Activity Wheel Running Reduces Escape Latency and Alters Brain Monoamine Levels After Footshock

Brain Research Bulletin, Vol. 42, No. 5, pp. 399–406, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00

PII S0361-9230(96)00329-2

Activity Wheel Running Reduces Escape Latency and Alters Brain Monoamine Levels After Footshock R. K. DISHMAN,* 1 K. J. RENNER,§ S. D. YOUNGSTEDT,* T. G. REIGLE,† B. N. BUNNELL,‡ K. A. BURKE,§ H. S. YOO,* E. H. MOUGEY\ AND J. L. MEYERHOFF\ Departments of *Exercise Science, †Pharmacology, and ‡Psychology, The University of Georgia, Athens, GA §Department of Biology, The University of South Dakota, Vermillion, SD \Division of Neurosciences, Walter Reed Army Institute of Research, Washington, DC [Received 26 March 1996; Accepted 11 September 1996] ABSTRACT: 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 (Ç50 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), 5hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5HIAA) 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. Q 1997 Elsevier Science Inc.

reported by humans after physical activity [50]. Studies of monoamines in exercising humans have reported increased concentrations of norepinephrine (NE) and its metabolite, 3-methoxy, 4-hydroxy phenethyleneglycol (MHPG) in plasma after acute physical activity [26] or elevated baseline levels of serotonin, i.e., 5-hydroxytryptamine (5-HT) in serum among trained athletes [66]. Such studies have limited meaning for understanding the effects of physical activity on brain monoaminergic systems. Estimates indicate that 25–60% of peripheral MHPG originates from brain NE under resting conditions [26], but increased NE in blood after physical activity comes mainly from spillover from cardiac and muscle sympathetic nerves [27], while the relative contributions by brain, blood platelets, and the intestinal wall to increased serum 5-HT after physical activity are not known. Moreover, an association between peripheral estimates of monoamine activity and mood after physical activity has not been established for humans [50]. This study uses an established experimental model of behavioral stress to examine the effects of chronic physical activity on brain monoaminergic systems implicated in depression and anxiety. Experimental models of anxiety and depression based on uncontrollable, inescapable stress have been developed in the rat using restraint [34] and forced swimming [59], but escape from uncontrollable foot shock is the most elaborated model [72]. Uncontrollable inescapable foot shock increases latency to escape from controllable foot shock administered 24–72 h later [73]. Escape-deficit after foot shock was reported 55 years ago by McCulloch and Bruner [46], who observed that rats exposed to foot shock had a deficit in escaping from water during trials administered on subsequent days. Hypotheses for explaining the escape-deficit have included motor, emotive, or learning mechanisms, with the most compelling model based on depletion of NE in cell bodies of the locus coeruleus (LC) [73]. A single session of high intensity uncontrollable foot shock leads to a decrease in brain NE [11,38,45], with less reliable decreases in brain 5-HT and dopamine (DA) levels [11,16,70,73]. In addition to lowered brain NE in the LC and terminal regions of the hippocampus, hypothalamus, and frontal cortex [2,71,73], uncontrollable foot shock leads to behavioral changes in the rat that

KEY WORDS: Exercise, Rat, NE, DA, 5-HT, ACTH, Prolactin.

INTRODUCTION Altered monoamine activity in the brain has been advanced as a plausible explanation for reductions in depression and anxiety

1 Requests for reprints should be addressed to Rod K. Dishman, Ph.D., Department of Exercise Science, The University of Georgia, Athens, GA 30602-3654.

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mimic features of human depression and/or anxiety [78]. These features include altered sleep, weight loss, anhedonia, and reductions in physical activity, eating, and sex behavior [74], which are reversed by agonists or antagonists of noradrenergic [63], dopaminergic [1], serotonergic [55,57], and GABAergic [56] receptors. In major depression [35] and panic disorder [33], brain monoaminergic systems, and glucocorticoids fail to restrain the hypothalamic–pituitary–adrenal (HPA) cortical axis response to stress. Brain serotonergic and noradrenergic activity during stress can activate the HPA cortical axis by stimulating the release of corticotropin releasing hormone or by direct effects on adrenocorticotropic hormone (ACTH) and corticosterone [15,32,58]. In this study, we examined behavioral and neurochemical effects of chronic physical activity using the escape-deficit model of uncontrollable foot shock. Decreased NE and increased 5-HT in rat brain have been reported after exhaustive swimming [6,49], but also from studies of nonexhaustive physical activity using forced swimming and running on a motorized treadmill or running in an activity wheel [26]. Studies of chronic exercise, conversely, have reported increased brain levels of NE, 5-HT, and DA [14]. The studies confounded exertion with other stressors including diet, cold, drugs, and electric shock [26]. We recently reported that chronic circadian activity wheel running increased NE levels in the pons-medulla [25], where LC-NE cell bodies are located [29]. The purpose of the study we report herein was to determine whether chronic activity wheel running attenuates the depletion of brain NE and the increase in escape latency from escapable foot shock that are induced by uncontrollable, inescapable foot shock delivered the previous day. Females were studied because of the higher prevalence of depression and anxiety reported by female humans and evidence for increased stress responsiveness of female compared with male rats [40]. We measured brain NE, DA, dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindole acetic acid (5-HIAA) in the locus coeruleus, the dorsal raphe, and terminal areas in the central amygdala and CA1 of the hippocampus. By assessing other systems that may modulate behavioral responses during stress, we tested the specificity of the LC-NE depletion hypothesis for the expected escape deficit. Levels and metabolites of monoamines do not measure neural events of release, reuptake, synthesis, or synaptic effects, but they can indicate brain regions where more direct probes can be targeted. Anxiety and analgesia, as well as learned helplessness or behavioral despair, may explain the escape deficit [22–24]; therefore, the midbrain central gray which plays a role in hippocampal-amygdalar models of anxiety [9,53] and analgesia [2], was assayed. Plasma ACTH and prolactin, as well as monoamines in the parvocellular area of the PVN and the arcuate nucleus, were assayed to determine if activity wheel running affected responses by the limbic–hypothalamic–pituitary adrenal (HPA) cortical axis during the escape testing [44]. MATERIALS AND METHODS Female Sprague–Dawley rats were obtained from Charles River at approximately 35 days of age (weight Ç 55 g) and allowed to adapt in individual cages for 2–3 weeks before being assigned to experimental conditions. The animals were handled and weighed daily and housed throughout the experiment in individual cages located equidistant from each other in a vivarium maintained at 23 { 27C with a 12 h light–dark cycle (lights on at 0700 h). Water and Purina lab chow were available ad lib except when the rats were removed from the home cages for testing. Animals were randomly assigned to cages with activity

wheels (n Å 12) or without activity wheels ( n Å 12) and to foot shock conditions in a 2 1 2 factorial design (two groups: activity wheel running vs. sedentary; and two conditions: controllable vs. uncontrollable foot shock). Activity Wheel Condition Each activity wheel animal was housed in a stainless steel cage with an activity wheel attached having a circumference of 113 cm (Lafayette Instruments, Lafayette, IN). Daily distance run by each animal was determined by multiplying the wheel circumference by the number of wheel revolutions indicated by a mechanical counter attached to the activity wheel. The mean ( {) daily distance run during the 9–12-week period was Ç1500 meters { 850 daily. Wheel resistance was calibrated weekly using a 100 g weight. In our lab wheel running has not resulted in a significant increase in oxidative capacity of locomotory muscle, an index of physical fitness, as measured by the activities of succinate dehydrogenase in slow oxidative muscle fibers [20,21,25]. Footshock Protocol After 9–12 weeks, animals were matched in pairs based on body mass. Activity wheel animals were also matched on running distance. An animal from each matched pair was randomly assigned in a counterbalanced manner to controllable or uncontrollable foot shock conditions 24 h after the final day of wheel activity. To control estrus and cirdadian effects, foot shock testing and sacrifice was staggered according to 4-day cycles, with order counterbalanced across groups and condition between 0730 and 1200 h. Animals remained in their home cage, but were locked out of the running wheel, during the 24 h preceding, and during, the 2 days of the foot shock trials. On the first day, each animal of a yoked pair was placed in a separate 28 1 20 1 21 cm Skinner box outside the vision of the other animal, where it received repeated 2 mA scrambled foot shock delivered by a Grayson–Stadler shock generator for a total of 360 s. Neither animal was restrained, but one animal of each pair could terminate the shock for both subjects by performing two bar presses (FR-2). The yoked animal of each pair had no control over the shock. A 30-s intertrial period was used. The maximum duration of a failed FR-2 trial was 30 s. The total session lasted Ç20–45 min. Latency of the FR-2 response after shock onset was similar for the activity wheel and sedentary groups ( Ç6 { 1.5 s) with similar reliabilities for each group across the FR-2 trials (Cronbach a Ç0.92). Fifteen learning trials each lasting 15 s, during which a single bar press ended the shock (FR-1), preceded the experimental FR-2 schedule. Footshock Escape Testing Escape testing was conducted individually 24 h later in a 61 1 20 1 20 cm shuttlebox divided by a 5.7 cm raised barrier. The floor was electrified by scrambled foot shock (1 mA) for a maximum of 15 s each trial with a 60-s intertrial period. Animals terminated the shock by crossing the barrier and returning. Testing lasted from Ç55–62 min. Escape latencies were summed for 50 trials. Immediately following the escape test, animals were killed by rapid decapitation using a guillotine. Brains were removed, frozen on dry ice, and stored at 0707C. Monoamine Assay Serial 300 mm sections of frozen brain were cut at 0107C in an IEC cryostat. The sections were freeze-mounted on glass slides and stored at 0707C. The following brain regions were

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PHYSICAL ACTIVITY ATTENUATES ESCAPE DEFICIT AFTER FOOTSHOCK microdissected using a dissecting microscope and a freezing stage as described by Palkovitz and Brownstein [52]: locus coeruleus (LC), dorsal raphe (DR), central amygdala (AC), hippocampus (CA1), the arcuate nucleus (ARC), the parvocellular paraventricular nucleus (PVN), and the midbrain central gray (MCG). Microdissection of the CA1 region of the hippocampus used alternate 300 mm slices starting at p 3000 and ending at p 6000. The tissue was expelled into 120 ml of acetate buffer (pH 5) containing 0.5 1 10 07 M of the internal standard, dihydroxybenzylamine (DHBA), and stored at 0707C until analyzed. Microdissected brain regions were assayed for concentrations of NE, DOPAC, DA, 5-HT, and 5-HIAA using high-performance liquid chromatography with electrochemical detection [60,61]. Tissue samples were thawed and centrifuged at 15,000 1 g for 2 min. A 60 ml aliquot of supernatant was treated with 2 ml of ascorbate oxidase (Boehringer–Mannheim, 2 mg/10 ml H2O) to minimize ascorbic acid contributions to the solvent front [47] and directly injected into a Waters chromatographic system (Waters Associates, Inc.). The tissue pellet was dissolved in 0.2 N NaOH and analyzed for protein content according to the method of Bradford [12]. Chromatographic separation was accomplished using a C-18, 4 mm radial compression cartridge and a mobile phase consisting of 8.4 g/l sodium acetate, 12 g/l citric acid, 150 mg/l sodium octanesulfonate (Eastman Kodak), 200 mg disodium EDTA, and 150 ml methanol. Electrochemical detection was provided by a laboratory built potentiostat and a glassy carbon electrode (Bioanalytical Systems) set at /0.7 V with respect to an Ag/ AgCl reference electrode. The pg/cm peak height of known concentrations of the standards were determined from the mean peak heights of three chromatograms for each respective standard. The DHBA internal standard was injected three times to determine the peak height for 100% sample recovery. Amine concentrations were calculated from the standard values and corrected for % recovery and injection volume using a Waters 730 Data Module. The calculation was (cm DHBA 100%/cm DHBA in sample) 1 pg/cm amine standard 1 respective cm amine peak height 1 (preparation volume/injection volume). The amine concentrations were divided by mg protein to yield pg amine/ mg protein. Blood Collection and Radioimmunoassay Procedures After the shuttle box testing, all animals were immediately transported to an adjacent room and decapitated without anesthesia. Trunk blood was collected in 5-ml plastic containers containing 0.3 ml of heparin. Blood was chilled on crushed ice for approximately 1 h prior to being spun at 2,000 1 g. The available plasma was pipetted into collection tubes containing 50 ml of aprotinin and stored at 0707C. Plasma concentrations of ACTH and prolactin were determined by radioimmunoassay (RIA) using a commercial kit (Incstar Corporation, Stillwater, MI, Cat. No. 24310) for ACTH [51]. Assay sensitivity was 5.0 pg/ml for ACTH. The within-assay coefficient of variation was 8.2% at 33.0 pg/ml and 2.0% at 112 pg/ml. The interassay coefficient of variation was 10.6% at 33.0 pg/ml and 6.4% at 109.0 pg/ml. Materials for the assay of prolactin were provided by the National Institutes of Health through the Rat Pituitary Hormone Distribution Program. Assay sensitivity was about 0.8 ng/ml. The intra- and interassay coefficients of variation were 6 and 12%. Muscle Collection and Analysis In order to determine if activity wheel running led to an exercise training effect of increased physical fitness, succinate dehydrogenase (SDH) activities were assayed as an indicant of the

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oxidative capacity of locomotory muscle [37]. Within 2 min of decapitation, the soleus muscle, comprised of slow oxidative fibers, was removed from the left hindlimb, frozen in liquid nitrogen, and stored at 0707 celsius. After homogenization of the muscle in a 33 mmol phosphate buffer (Ph 7.4), SDH activity was quantified using spectrophotometry (Bausch and Lomb Spectronic model 1001) by following cytochrome c reduction at 550 nm and 257C [18]. Statistical Analysis To test hypotheses about group differences on escape latency, brain monoamine concentrations, and plasma ACTH and prolactin concentrations, two planned contrasts of means were conducted. First, the animals exposed to uncontrollable foot shock were compared between the activity wheel vs. the sedentary groups. This contrast tested the effect of activity wheel running on responses to uncontrollable stress. Second, the activity wheel vs. the sedentary groups were compared after averaging the responses within each group across the controllable and the uncontrollable foot shock conditions. This contrast tested the main effect of activity wheel running on responses to foot shock, regardless of controllability. The contrasts were conducted by ttests (SPSS Inc., Chicago, IL) with bonferonni adjustment of alpha per dependent variable (p õ 0.025). Changes in body mass were analyzed by a 2 (activity wheel vs. sedentary groups) 1 10 (weeks 1–10) mixed model ANOVA with repeated measures on weeks to examine whether trends differed, which might explain faster foot shock latencies for activity wheel vs. sedentary animals. Group differences in SDH were compared by an independent t-test. Missing data and outliers ( ú 2 SD) were 1% of the observations and were replaced by the cell mean. Values in the text are means { SD. RESULTS Latency to escape foot shock in the shuttle box was faster for animals that had daily access to activity wheels compared with sedentary animals. Also, NE concentration was higher in the locus coeruleus and dorsal raphe for activity wheel animals compared with sedentary animals. Conversely, 5-HIAA concentration in CA1 and 5-HIAA and 5-HT concentrations in AC were higher for sedentary animals compared with activity wheel animals. Plasma ACTH and prolactin concentrations did not differ among groups or conditions, despite higher DA concentration in the arcuate nucleus and higher DOPAC/DA and higher 5-HIAA/ 5-HT concentration in the PVN for sedentary compared with activity wheel (AW) animals. Monoamine levels in the central gray did not differ among the groups or conditions. Escape Latency Escape latency (s) was 34% faster for yoked AW vs. yoked sedentary, t(20) Å 2.3 p õ 0.02, and 27% faster for AW vs. sedentary, t(20) Å 2.33 p õ 0.02 (Fig. 1). Norepinephrine and Dopamine In the LC, NE concentration (pg/ mg) was 61% higher for yoked AW vs. yoked sedentary, t(20) Å 3.2 p õ 0.001, and 49% higher for AW vs. sedentary, t(20) Å 2.9 p õ 0.001. In the DR, NE concentration was 44% higher for yoked AW vs. yoked sedentary, t (20) Å 4.1 p õ 0.001, and 27% higher for AW vs. sedentary, t(20) Å 2.7 p õ 0.01. In the ARC, DA concentration was 52% higher in yoked sedentary vs. yoked AW, t(20) Å 2.92 p õ 0.01, and 32% higher for sedentary vs. AW, t(20) Å 2.5 p õ 0.01. In the PVN, DOPAC/DA concentration was 50% higher

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FIG. 1. Latency to escape foot shock in a shuttle box (sum of 50 trials) among matched pairs of rats assigned to controllable or uncontrollable, inescapable foot shock on the previous day. Rats that had 24 h access to activity wheels for 9–12 weeks had shorter escape latency after uncontrollable foot shock and when escape latency was averaged across the controllable and uncontrollable foot shock conditions. Values are means { SEM.

for sedentary vs. AW, t (20) Å 2.6 p õ 0.01. There were no group or condition differences in the MCG (see Table 1). 5-HT and 5-HIAA The concentration of 5-HIAA (pg/ mg) in the CA1 hippocampal area was 31% higher for yoked sedentary vs. yoked AW, t(20) Å 2.2 p õ0.02. In the AC, 5-HIAA concentrations was 30% higher in yoked sedentary vs. yoked AW, t(20) Å 2.0 p õ 0.025, and 33% higher in sedentary vs. AW, t (20) Å 3.09 p õ 0.01. The concentration of 5-HT (pg/ mg) in the AC was 28% higher in sedentary vs. AW, t (20) Å 2.5 p õ 0.01. The 5-HIAA/ 5-HT concentration ratio in the PVN was 17% higher in the sedentary vs. AW, t (20) Å 2.3 p õ 0.02. There were no group or condition effects in MCG (see Table 1). ACTH and Prolactin Concentration levels of ACTH (pg/ml) were not different between yoked AW vs. yoked sedentary, t (20) Å 0.5 p ú 0.60, or between AW vs. sedentary, t (20) Å 0.3 p ú 0.70. Concentration levels of Prolactin (ng/ml) were not different between yoked AW and yoked sedentary, t (20) Å 0.38 p ú 0.70, or between AW vs. sedentary, t (20) Å 0.1 p ú 0.90 (Fig. 2). Body Mass A significant time effect, indicated that body mass increased equally, F(8,24) Å 131.6, p õ 0.001, for both groups from week 1 (114 { 18 g) through week 9 (274 { 128 g) of the experiment with no effects for treatment or the treatment 1 time interaction. Succinate Dehydrogenase Activity Succinate dehydrogenase activity ( mmol cytochrome c reduced/min/g wet wt) in hindlimb soleus muscle of the AW animals (3.1 { 0.85) was not different from that of the sedentary animals (3.0 { 0.66), t(22) Å 0.6, p ú 0.50. DISCUSSION Chronic running on an activity wheel attenuated the escape deficit induced by uncontrollable foot shock. The effect was as-

sociated with higher concentrations of norephinephrine in locus coeruleus and dorsal raphe for activity wheel animals compared with sedentary animals. These results were independent of an exercise training effect on oxidative capacity of locomotory muscle or changes in body mass. Our sample size was not chosen for a statistical test of the interaction between experimental groups and foot shock controllability. Nonetheless, among sedentary animals prior exposure to uncontrollable foot shock tended to be associated with a longer escape latency, lower norephinephrine in the locus coeruleus and dorsal raphe, and higher ACTH and prolactin, while responses by wheel runners to the escape testing did not differ according to controllability of the prior foot shock. Because performance during the controllable and uncontrollable foot shock trials did not differ between the active and sedentary groups, the effects of wheel running on responses to the shuttle box escape testing do not appear explainable by learning. Though we did not use a no-stress control group for comparison, our results suggest that chronic activity wheel running protects against the depletion of NE induced by uncontrollable foot shock in sedentary animals [11,38,42,45,73]. Higher NE in LC for the activity wheel group is consistent with other studies showing increased brain levels of NE after chronic treadmill running or activity wheel running [14,25]. Our description of monoamine levels indicate brain regions where probes for monoamine synthesis and release or neural discharge can be targeted for enzymology, microdialysis, or electrophysiology. In addition to changes in NE, the concentration of 5-HT was higher in the central amygdala of sedentary animals. Similar findings of higher 5-HT from the limbic forebrain, which contained amgydala nuclei, were reported in a comparison of sedentary vs. chronic wheel runners without acute stress [36]. In contrast with that report, levels of 5-HIAA in the present study were elevated in the central amygdala and the CA1 region of hippocampus in the sedentary animals, suggesting an increase in 5-HT metabolic activity [39]. The higher hippocampal 5-HT activity in the sedentary animals is not explainable by a differential downregulation by glucocorticoids, because glucocorticoids do not increase presynatptic 5-HT turnover in the CA1 area [48]. Higher 5-HT activity in the CA1 area and the central amygdala in the sedentary animals is consistent with studies that indicate a modulating effect of serotonin on the neuronal activity of the hippocampus and amygdala during stress [31,53,69]. Tricyclic antidepressant drugs that inhibit 5-HT reuptake increase postynaptic sensitivity to 5-HT in the hippocampus and amygdala [10,69]. Hence, it is important for future studies to determine the effect of wheel running on brain 5-HT, as well as adrenergic, receptor-effectors. The aforementioned effects may be related to the comparatively higher levels of NE in the LC of activity wheel animls. Lower NE in the DR for the sedentary animals is consistent with a possibly reduced potential for LC-NE inhibition of the DR concomitant with LC-NE depletion. Raphe cell bodies are densely innervated with noradrenergic cell terminals [7], NE inhibits raphe neurons [19,41], and it is believed that LC–noradrenergic and the DR–serotonergic systems are reciprocally innervated [3,4,17,30,31,43,64,65]. The absence of group and condition effects for monoamine levels in the central gray suggests that the escape deficit by the sedentary animals is not explained specifically by anxiety [10,53] or analgesia [2], but such an interpretation is limited to our methods and the time sampled. Though the chronic effects of physical activity and stress on brain noradrenergic and serotonergic receptor-effectors are not known, it is plausible that physical activity, like other chronic stressors, affects monoamine systems in ways qualitatively similar to the effects of pharmacological interventions [5]. Acute cold exposure and treadmill running lead to decreased brain NE

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TABLE 1 CONCENTRATIONS [pg/mg] OF MONOAMINES AND METABOLITES IN MICRODISSECTED NUCLEI OF RAT BRAIN AFTER ESCAPABLE FOOTSHOCK IN ACTIVITY WHEEL (AW) OR SEDENTARY (S) ANIMALS EXPOSED TO CONTROLLABLE (C) OR UNCONTROLLABLE (U) FOOTSHOCK THE PREVIOUS DAY Brain Area

Locus coeruleus

Dorsal raphe

Central amygdala

Hippocampus (CA1)

Arcuate nucleus

Paraventricular nucleus

Midbrain central gray

Group

Condition

NE

DA

DOPAC

DOPAC/DA

AW AW S S AW AW S S AW AW S S AW AW S S AW AW S S

C U C U C U C U C U C U C U C U C U C U

23.8 24.8 17.3 15.4 20.5 19.0 17.8 13.2 6.4 6.8 8.4 7.0 2.9 2.3 3.1 3.0 24.3 21.1 19.8 21.5

(2.9)† (6.9)†‡ (6.6) (2.3) (5.8)* (1.8)*‡ (4.6) (3.0) (1.2) (1.3) (1.8) (2.3) (0.36) (0.48) (1.0) (0.67) (3.4) (2.7) (2.2) (6.9)

2.8 2.5 2.0 2.8 4.2 4.0 3.1 3.7 6.1 5.8 7.6 6.2 0.09 0.13 0.08 0.13 10.8 10.1 12.1 15.4

(0.46) (0.98) (0.33) (1.25) (0.99) (0.75) (1.1) (0.98) (2.9) (1.9) (3.1) (2.5) (0.06) (0.12) (0.08) (0.11) (4.9) (3.1) (2.9)§ (2.3)§Ø

7.8 8.1 6.9 9.2 1.7 1.5 1.0 1.6 1.5 1.3 1.6 1.6

(5.4) (4.6) (1.9) (6.6) (0.94) (0.42) (0.48) (0.56) (0.67) (0.25) (0.63) (1.02)

2.6 3.1 3.5 2.9 0.39 0.38 0.32 0.43 0.27 0.24 0.24 0.25

(1.4) (0.73) (0.95) (1.4) (0.20) (0.09) (0.17) (0.09) (0.09) (0.06) (0.11) (0.07)

5.4 5.0 7.0 8.2

(3.5) (0.7) (3.2) (3.9)

0.50 0.54 0.62 0.55

AW AW S S AW AW S S

C U C U C U C U

39.4 52.6 38.3 42.3 9.1 7.6 6.6 9.1

(5.8) (16.4) (10.9) (9.2) (1.7) (2.1) (0.64) (2.5)

6.4 7.6 6.3 6.6 2.3 2.0 2.0 2.0

(1.6) (2.2) (1.3) (1.2) (0.35) (0.49) (0.27) (0.42)

0.75 1.2 1.1 1.3 0.69 0.49 0.53 0.72

(0.52) (0.44) (0.19) (0.28) (0.14) (0.16) (0.14) (0.27)

0.10 0.16 0.19 0.20 0.31 0.26 0.28 0.32

5-HT

5-HIAA

5-HIAA/5-HT

(0.17) (0.19) (0.34) (0.29)

14.5 14.2 14.5 13.1 54.1 55.6 45.0 54.5 8.1 7.6 10.9 9.2 2.5 2.0 2.6 2.6 6.7 5.3 6.2 6.1

(2.4) (2.6) (2.6) (3.9) (7.5) (10.7) (6.0) (15.5) (1.7) (2.0) (2.1)§ (2.6)§ (0.43) (0.57) (1.5) (0.70) (1.3) (1.0) (1.5) (0.9)

17.5 19.4 21.0 17.3 60.4 67.3 53.8 65.3 10.6 10.2 14.3 13.3 9.4 7.4 8.6 9.7 11.3 9.6 11.1 10.4

(3.2) (1.8) (3.3) (6.0) (7.2) (12.2) (7.6) (18.9) (2.1) (2.7) (2.5)§ (3.2)§# (0.9) (1.3) (2.4) (1.9)# (2.7) (1.5) (2.5) (3.1)

1.22 1.40 1.47 1.30 1.12 1.22 1.21 1.20 1.32 1.37 1.33 1.47 3.8 4.0 3.8 3.9 1.73 1.90 1.85 1.70

(0.19) (0.24) (0.15) (0.17) (0.07) (0.09) (0.20) (0.03) (0.19) (0.32) (0.16) (0.10) (0.55) (0.78) (1.16) (0.77) (0.59) (0.68) (0.36) (0.34)

(0.07) (0.06) (0.06)§ (0.04)§ (0.07) (0.11) (0.10) (0.08)

12.5 10.7 11.9 10.8 13.9 12.6 12.2 13.4

(1.7) (2.7) (1.9) (1.4) (2.5) (4.4) (2.1) (2.6)

14.5 14.4 17.4 16.3 17.4 15.1 16.1 18.1

(1.8) (3.5) (2.8) (3.2) (2.2) (3.6) (1.3) (3.4)

1.17 1.37 1.47 1.51 1.28 1.25 1.37 1.36

(0.14) (0.28) (0.21)** (0.24)** (0.24) (0.20) (0.37) (0.21)

Mean (S. D.), n Å 6. * AW higher than S, p õ 0.01. † AW higher than S, p õ 0.001. ‡ AW U higher than S U, p õ 0.001. § S higher than AW, p õ 0.01. Ø S U higher than AW U, p õ 0.01. # S U higher than AW U, p õ 0.025. ** S higher than AW, p õ 0.02.

in the LC and its terminal areas [67]. There are concomitant increases in noradrenergic cell bodies of DHPG (the deaminated intraneuronal metabolite of NE following NE reuptake), indicative of increased NE release, and decreases in DOPAC (the deaminated intraneuronal metabolite of dopamine), indicative of reduced dopamine metabolism or reduced conversion of dopamine to NE by dopamine hydroxylase in storage vesicles [28]. These findings suggest that depletion of NE in the LC during acute stress is due to the inability of NE synthesis (beyond the rate-limiting step of tyrosine hydroxylation) to keep pace with release. By contrast, chronic (14–30 days) stress leads to increased activity of tyrosine hydroxylase in LC-NE terminal areas in hippocampus and frontal cortex, indicative of increased synthesis of dopamine and NE [28]. Exercise studies are needed to examine specific aspects of synthesis (e.g., expression of mRNA for tyrosine hydroxylase) and metabolism of these neuromodulators. Stone [67] compared acute running stress with injections of reserpine or a methyl- p-tyrosine, which inhibit NE storage and synthesis, respectively. Running stress did not alter storage

of NE, so it is likely that the NE depletion in the running condition was derived from newly synthesized NE and not from reuptake mechanisms. This conclusion appears also to be true for 5-HT. Chaouloff [13] has reported that acute treadmill running is accompanied by increases in brain tryptophan and the synthesis and metabolism of 5-HT. Our finding of similar ACTH levels after prior exposure to controllable or uncontrollable shock agrees with past research [44]. Also, our results agree with other research using inescapable shock that reported increased DA and DOPAC levels only in the PVN and the arcuate nucleus [78]. Nonetheless, ACTH levels did not differ between the activity wheel and sedentary animals, despite higher DA in the arcuate nucleus and higher DOPAC/DA in the PVN for the sedentary group. Activation of brain serotonergic and adrenergic systems increase plasma ACTH by stimulating secretion of corticotropin releasing factor from the parvocellular area of the PVN [8,58] or by direct effects on ACTH and corticosterone [15], though the effects of DA and NE are controversial [58]. Inhibition of DA activity in the ar-

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DISHMAN ET AL. Administration of the benzodiazepine agonist diazepam to rats prior to inescapable shock attenuates the escape deficit 24 h later [24]. Also, behavioral responses similar to those induced by inescapable foot shock are seen after injection of the benzodiazepine inverse-agonist, b-carboline [23]. It is important to distinguish the ways anxiety and depression may be distinguished by responses to inescapable foot shock because they are related disorders among humans and may represent a common continuum [54]. In summary, chronic activity wheel running attenuated the increased latency to escape foot shock and the reduction in norepinephrine levels in the locus coeruleus and dorsal raphe observed concomitantly after exposure to both uncontrollable and controllable foot shock. These changes were accompanied by altered levels or metabolic activity of serotonin in ascending norepinephrine terminal regions. These findings provide experimental evidence using an animal model to support reports by humans that physical activity reduces depression and anxiety. Furthermore, the findings suggest that chronic activity wheel running by young female rats is accompanied by adaptations in brain monoamine systems that modulate the attenuated escape deficit. ACKNOWLEDGEMENTS

This work was supported by University of Georgia Research Foundation to R.K.D. and USPHS Grant MH-44893 to K.J.R. We thank Cliff H. Summers for his helpful comments. Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NIH publication 86-23, 1985 edition. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense (para. 4-3, AR 360-5).

REFERENCES FIG. 2. Plasma [concentrations] of adrenocorticotrophin (ACTH) (pg/ ml) and prolactin (ng/ml) after about 1 h of foot shock escape in a shuttle box. Activity wheel and sedentary rats are compared. Matched pairs had either controllable or uncontrollable foot shock on the previous day. There were no effects of activity wheel running or controllability of foot shock on either ACTH or prolactin, p ú 0.10. Values are means { SEM.

cuate nucleus permits the release of prolactin [8], while 5-HT and NE can modulate prolactin release via the stimulatory effects of thyrotropin releasing hormone [8]. The ACTH levels observed were five- to sixfold higher than we have reported elsewhere for unstressed females of this strain [76]. This observation suggests that the intensity and duration of the foot shock maximized ACTH secretion, masking group or condition differences, or it suggests that secretagogues other than monoamines released ACTH. Prolactin levels were elevated, though not markedly so, above levels typically seen in female rats in the absence of stress [40,76,77]. We are unaware of reports of prolactin responses to the shuttle box test, but we reported elsewhere no effects of activity wheel running or controllability of foot shock on ACTH or prolactin levels after foot shock in a study of male Sprague– Dawley rats [21]. Although the foot shock escape deficit model is largely isomorphic with shared features of human depression, its construct validity for depression remains unclear, because most of those features also are common to anxiety disorders. Early findings that tricyclic antidepressants, but not anxiolytics, reversed the escape deficit [62] have not been supported by later studies, which show similar efficacy for anxiolytics and atypical antidepressants [78].

1. Anisman, H.; Suissa, A.; Sklar, L. S. Escape deficits induced by uncontrollable stress: Antagonism by dopamine and noradrenaline agonists. Behav. Neural Biol. 28:34–47; 1980. 2. Anisman, H.; Zacharko, R. M. Behavioral and neurochemical consequences of stressors. In: Kelly, D. D., ed. Stress-induced analgesia. New York: New York Academy of Sciences; 1986:205–225. 3. Aston–Jones, G. Potent interactions between NE and 5-HT systems. Biol. Psychiatry 35(9) :621; 1994. 4. Aston–Jones, G.; Akaoka, H.; Chariety, P.; Chouvet, G. Serotonin selectively attenuates glutamate-evoked activation of noradrenergic locus coeruleus neurons. J. Neurosci. 11:760–769; 1991. 5. Baldesserini, R. J. Current status of antidepressants: Clinical pharmacology and therapy. J. Clin. Psychol. 50:117–126; 1989. 6. Barchas, J. D.; Friedman, D. X. Brain amines: Response to physiological stress. Biochem. Pharmacol. 12:1232–123; 1963. 7. Baraban, J. M.; Aghajanian, G. K. Noradrenergic innervation of serotonergic neurons in the dorsal raphe: Demonstration by electron microscopic autoradiography. Brain Res. 204:1–11; 1981. 8. Bennett, G. W.; Whitehead, S. A. Mammalian neuroendocrinology. New York: Oxford University Press; 1983:119–250. 9. Blanchard, D. C.; Williams, G.; Lee, E. M. C.; Blanchard, R. J. Taming of wild Rattus norvegicus by lesions of the mesencephalic central gray. Physiol. Psychol. 9:157; 1981. 10. Bleir, P.; Montigny, C.; Chaput, Y. Modifications of the serotonin system by antidepressant treatments: Implications for the therapeutic response in major depression. J. Clin. Psychopharmacol. 7:24S– 35S; 1987. 11. Bliss, E. L.; Ailion, J.; Zwanziger, J. Metabolism of norepinephrine, serotonin, and dopamine in rat brain with stress. J. Pharmacol. Exp. Ther. 164:122–133; 1968. 12. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram concentrations of protein utilizing the principle of protein-dye binding. Anal. Chem. 72:248–254; 1976.

/ 2a36 2282 Mp 404 Thursday Mar 06 08:00 AM EL–BRB (v. 42, no. 4) 2282

PHYSICAL ACTIVITY ATTENUATES ESCAPE DEFICIT AFTER FOOTSHOCK 13. Chaouloff, F. Effects of acute physical exercise on central serotonergic systems. Med. Sci. Sports Exerc. 29(1); 1997. 14. Chaouloff, F. Physical exercise and brain monoamines: A review. Acta Physiol. Scand. 137:1–13; 1989. 15. Chaouloff, F. Physiopharmacological interactions between stress hormones and central serotonergic systems. Brain Res. Rev. 18:1– 32; 1993. 16. Cicardo, V. H.; Carbone, S. E.; Carneiro de Rondina, D.; Mastronardi, I. O. Stress by forced swimming in the rat: Effects of mianserin and moclobemide on gabaergic-monoaminergic systems in the brain. Comp. Biochem. Physiol. 83:133–135; 1986. 17. Clement, H. W.; Gemsa, D; Wesemann, W. The effect of adrenergicdrugs on serotonin metabolism in the nucleus-raphe-dorsalis of the rat, studied by in vivo voltammetry. Eur. J. Pharmacol. 217:43–48, 1992. 18. Cooperstein, S. J.; Lazarow, A.; Kurfess, N. J. A microspectrophotometric method for the determination of succinic dehydrogenase. J. Biol. Chem. 186:129–139; 1950. 19. Couch, J. R. Responses of neurons in the raphe nucleus to serotonin, norepinephrine and acetylcholine and their correlations with an excitatory synaptic input. Brain Res. 19:137–150; 1970. 20. Dishman, R. K.; Dunn, A. L.; Youngstedt, S. D.; Davis, J. M.; Burgess, M. L.; Wilson, S. P.; Wilson, M. A. Increased open-field locomotion and decreased striatal GABAA binding after activity wheel running. Physiol. Behav. 60:699–705; 1996. 21. Dishman, R. K.; Warren, J. M.; Youngstedt, S. D.; Yoo, H.; Bunnell, B. N.; Mougey, E. H.; Meyerhoff, J. L.; Jaso–Friedmann, L.; Evans, D. L. Activity wheel running attenuates suppression of Natural Killer Cell activity after foot shock. J. Appl. Physiol. 78:1547–1554; 1995. 22. Drugan, R. C.; Maier, S. F. Analgesic and opioid involvement in the shock-elicited activity and escape deficits produced by inescapable shock. Learn. Motiv. 14:30–48; 1983. 23. Drugan, R. C.; Maier, S. F.; Skolnick, P.; et al. An anxiogenic benzodiazepine receptor ligand induces learned helplessness. Eur. J. Pharmacol. 113:453–457; 1985. 24. Drugan, R. C.; et al. Librium prevents the analgesia and shuttlebox escape deficit typically observed following inescapable shock. Pharmacol. Biochem. Behav. 21:749–754; 1984. 25. Dunn, A. L.; Reigle, T. G.; Youngstedt, S. D.; Armstrong, R. B.; Dishman, R. K. Brain norepinephrine and metabolites after treadmill training and wheel running in rats. Med. Sci. Sports Exerc. 28:204– 209; 1996. 26. Dunn, A. L.; Dishman, R. K. Exercise and the neurobiology of depression. Exerc. Sport Sci. Rev. 19:41–98; 1991. 27. Esler, M.; Jennings, G.; Lambert, G.; Meredith, I.; Horne, M. Overflow of catecholamine neurotransmitters to the circulation: Source, fate, and functions. Physiol. Rev. 70:963–985; 1990. 28. Fillenz, M. Noradrenergic neurons. Cambridge, England: Cambridge University Press; 1990:1–238. 29. Foote, S. L.; Bloom F. E.; Aston–Jones, G. Nucleus locus ceruleus: New evidence of anatomical and physiological specificity. Physiol. Rev. 63:844–914; 1983. 30. Frankfurt, M.; McKittrick, C. R.; Luine, V. N. Short-term fluoxetine treatment alters monoamine levels and turnover in discrete brain nuclei. Brain Res. 650:127–132; 1994. 31. Frazer, A. Regionally selective effects in brain of typical and atypical antidepressants. In: Neurobiology of affective disorders. New York: Raven Press; 1993:17–21. 32. Fuller, R. W. Serotonin receptors involved in regulation of pituitary– adrenocortical function in rats. Behav. Brain Res. 73:215–219; 1996. 33. Geracioti, T. D., Jr.; Kalogeras, K. T.; Pigott, T. A.; Demitrack, M. A.; Altemus, M.; Chrousos, G. P.; Gold, P. W. Anxiety and the hypothalamic–pituitary–adrenal axis. In: Burrows, G. D.; Roth, M.; Noyes, R., Jr. Handbook of anxiety, vol. 3. The neurobiology of anxiety. Amsterdam: Elsevier; 1990:355–364. 34. Glavin, G. B.; Pare, W. P.; Sandbak, T.; Bakke, H.-K.; Murison, B. Restraint stress in biomedical research: An update. Neurosci. Biobehav. Rev. 18:223–249; 1994. 35. Gold, P. W.; Goodwin, F. K.; Chrousos, G. P. Clinical and biochemical manifestations of depression, related to the neurobiology of stress (second of two parts). N. Engl. J. Med. 319:413–420; 1988.

405

36. Hoffmann, P.; Elam, M.; Thoren, P; Hjorth, S. Effects of long-lasting voluntary running on the cerebral levels of dopamine, serotonin and their metabolites in the spontaneously hypertensive rat. Life Sci. 54:855–861; 1994. 37. Holloszy, J. O. Biochemical adaptations in muscle: Effects of exercise on mitochondrial O2 uptake and respiratory enzyme activity in skeletal muscle. J. Appl. Physiol. 242:2278–2282; 1967. 38. Iimori, K.; Tanaka, M.; Hohno, Y.; Ida, Y.; Nakagawa, R.; Hoaki, Y.; Tsuda, A.; Nagasaki, N. Psychological stress enhances noradrenergic turnover in specific brain regions in rats. Pharmacol. Biochem. Behav. 16:637–640; 1982. 39. James, M. D.; Hole, D. R.; Wilson, C. A. Differential involvement of 5-hydroxytryptamine (5HT) in specific hypothalamic areas in the mediation of steroid-induced changes in gonadotrophin release and sexual behaviour in female rats. Neuroendocrinolgy 49:561–569; 1989. 40. Kant, G. J.; Lenox. R. H.; Bunnell, B. N.; Mougey, E. H.; Pennington, L. L.; Meyerhoff, J. L. Comparision of stress response in male and female rats: Pituitary cyclic AMP and plamsa prolactin, growth hormone and corticosterone. Psychoneuroendocrinology 8:421– 428; 1983. 41. Koyama, Y; Kayama, Y. Mutual interactions among cholinergic, noradrenergic and serotonergic neurons studied by ionophoresis of these transmitters in rat brain-stem nuclei. Neuroscience 55:1117– 1126; 1993. 42. Lenhart, H.; Reinstein, D.; Strowbridge, B.; Wurtman, R. Neurochemical and behavioral consequences of acute, uncontrollable stress: Effects of dietary tyrosine. Brain Res. 303:215–223; 1984. 43. Luppi, P. H.; Aston–Jones, G.; Akaoka, H.; Chouvet, G.; Jouvet, M. Afferent-projections to the rat locus-coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin-B subunit and phaseous-vulgaris-leukoagglutinin. Neuroscience 65:119–160; 1995. 44. Maier, S. F.; Ryan, S. M.; Barksdale, C. M.; Kalin, N. H. Stressor controllability and the pituitary-adrenal system. Behav. Neurosci. 100:669; 1986. 45. Maynert, E. W.; Levi, R. Stress-induced release of brain norepinephrine and its inhibition by drugs. J. Pharmacol. Exp. Ther. 143:90–95; 1964. 46. McCulloch, T. L.; Bruner, J. S. The effect of electric shock upon subsequent learning in the rat. J. Psychol. 7:333–336; 1939. 47. McKay, L.; Bradberry, C.; Oke, A. Ascorbic acid oxidase speeds up the analysis for catecholamines, indoleamines and their metabolites in brain tissue using high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 311:167–169; 1984. 48. Mendelson, S.; McEwen, B. S. Autoradiographic analyses of the effects of adrenalectomy and corticosterone on 5-HT 1A and 5-HT 1B receptors in the dorsal hippocampus and cortex of the rat. Neuroendocrinology 55:444–450; 1992. 49. Moore, K. E.; Lariviere, E. W. Effects of stress and d-amphetamine on rat brain catecholamines. Biochem. Pharmacol. 13:1098–1100; 1964. 50. Morgan, W. P., ed. Physical activity and mental health. Washington, DC: Taylor & Francis; 1996. 51. Mougey, E. H.; Lambe, L. M.; Meyerhoff, J. L.; Kant, J. Extraction procedure for plasma ACTH. Soc Neurosci. Abstr. 14:445; 1988. 52. Palkovits, M.; Brownstein, M. J. Maps and guide to microdissection of the rat brain. New York: Elsevier; 1988. 53. Panksepp, J. The neurochemistry of behavior. Annu. Rev. Psychol. 37:77; 1986. 54. Paul, S. M. Anxiety and depression: a common neurobiological substrate? J. Clin. Psychiatry Suppl. 49(10):13–16; 1988. 55. Petty, F.; Sherman, A. D. Regional aspects of the prevention of learned helplessness by desipramine. Life Sci. 26:1447–1452; 1980. 56. Petty, F.; Sherman, A. D. GABAergic modulation of learned helplessness. Pharmacol. Biochem. Behav. 15:567–570; 1981. 57. Petty, F.; Sherman, A. D. Learned helplessness induction decreases in vivo cortical serotonin release. Pharmacol. Biochem. Behav. 18:649–651; 1983. 58. Plotsky, P. M.; Cunningham, E. T., Jr.; Widmaier, E. P. Catecholaminergic modulation of corticotropin-releasing factor and adrenocorticotropin secretion. Endocr. Rev. 10:437–458; 1989.

/ 2a36 2282 Mp 405 Thursday Mar 06 08:00 AM EL–BRB (v. 42, no. 4) 2282

406

DISHMAN ET AL.

59. Porsolt, R. D.; Bertin, A.; Blavet, N.; Deniel, M.; Jalfre, M. Immobility induced by forced swimming in rats: Effects of agents which modify central catecholamine and serotonin activity. Eur. J. Pharmacol. 57:201–210; 1979. 60. Renner, K.; Luine, V. Determination of monoamines in brain nuclei by high performance liquid chromatography with electrochemical detection: Young vs. middle-aged rats. Life Sci. 34:2077–2083; 1984. 61. Renner, K.; Luine, V. Analysis of temporal and dose-dependent effects of estrogen on monoamines in brain nuclei. Brain Res. 336:64–71; 1986. 62. Sherman, A. D.; Petty, F. Additivity of neurochemical changes in learned helplessness. Behav. Neural. Biol. 35:344–353; 1982. 63. Sherman, A. D.; Sacwuitne, J. L.; Petty, F. Selectivity of the learned helplessness model of depression. Pharmacol. Biochem. Behav. 16:449–454; 1982. 64. Shopsin, B.; Gershon, S.; Goldstein, M.; Friedman, E.; Wilk, S. Use of synthesis inhibitors in defining a role for biogenic amines during imipramine treatment in depressed patients. Psychopharmacology (Berlin) 1:239–249; 1975. 65. Shiekhattar, R; Aston–Jones, G. Sensory responsiveness of brain noradrenergic neurons is modulated by endogenous brain-serotonin. Brain Res. 623:72–76; 1993. 66. Soares, J.; Naffah–Mazzacoratti, M. G.; Cavalheiro, E. A. Increased serotonin levels in physically trained men. Braz. J. Med. Biol. Res. 27:1635–1638; 1994. 67. Stone, E. A. Accumulation and metabolism of NE in rat hypothalamus after exhaustive stress. J. Neurochem. 21:589–601; 1973. 68. Stone, E. A.; Platt, J. E. Brain noradrenergic receptors and resistance to stress. Brain Res. 237:405–414; 1982. 69. Wang, R. Y.; Aghajanian, G. K. Enhanced sensitivity of amygdaloid neurons to serotonin and norepinephrine after chronic antidepressant treatment. Eur. J. Pharmacol. 74:27–35; 1981. 70. Watson, S. J. The brain’s stress axis and affective disorders: Basic and clinical studies. In: Neurobiology of affective disorders. New York: Raven Press; 1993:59–60.

71. Weiss, J. M.; Glazer, H. I. Effects of acute exposure to stressors on subsequent avoidance-escape behavior. Psychosom. Med. 3:499– 521; 1975. 72. Weiss, J. M.; Glazer, H. I.; Pohorecky, L. A.; Brick, J.; Miller, N. E. Effects of chronic exposure to stressors on avoidance-escape behavior and on brain norepinephrine. Psychosom. Med. 37:522–534; 1975. 73. Weiss, J. M.; Glazer, H. I.; Pohorecky, L. A. Coping behavior and neurochemical changes: An alternative explanation for the original ‘‘learned helplessness’’ experiments. In: Serban, G.; Kling, A., eds. Animal models in human psychobiology. New York: Plenum Press; 1976:141–173. 74. Weiss, J. M.; Goodman, P. A.; Losito, B. G.; Corrigan, S.; Charry, J. M.; Bailey, W. H. Behavioral depression produced by an uncontrollable stressor: Relationship to norepinephrine, dopamine, and serotonin levels in various regions of rat brain. Brain Res. 3:167–205; 1981. 75. Weiss, J. M.; Simson, P. G. Electrophysiology of the locus coeruleus: Implications for stress-induced depression. In: Koob, G. F.; Ehlers, C. L.; Kupfer, D. J., eds. Animal models of depression. Boston: Birkhauser; 1989:111–134. 76. White–Welkley, J. E.; Bunnell, B. N.; Mougey, E. H.; Meyerhoff, J. L.; Dishman, R. K. Treadmill exercise training and estradiol differentially modulate hypothalamic–pituitary–adrenal cortical responses to acute running and immobilization. Physiol. Behav. 57:533–540; 1995. 77. White–Welkley, J. E.; Warren, G. L.; Bunnell, B. N.; Mougey, E. H.; Meyerhoff, J. L.; Dishman, R. K. Treadmill exercise training and estradiol increase plasma ACTH and prolactin after novel foot shock. J. Appl. Physiol. 80:931–939; 1996. 78. Zacharko, R. M.; Anisman, H. Pharmacological, biochemical, and behavioral analyses of depression. In: Koob, G. F.; Ehlers, C. L.; Kupfer, D. J., eds. Animal models of depression Boston: Birkhauser; 1989:204–238.

/ 2a36 2282 Mp 406 Thursday Mar 06 08:00 AM EL–BRB (v. 42, no. 4) 2282