to validate our findings of social stress induced Y‐maze impairment. We measured performance of mice in 5‐ and 9‐day test paradigms previously used in rats and mice, respectively. Selecting features from each paradigm, we implemented a 5‐d test (pre‐training, training (4 trials/d/3 d) and probe testing for assessment of spatial memory in mice. Stress consisted of placing each test mouse in a stainless steel perforated box (25.5 cm × 21.5 cm × 16.5 cm) within an aggressors home cage for 6 h/d for 21 d; direct agonistic encounters occurred randomly throughout stress periods. Barnes maze pre‐training (habituation) was on day 21 of the stress exposures. In a preliminary experiment, mice that habituated following their last stressor performed poorly relative to unstressed and to those not habituated prior to the last stressor, as demonstrated by a greater latency to escape and more errors. We conclude that acute stress in a chronic stress paradigm may impair spatial memory acquisition.

Effects of phoneme characteristics on TEO feature-based automatic stress detection in speech

A major challenge of automatic speech recognition systems found in many areas of todays society is the ability to overcome natural phoneme conditions that potentially degrade performance. In this study, we discuss the effects of two critical phoneme characteristics, decreased vowel duration and mismatched vowel type, on the performance of automatic stress detection in speech using Teager energy operator features. We determine the scope and magnitude of these effects on stress detection performance and propose an algorithm to compensate for vowel type and duration shortening on stress detection performance using a composite phoneme decision scheme, which results in relative error reductions of 24% and 39% in the non-stress and stress conditions, respectively.

Repeated exposure to sublethal doses of the organophosphorus compound VX activates BDNF expression in mouse brain.

The highly toxic organophosphorus compound VX [O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonate] is an irreversible inhibitor of the enzyme acetylcholinesterase (AChE). Prolonged inhibition of AChE increases endogenous levels of acetylcholine and is toxic at nerve synapses and neuromuscular junctions. We hypothesized that repeated exposure to sublethal doses of VX would affect genes associated with cell survival, neuronal plasticity, and neuronal remodeling, including brain-derived neurotrophic factor (BDNF). We examined the time course of BDNF expression in C57BL/6 mouse brain following repeated exposure (1/day × 5 days/week × 2 weeks) to sublethal doses of VX (0.2 LD(50) and 0.4 LD(50)). BDNF messenger RNA expression was significantly (p < 0.05) elevated in multiple brain regions, including the dentate gyrus, CA3, and CA1 regions of the hippocampal formation, as well as the piriform cortex, hypothalamus, amygdala, and thalamus, 72 h after the last 0.4 LD(50) VX exposure. BDNF protein expression, however, was only increased in the CA3 region of the hippocampus. Whether increased BDNF in response to sublethal doses of VX exposure is an adaptive response to prevent cellular damage or a precursor to impending brain damage remains to be determined. If elevated BDNF is an adaptive response, exogenous BDNF may be a potential therapeutic target to reduce the toxic effects of nerve agent exposure.

Stress Level Classification of Speech Using Euclidean Distance Metrics in a Novel Hybrid Multi-Dimensional Feature Space

Presently, automatic stress detection methods for speech employ a binary decision approach, deciding whether the speaker is or is not under stress. Since the amount of stress a speaker is under varies and can change gradually, a reliable stress level detection scheme becomes necessary to accurately assess the condition of the speaker. Such a capability is pertinent to a number of applications, such as for those personnel in law enforcement positions. Using speech and biometric data collected from a real-world, variable-stress level law enforcement training scenario, this study illustrates two methods for automatically assessing stress levels in speech using a hybrid multi-dimensional feature space comprised of frequency-based and Teager energy operator-based features. The first approach uses a nearest neighbor-type clustering scheme at the vowel token level to classify speech data into one of three levels of stress, yielding an overall error rate of 50.5%. The second approach employs accumulated Euclidean distance metric weighting at the sentence-level to yield a relative improvement of 12.1% in performance