James L. Meyerhoff

REGIONAL γ‐AMINOBUTYRIC ACID LEVELS IN RAT BRAIN DETERMINED AFTER MICROWAVE FIXATION

—GABA levels in rat whole brain were compared following three methods of sacrifice: rapid microwave fixation, decapitation into liquid nitrogen, and decapitation at 20°C. Levels were shown to be identical in animals sacrificed by microwave fixation and decapitation into liquid nitrogen. In contrast, rats decapitated at 20°C had 18 per cent higher GABA levels when determined immediately post‐mortem and 48 per cent higher levels after 30 min at 20°C. Microwave treatment prevented these post‐mortem increases. The increase in GABA after decapitation at 20°C was even greater in hypothalamus than in whole brain.

Habituation to repeated stress is stressor specific

Rats were exposed to 15 min of restraint or footshock or forced running in an activity wheel once a day for 10 days. Control groups were handled only. On the 11th day, rats from each stressor group and controls were exposed to 15 min of one stressor in a crossed design such that all combinations of one chronic stressor and one acute stressor were performed. Rats were sacrificed immediately following removal from their home cage or after 15 min stressor exposure on the 11th day and plasma corticosterone and prolactin and pituitary cyclic AMP levels were determined. There were no measured differences in these stress indices among groups of rats sacrificed immediately upon removal from their home cage on day 11 regardless of previous history on days 1 through 10. Plasma corticosterone and plasma prolactin and pituitary cyclic AMP levels were elevated in all rats exposed to any of the three stressors immediately prior to sacrifice as compared to all rats not exposed to stress immediately before sacrifice. However, plasma prolactin and pituitary cyclic AMP responses to each of the 3 stressors were attenuated in rats which had previous exposure to that specific stressor as compared to rats which had previous experience with a different or no stressor. We conclude that habituation results from behavioral experience with a particular stressor rather than biochemical adaptation resulting from repeated challenge to hormonal and neurochemical systems responsive to stress.

Comparison of stress response in male and female rats: pituitary cyclic AMP and plasma prolactin, growth hormone and corticosterone.

Three potent stressors (forced running, immobilization, and footshock) were found to increase levels of cyclic AMP in the pituitaries of both female and male rats. The pituitary cyclic AMP response in females was generally similar to that observed in males. The tested stressors elevated both plasma corticosterone and prolactin and decreased plasma growth hormone. Plasma corticosterone rose more rapidly in females than in males following stress. Control growth hormone levels were higher in male rats. There was no clear cause and effect relationship between elevations of pituitary cyclic AMP and changes in plasma levels of prolactin, corticosterone, and growth hormone.

Behavioral and neurochemical effects of intraventricular AF64A administration in rats

Ethylcholine aziridinium ion solution (AF64A), a putative specific cholinergic neurotoxin, was injected bilaterally into the lateral ventricles of rats. Following administration of 3 or 6 nmoles of AF64A, drinking and eating were depressed but returned to normal levels after several days; increased activity in the 6 nmole group persisted throughout the 21 days of observations. Performance on complex place and cue tasks indicated that injected animals were impaired in reference memory only on the place task, but working memory was impaired on both tasks. Neurochemical measurements in a separate group of animals one week after AF64A injections found large depletions of acetylcholine in hippocampus and corpus striatum, but not depletions of norepinephrine (hippocampus) or dopamine (striatum). Histological examination of the injection site revealed extensive damage to the fimbria-fornix similar to that seen after electrolytic lesions. Since the behavorial and neurochemical changes are similar to those previously found following fimbria-fornix lesions, it is concluded that the present results are possibly due to non-specific lesion effects of the neurotoxin rather than a specific effect on cholinergic systems.

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.

Acute and repeated exposure to social conflict in male golden hamsters : increases in plasma POMC-peptides and cortisol and decreases in plasma testosterone

The purpose of the present study was to characterize the hormonal response of dominant and submissive male hamsters to acute and repeated exposure to social conflict. We found that submissive, but not dominant, males exhibited elevated plasma levels of adrenocorticotropin (ACTH), cortisol, and beta-endorphin (beta-EP) following one exposure to an agonistic encounter. After five exposures to a dominant opponent, submissive males showed smaller, but still significant, elevations in these plasma hormones. After nine exposures, submissive hamsters showed significant elevations only in plasma ACTH and beta-EP. Plasma testosterone was significantly suppressed in submissive males that fought nine times. We conclude that hamsters are a useful species with which to study the neuroendocrine correlates of social behavior.

Diurnal Variation in Neuroendocrine Response to Stress in Rats: Plasma ACTH, β-Endorphin, β-LPH, Corticosterone, Prolactin and Pituitary Cyclic AMP Responses

The effects of restraint stress applied at different times of the day on levels of five stress-responsive plasma hormones (ACTH, β-endorphin, β-LPH, corticosterone and prolactin) and pituitary cyclic AMP levels were assessed. Different groups of rats were subjected to 15 min of restraint stress at 2-hour intervals over a 24-hour period. Rats were sacrificed immediately upon removal from their home cage (controls) or immediately following restraint (stressed). The time of day of stress exposure markedly affected the stress responses measured. Generally, responses to stress applied at the beginning of the dark cycle (18.00) were less than those seen following stress applied at the beginning of the light cycle (06.00). Stress at 06.00 increased levels of pituitary cyclic AMP 10-fold, while stress applied at 18.00 did not significantly increase pituitary cyclic AMP levels. In stressed rats, high correlations were seen among levels of hormones derived from the common precursor, proopiomelanocortin (ACTH, β-endorphin, β-LPH) and between these hormones and levels of pituitary cyclic AMP. These findings support the hypothesis that pituitary cyclic AMP is involved in the stress-induced release or synthesis of the pituitary hormones ACTH, β-endorphin, and β-LPH.

Effects of repeated stress on pituitary cyclic AMP, and plasma prolactin, corticosterone and growth hormone in male rats

The effects of five putative stressors (saline injection, cold exposure, forced running, immobilization, and footshock) on levels of pituitary cyclic AMP, plasma prolactin, corticosterone and growth hormone were examined. In naive rats exposed to 15 min of these stressors for the first time, running, immobilization and footshock increased levels of pituitary cyclic AMP, plasma corticosterone and prolactin and decreased growth hormone, typical of stress response in the rat. Cold exposure only increased corticosterone and saline injection did not affect any measured parameter. In rats chronically exposed to the same stressor (once a day for 15 min) for 10 days immediately prior to the experiment, an attenuated pituitary cyclic AMP and plasma prolactin response was seen upon application of 15 min of that stressor on the day of the experiment, compared to the responses observed in the naive rats.

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.

Graded footshock stress elevates pituitary cyclic AMP and plasma β-endorphin, β-LPH, corticosterone and prolactin

Abstract Male rats were subjected to 15 min of various intensities of footshock current (0.0, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2mA) on a variable interval schedule with an average intershock interval of 30 sec (30 shocks/15 min session). Each shock lasted 5 sec. Animals were sacrificed immediately after being removed from the shock box. Two similar studies were conducted. In the first experiment, rats were sacrificed by microwave irradiation and pituitary cyclic AMP levels were determined. In the second study, rats were decapitated and plasma hormones (prolactin, corticosterone, β-endorphin, β-LPH) were measured by radioimmunoassay. Although all biochemical indices of stress measured increased as shock intensity increased, some differences among the substances measured were observed with respect to threshold intensity, range of proportional response and maximal response.