Genes and proteins of the Fos family are used as markers of neuronal activity and can be modulated by stress. This study investigated whether social defeat (SD) or exposure to an olfactory cue associated with the SD experience activated Fos and FosB/DeltaFosB (ΔFosB) expression in brain regions implicated in the development of post-traumatic stress disorder. Mice exposed to acute SD showed more Fos positive cells in the basolateral amygdala (BLA), CA1 of the hippocampus and the medial prefrontal cortex (mPFC) 1h after SD, and had greater expression of the more persistent FosB/ΔFosB protein in the BLA 24 h after SD compared to controls. Mice exposed to an olfactory cue 24 h or 7 days after SD had higher levels of Fos expression in all three regions 1h after exposure to the cue, and displayed increased avoidance behavior compared to controls. While the avoidance response dissipated with time (less at 7 day vs 24 h after social defeat), Fos expression in the mPFC and CA1 in response to an olfactory cue was greater at 7 days relative to 24 h after social defeat. The results suggest additional processing of the cue-stress association and may provide further support for a role of the mPFC in fear inhibition. These findings may have implications for brain regions and circuitry involved in the avoidance of cues associated with a stressful event that may lead to context-dependent adaptive or maladaptive behavior.

Police trainees who were ready to graduate from the Federal Law Enforcement Training Center (FLETC) volunteered, under an IRB-approved protocol, to participate in an exercise designed to evaluate their survivability. In a highly stressful interactive scenario, which included hostage situation arising during a staged domestic dispute, performance was evaluated for a range of responses, including: management of a weapon malfunction, shooting judgment and accuracy, communications, and recall of events. Heart rate, blood pressure and salivary levels of the stress-responsive hormone cortisol, were elevated after the domestic dispute, as were scores on the Spielberger State Anxiety and State Anger Questionnaires (Spielberger, et al. 1983). Nineteen percent of subjects shot the hostage, a failure rate that falls in the reported range of friendly fire casualties in military combat. Ninety-seven percent failed to meet the criterion of 70% of their rounds hitting the suspect. Many of the students fired blindly, from the minimal cover available. The majority of the students failed to correctly manage a weapon malfunction. During a formal interview after the domestic dispute, only 43% of trainees could accurately describe their shot placement, and only 57% could accurately identify the exact moment when the situation and doctrine first justified the use of lethal force. In the present study, significant elevations in salivary levels of the stress hormone cortisol were noted after the shooting episode. Of particular interest, subjects achieving passing scores during the two most challenging events had significantly higher heart rates during those episodes. This suggests that differences in arousal or sympathetic/parasympathetic balance could be effective predictors of performance decrement. The Spielberger Trait Anger Scale showed an association with shot placement and performance during the gunfight, as well as with overall performance scores.

Glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), which is also known as N-acetylated-alpha-linked acidic dipeptidase (NAALADase), hydrolyses the endogenous acidic dipeptide N-acetylaspartylglutamate (NAAG), yielding N-acetyl-aspartate and glutamate. Inhibition of this enzyme by 2-(phosphonomethyl) pentanedioic acid (2-PMPA) has been shown to protect against ischemic injury to the brain and hypoxic and metabolic injury to neuronal cells in culture, presumably by increasing and decreasing the extracellular concentrations of NAAG and glutamate, respectively. Since both NAAG and GCP II are found in especially high concentrations in the spinal cord, injuries to the spinal cord involving pathophysiological elevations in extracellular glutamate might be particularly responsive to GCP II inhibition. Lumbar subarachnoid injections of dynorphin A in rats cause ischemic spinal cord injury, elevated extracellular glutamate and a persistent hindlimb paralysis that is mediated through excitatory amino acid receptors. We therefore used this injury model to evaluate the protective effects of 2-PMPA. When coadministered with dynorphin A, 2-PMPA significantly attenuated the dynorphin A-induced elevations in cerebrospinal fluid glutamate levels and by 24 h postinjection caused significant dose-dependent improvements in motor scores that were associated with marked histopathological improvements. These results indicate that 2-PMPA provides effective protection against excitotoxic spinal cord injury.

We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH2, His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior.

RATIONALE:Long-term individual housing increases aggressive behavior in mice, a condition termed isolation-induced aggression; this aggressiveness is reduced by some antidepressants and anxiolytics. NMDA antagonists also inhibit isolation-induced aggression in mice. The enzyme N-acetylated-alpha-linked acidic dipeptidase (NAALADase) hydrolyzes the neurotransmitter N-acetylaspartylglutamate (NAAG) to form glutamate and N-acetylaspartate; NAAG acts as a partial NMDA agonist as well as a full agonist at the presynaptic metabotropic glutamate receptor 3 (mGluR3), where it acts to reduce glutamate release.OBJECTIVE:We postulated that NAALADase inhibition would reduce isolation-induced aggression in mice.METHODS:We tested whether acute exposure to the NAALADase inhibitor 2-[[hydroxy[2,3,4,5,6-pentafluorophenyl)methyl]phosphinyl]methyl] pentanedioic acid (GPI-5232), administered 30 min prior to a social interaction test, would inhibit aggressive behavior in SJL mice that had been individually housed long term.RESULTS:Administration of GPI-5232 (30 mg/kg, IP) inhibited initiation of aggressive behavior, indicated by greater latencies to display tail-rattling, attack and biting, and by fewer mice initiating aggressive behavior, compared to mice that received vehicle. In addition, GPI-5232 treated mice had fewer tail-rattling responses to a non-aggressive conspecific.CONCLUSIONS:The effectiveness of GPI-5232 in this animal model suggests that NAALADase inhibition may be a novel therapeutic approach to reduce or inhibit heightened aggressiveness, and possibly to treat aggressive behavior associated with psychiatric disorders.

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 aggressor's 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.

Corticotropin-releasing hormone (CRH) is believed to play an important role in the regulation of behavioral responses to stress. CRH(1) receptor antagonists may reduce stress responsivity. Stress increases CRH in the amygdala, important in memory consolidation. We hypothesized that infusion of a CRH(1) 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 CRH(1) antagonist antalarmin (0.25 microg/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 CRH(1) receptors in the amygdala may prevent the development of exaggerated fear responses in stressed mice.

In mice, the neuropeptide arginine-8-vasopressin (AVP) induces excessive grooming, scratching, and hyperactivity when administered intracerebroventricularly. In hamsters, AVP infusion into the medial preoptic area/anterior hypothalamus (MPOA/AH) increases flank marking and flank mark grooming. We measured the behavioral effects of administration of AVP (0, 1, and 10 ng/250 nl) into the preoptic area (POA) of male C57BL/6 mice. Administration of AVP into the POA induced robust effects on grooming, including increased hindleg scratching and face washing. Rearing and olfactory investigation were inhibited by AVP into the POA. These findings indicate that the POA is one site in which AVP induces grooming behavior in mice.

We have studied the neuroanatomic extent of electroconvulsive (ECS)-responsive prepro-TRH and TRH-related gene expression and its possible interaction with forced swimming. Young adult male Wistar rats were treated in a 2×2 Latin square protocol of swimming, no swimming, three daily ECS or sham ECS. Sixteen different brain regions were dissected and immunoreactivity measured for TRH (pGlu–His–Pro–NH2); TRH–Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP (pGlu–Glu–Pro–NH2). ECS, in addition to elevating TRH–immunoreactivity (TRH–IR), TRH–Gly–IR, Ps4–IR and EEP–IR levels in the limbic regions, as we have previously reported, also significantly increased Ps4–IR levels in hypothalamus, posterior cingulate and lateral cerebellum, and increased TRH–Gly–IR levels in hypothalamus. Interestingly, the combination of ECS and swimming significantly reduced the levels of TRH–Gly–IR in the anterior cingulate compared to the sham ECS-no swim group. The combined use of high-pressure liquid chromatography and the EEP radioimmunoassay (RIA) revealed that pGlu–Tyr–Pro–NH2 and/or pGlu–Phe–Pro–NH2 occur in amygdala, anterior cingulate, frontal cortex, entorhinal cortex, lateral cerebellum and striatum and make a substantial contribution to the EEP–IR and TRH–IR. We conclude that ECS can alter the expression and secretion of TRH-related peptides in the hypothalamus, cingulate and lateral cerebellum. Such effects have not previously been reported in these limbic and extra-limbic regions which are increasingly implicated in the autonomic, behavioral and volitional changes which accompany severe depression and its treatment.

This study extended to treadmill exercise training our prior report (Dishman RK, Warren JM, Youngstedt SD, Yoo H, Bunnell BN, Mougey EH, Meyerhoff JL, Jaso-Friedmann L, and Evans DL. J Appl Physiol 78: 1547-1554, 1995) that activity wheel running abolished the suppression of footshock-induced natural killer (NK) cell cytolysis. Twenty-four male Fischer 344 rats were assigned to one of three groups (n = 8, all groups): 1) a home-cage control group, 2) a sedentary treatment group, or 3) a treadmill-running group (0 degrees incline, 25 m/min, 35 min/day, 6 days/wk). After 6 wk, the treadmill and sedentary groups received 2 days of footshock. Splenic NK cytotoxicity was determined by standard 4-h (51)Cr release assay. Percentages of lymphocytes were determined by flow cytometry. Plasma levels of ACTH, corticosterone, and prolactin concentration were measured by radioimmunoassay. After footshock, percentage of lysis relative to home-cage controls was 40% and 80% for sedentary and treadmill-trained animals, respectively (P < 0.05). Our results indicate that the protective effect of chronic exercise on innate cellular immunity in the Fischer 344 male rat is not restricted to activity wheel running, nor is it explained by elevations in basal NK activity, increased percentages of splenic NK and cytotoxic T cells, or increased plasma levels of ACTH, corticosterone, and prolactin.

Social stress induces robust behavioral and physiological changes, some of which may alter the responsiveness to pharmacological agents, including diazepam (DZP). We used a resident–intruder paradigm to (1) develop a comprehensive ethogram of behavioral changes following social defeat (SD) in the socially reactive strain, DBA/2 male mice, (2) determine whether acute exposure of DBA/2 mice to low-dose DZP would induce flight or aggressive behavior, both of which have been observed in other rodent models and (3) to test whether prior social stress affects responses to DZP. Behavioral responses to a nonaggressive intruder (NAI) mouse 24 h post-SD were measured in resident subject mice exposed to DZP (0, 0.5, 2.0 mg/kg, ip) either prior to the resident–intruder test (Experiment 1) or immediately post-SD (Experiment 2); control mice were not defeated (NOSD). In general, SD mice displayed increased passive and active avoidance, defense, immobility, and risk assessment relative to NOSD mice. In Experiment 1, mice treated acutely with 0.5 mg/kg DZP had more approach and flight behavior, while those treated with 2.0 mg/kg DZP had more avoidance than vehicle-treated mice, independent of SD. In Experiment 2, acute DZP (2 mg/kg) induced effects 24 h later, possibly secondary to withdrawal. In a nonsocial context (Experiment 3), DZP increased exploratory activity.

Retinal tissue is subject to ischemia from diabetic retinopathy and other conditions that affect the retinal vasculature such as lupus erythematosus and temporal arteritis. There is evidence in animal models of reversible ischemia that a therapeutic window exists during early recovery when agents that reduce glutamate activity at its receptor sites can rescue neurons from injury. To model ischemia, we used sodium cyanide (NaCN), to inhibit oxidative metabolism, and 2-deoxyglucose (2-DG), to inhibit glycolysis. Dissociated rabbit retina cells were studied to evaluate the potential neuroprotective effects of N-acetyl-aspartyl-glutamate (NAAG), which competes with glutamate as a low-potency agonist at the NMDA receptor complex. N-acetylated alpha-linked acidic dipeptidase (NAALADase; the NAAG-hydrolyzing enzyme) is responsible for the hydrolysis of NAAG into glutamate, a neurotransmitter and potent excitotoxin, and N-acetylaspartate, 2-Phosphonyl-methyl pentanedioic acid (PMPA) and beta-linked NAAG (beta-NAAG), inhibitors of NAALADase, were also tested, since inhibition of NAALADase could reduce synaptic glutamate and increase the concentration of NAAG. We found that metabolic inhibition with NaCN/2-DG for I hour caused 50% toxicity as assessed with the MTT assay. Go-treatment with NAAG resulted in dose-dependent protection of up to 55% (p < 0.005). When the non-hydrolyzable, NAALADase inhibitor beta-NAAG was employed, dose-dependent protection of up to 37% was observed (p < 0.001). PMPA also showed 48% protection (p < .05 - .001) against these insults. These data suggest that NAAG may antagonize the effect of glutamate at the NMDA receptor complex in retina. Inhibition of NAALADase by PMPA and beta-NAAG may increase the activity of endogenous NAAG.

Acute social defeat (SD) leads to transient and persistent physiological and behavioral changes. We examined the effects of acute SD on territorial urine marking and ultrasonic courtship vocalizations in DBA/2 male mice. Both behaviors are considered androgen dependent and are influenced by social status, with dominant mice displaying more of both behaviors. In Experiment 1, male mice that received SD displayed prolonged inhibition of territorial urine marking, relative to nondefeated control mice (NOSD). In addition, territorial marking increased with repeated tests. In Experiment 2, male mice that received 3 successive days of SD displayed fewer ultrasonic courtship vocalizations at 30 min. post-SD1 and 30 min. post-SD2, relative to NOSD mice. In Experiment 2, we also observed decreased territorial marking 4 weeks post-SD. In sum, SD induced prolonged inhibition of territorial marking, but had only transient effects on ultrasonic courtship vocalizations, suggesting that different mechanisms may mediate the maintenance of these behaviors.

The NMDA receptor complex is thought to be altered in kindling, an animal model for complex partial epilepsy. This receptor complex has several modulatory sites including those for glutamate, glycine and polyamines with activation resulting in altered cation channel opening. Two NMDA receptor effectors, ifenprodil and arcaine, were evaluated for effects on the acquisition of electrical kindling of the amygdala. Rats were administered 0, 3.2, 10, 32 and 100 μg of ifenprodil or 0, 32 or 100 μg of arcaine, intracerebroventricularly, 10 min before a daily kindling stimulus. Ifenprodil, at low doses, enhanced kindling acquisition, while the highest dose, 100 μg, inhibited kindling. Arcaine increased the number of trials required to reach fully generalized (stage 5) seizures at the 100 μg dose. Since these agents had mixed actions on kindling development, it is unclear whether these or similar NMDA effectors would be useful in the modulation of complex partial seizures.

In the early stages of an agonistic encounter between mice, the loser of the conflict initially exhibits pronounced flight and escape attempts. When exposure to attack is prolonged, however, defeated mice display a decrease in these active defenses and become increasingly passive. The generality of such defeat-induced passivity was investigated in the present study by examining acutely defeated mice in the forced swim test, a nonsocial but threatening environment that normally engenders high levels of escape behavior. In the present experimental model, highly aggressive male C57BL/6 mice were used to defeat smaller male intruders of the DBA/2 strain in a series of brief (2-min) encounters spaced 2 min apart. In Experiment 1, DBA/2 mice were administered four defeat encounters and were then given a 10-min swim test following a postdefeat delay of 0, 30, or 60 min. Activity in the swim test was determined both by a novel, automated device that detected movement-induced perturbations in the water medium via infrared beam interruptions and by the standard observational measurement of percent time immobile. Both activity measures indicated a significant suppression effect of defeat on swim activity, the magnitude of which declined steadily as the delay between the defeat encounters and the swim test increased. In Experiment 2, DBA/2 mice experienced zero, one, two, three, or four defeat encounters spaced 2 min apart and were administered the swim test 30 min following the last defeat encounter. Activity in the swim test decreased as the number of defeat encounters increased. The present findings suggest that defeat-induced passivity in mice is of a global nature, persisting in other threatening environments in which high levels of escape activity would normally be observed. Similarities between the parameters of the present phenomenon and those of opioid-mediated defeat analgesia are discussed. Aggr. Behav. 24:257–269, 1998. © 1998 Wiley-Liss, Inc.

We examined whether chronic circadian physical activity attenuates hypothalamic-pituitary-adrenal hormone responses after footshock with or without cage-switch stress. Young (45 g) male Fischer 344 rats were randomly assigned to individual suspended home cages (HC) or cages with activity wheels (AW) (12 h:12 h light-dark photoperiod). After 6 weeks, each animal from a pair matched on mass (HC and AW) and average weekly running distance (AW) was randomly assigned to controllable or uncontrollable footshock on 2 days separated by 24 h. Half the animals were returned to the HC after the first day of shuck, and half were switched to a new shoebox cage. One animal of each pair could end the shock for both rats by performing an FR-2 lever press. The yoked animal could not control the shock. After shock on Day 2, trunk blood was collected after decapitation. Plasma adrenocorticotrophin (ACTH), corticosterone, and prolactin were determined by radioimmunoassay. ANOVA for a 2 Group (AW vs. sedentary) x 2 Test (controllable vs. uncontrollable shock) x 2 Condition (HC vs. cage-switch) design indicated a Group x Test x Condition effect [F(1, 48) = 5.07, p = 0.03] and a Test main effect [F(1, 47) = 6.93, p = 0.01] for ACTH. ACTH was higher for sedentary animals after uncontrollable footshock under cage-switch conditions and higher after uncontrollable versus controllable footshock when averaged across groups and cage conditions. No effects were found for corticosterone or prolactin. Our results extend to activity wheel running prior findings of a cross-stressor attenuation in plasma [ACTH] in response to cage-switch after treadmill exercise training, though the cross-stressor effect was additive with footshock. Consistent with our prior reports, the cross-stressor effect of wheel running was not apparent after footshock administered under home-cage conditions. (C) 1998 Elsevier Science Inc.

We reported (J Appl Physiol, 78: 1547-1554, 1995) that 6 wk of circadian activity wheel running blocked the decrease in splenic Natural Killer Cell cytolysis induced by footshock, with no effects on baseline cytotoxicity. The study not determine whether an enriched environment or adaptations to exertion explained the results. The present study extended our test to exercise training. Male Fischer rats (60 d) were assigned randomly to: 1) a sedentary home-cage control group (n=8), 2) a sedentary treatment group (n=8), or 3) a treadmill exercise training group (n=8) (25 m/min, zero°, 45 min/d, 6 d/wk). After 6 wk, the treadmill and sedentary groups received uncontrollable footshock on 2 days separated by 24 h. Each day, 6 min of scrambled shock was delivered in bouts of 3-30 s, with a 30-s intershock period. Spleens and trunk blood were collected 30 min after shock on day 2. Cytotoxicity was determined by standard 4-h 51Cr release assay using YAC-1 target cells. B, CD8, CD4, THY1.1 and NK cell% were determined by flow cytometry. Plasma [levels] of[ACTH], [corticosterone], and [prolactin] were assayed by RIA. A group effect was found for effector:target ratio, F(2,21)=6.82 p=0.005. After footshock% lysis relative to HC controls was ≈ 40% for sedentary animals compared with≈ 80% for treadmill exercise trained animals. One-way ANOVA with Duncan post hoc tests indicated that [ACTH] and [corticosterone] were elevated after footshock compared with home-cage controls, p < 0.05, but did not differ by group. Groups did not differ on splenic cell percentages. Our results indicate that treadmill exercise training attenuates the suppression of splenic NK cytolysis induced by footshock. Thus, the apparently protective effect of chronic exercise on natural immunity in the Fischer male rat is not merely explainable by an enriched environment compared with standard husbandry.