Molecular indicators of stress-induced neuroinflammation in a mouse model simulating features of post-traumatic stress disorder.

A social-stress mouse model was used to simulate features of post-traumatic stress disorder (PTSD). The model involved exposure of an intruder (male C57BL/6) mouse to a resident aggressor (male SJL) mouse for 5 or 10 consecutive days. Transcriptome changes in brain regions (hippocampus, amygdala, medial prefrontal cortex and hemibrain), blood and spleen as well as epigenome changes in the hemibrain were assayed after 1- and 10-day intervals following the 5-day trauma or after 1- and 42-day intervals following the 10-day trauma. Analyses of differentially expressed genes (common among brain, blood and spleen) and differentially methylated promoter regions revealed that neurogenesis and synaptic plasticity pathways were activated during the early responses but were inhibited after the later post-trauma intervals. However, inflammatory pathways were activated throughout the observation periods, except in the amygdala in which they were inhibited only at the later post-trauma intervals. Phenotypically, inhibition of neurogenesis was corroborated by impaired Y-maze behavioral responses. Sustained neuroinflammation appears to drive the development and maintenance of behavioral manifestations of PTSD, potentially via its inhibitory effect on neurogenesis and synaptic plasticity. By contrast, peripheral inflammation seems to be directly responsible for tissue damage underpinning somatic comorbid pathologies. Identification of overlapping, differentially regulated genes and pathways between blood and brain suggests that blood could be a useful and accessible brain surrogate specimen for clinical translation.

Species-specific preferences of German recreational anglers for freshwater fishing experiences, with emphasis on the intrinsic utilities of fish stocking and wild fishes.

To answer the question, whether anglers have an intrinsic preference for stocking or a preference for catch outcomes (e.g. catch rates) believed to be maintained by stocking, a discrete choice experiment was conducted among a sample of anglers (n = 1335) in Lower Saxony, Germany. After controlling for catch aspects of the fishing experience, no significant influence of two stocking attributes (stocking frequency and composition of the catch in terms of wild v. hatchery fishes) on the utility gained from fishing was found for any of the freshwater species that were studied. It was concluded that the previously documented large appreciation of fish stocking by anglers may be indicative of an underlying preference for sufficiently high catches rather than reflect an intrinsic preference for stocking or the catching of wild fishes per se.

Olfactory cues increase avoidance behavior and induce Fos expression in the amygdala, hippocampus and prefrontal cortex of socially defeated mice.

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.

Accelerated Barnes maze test in mice for assessment of stress effects on memory.

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 x 21.5 cm x 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.

A CRH1 antagonist into the amygdala of mice prevents defeat-induced defensive behavior.

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.

Reduced isolation-induced aggressiveness in mice following NAALADase inhibition.

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.

Vasopressin into the preoptic area increases grooming behavior in 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.

In vitro neuroprotection against glutamate-induced toxicity by pGlu-Glu-Pro-NH(2) (EEP).

EEP is a tripeptide structurally similar to thyrotropin releasing hormone (TRH) and, like TRH, it is found in the mammalian brain. TRH has been found to increase in brain regions after seizures and to be neuroprotective. EEP has also been shown to increase in brain regions following seizure activity. We therefore sought to determine whether the similarities between these two peptides might be extended to include neuroprotection. Both TRH and EEP were found to be neuroprotective in vitro against an excitotoxic insult. Interestingly, the two tripeptides appeared to have different mechanisms of action. Even though EEP was as much as four times more neuroprotective than TRH, its ability to reduce glutamate-stimulated increases in intraneuronal Ca(2+) was about half that of TRH.

Electroconvulsive seizures modulate levels of thyrotropin releasing hormone and related peptides in rat hypothalamus, cingulate and lateral cerebellum.

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 2x2 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-NH(2)); TRH-Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP (pGlu-Glu-Pro-NH(2)). 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-NH(2) and/or pGlu-Phe-Pro-NH(2) 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.

Treadmill exercise training blunts suppression of splenic natural killer cell cytolysis after footshock.

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.

Effects of social defeat and of diazepam on behavior in a resident-intruder test in male DBA/2 mice.

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.

Evaluating an alternative oral regimen for the treatment of polymyalgia rheumatica.

It is often recommended that patients with polymyalgia rheumatica (PMR) be started with 15 mg/day or more of prednisone for at least 1 month, and then continually tapered until they stop taking prednisone. A much shorter time at 15 mg/day and a prolonged period at 5 mg/day may be as efficacious and lead to shorter treatment courses. I had 22 patients meeting criteria for PMR and started with a regimen of 15 mg/day for 3 days, 10 mg/day for 7 days, 5 mg/day for 1 year, and then tapered at 1 mg/day/month. I had 15 patients rapidly tapered to 5 mg, whereas for 7 a rapid taper failed. Among 14 patients followed to the end of treatment, 10 patients successfully stopped taking prednisone without a relapse (mean prednisone dose 1864 mg at 415 days) and 4 additional patients relapsed but were tapered off prednisone later (mean = 2365 mg at 525 days for all 16 patients). The ability to be tapered off prednisone was not related to the ability to be rapidly tapered to 5 mg/day (p = NS). One patients is taking 3 mg/day at 53 months, and 1 required hydroxychloroquine therapy to be tapered to 5 mg/day. There was 6 patients lost to follow-up. These results suggest that a rapid taper of prednisone dose from 15 to 5 mg is possible in many patients with PMR and that a subsequent year of 5 mg/day before further tapering may result in a lower total dose of prednisone than with many previously recommended regimens.

Social stress effects on territorial marking and ultrasonic vocalizations in mice.

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.

Ifenprodil and arcaine alter amygdala-kindling development.

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 microg of ifenprodil or 0, 32 or 100 microg of arcaine, intracerebroventricularly, 10 min before a daily kindling stimulus. Ifenprodil, at low doses, enhanced kindling acquisition, while the highest dose, 100 microg, inhibited kindling. Arcaine increased the number of trials required to reach fully generalized (stage 5) seizures at the 100 microg 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.

Activity wheel running blunts increased plasma adrenocorticotrophin (ACTH) after footshock and cage-switch stress.

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 shock, 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.

Heart rate and blood pressure resting levels and responses to generic stressors in Vietnam veterans with posttraumatic stress disorder.

Resting heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured on 3 successive mornings in the homes of drug-free Vietnam combat veterans, classified on the basis of DSM-III-R criteria into current posttraumatic stress disorder (PTSD; n = 20) or non-PTSD (n = 15). Responses to three generic stressor challenges (orthostatic, mental arithmetic, and cold pressor) were also measured. In the orthostatic stressor condition, DBP increased over time in the non-PTSD, but not in the PTSD, veterans, suggesting a paradoxically reduced autonomic response in PTSD. There were no other significant group differences in resting levels or responses to any of the challenges for any measure.

Thyrotropin-releasing hormone (TRH) is markedly increased in the rat brain following soman-induced convulsions.

Soman is an organophosphorus (OP) compound which irreversibly inhibits acetylcholinesterase (AChE), the primary synaptic inactivator of acetylcholine. Resultant excessive cholinergic activity elicits generalized convulsions and brain lesions. Recent evidence suggests that other neurotransmitter/neuromodulator systems may be affected by the OP compounds as well. Since we have shown that both electrically and chemically induced seizures cause significant and prolonged increases in the neuropeptide thyrotropin-releasing hormone (TRH) in epileptogenic sites, we examined soman-induced convulsion effects on CNS TRH. Rats were injected with either soman (100 microg/kg SC; equivalent to 0.9 LD50) or saline and observed for convulsive activity. Forty-eight hours post injection, dramatic increases of TRH over control levels were seen in frontal cortex (30-fold), pooled cortex (24-fold), hippocampus (16-fold), piriform cortex (14-fold), entorhinal cortex (11-fold), and amygdala (2-fold). No change was observed in either hypothalamus or pituitary. Our results demonstrate, for the first time, a substantial effect of an OP on a specific neuropeptide system in vivo. The neurochemical and behavioral consequences of the soman-induced increases in TRH, especially in the frontal cortex, are presently unknown. Clearly, much more work is required to discern the exact role TRH has following soman exposure.

Activity wheel running reduces escape latency and alters brain monoamine levels after footshock.

We examined the effects of chronic activity wheel running on brain monoamines and latency to escape foot shock after prior exposure to uncontrollable, inescapable foot shock. Individually housed young (approximately 50 day) female Sprague-Dawley rats were randomly assigned to standard cages (sedentary) or cages with activity wheels. After 9-12 weeks, animals were matched in pairs on body mass. Activity wheel animals were also matched on running distance. An animal from each matched pair was randomly assigned to controllable or uncontrollable inescapable foot shock followed the next day by a foot shock escape test in a shuttle box. Brain concentrations of norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were assayed in the locus coeruleus (LC), dorsal raphe (DR), central amygdala (AC), hippocampus (CA1), arcuate nucleus, paraventricular nucleus (PVN), and midbrain central gray. After prior exposure to uncontrollable foot shock, escape latency was reduced by 34% for wheel runners compared with sedentary controls. The shortened escape latency for wheel runners was associated with 61% higher NE concentrations in LC and 44% higher NE concentrations in DR compared with sedentary controls. Sedentary controls, compared with wheel runners, had 31% higher 5-HIAA concentrations in CA1 and 30% higher 5-HIAA concentrations in AC after uncontrollable foot shock and had 28% higher 5-HT and 33% higher 5-HIAA concentrations in AC averaged across both foot shock conditions. There were no group differences in monoamines in the central gray or in plasma prolactin or ACTH concentrations, despite 52% higher DA concentrations in the arcuate nucleus after uncontrollable foot shock and 50% higher DOPAC/DA and 17% higher 5-HIAA/5-HT concentrations in the PVN averaged across both foot shock conditions for sedentary compared with activity wheel animals. The present results extend understanding of the escape-deficit by indicating an attenuating role for circadian physical activity. The altered monoamine levels suggest brain regions for more direct probes of neural activity after wheel running and foot shock.