The Japanese Journal of Psychiatry and Neurology, Vol. 46, No. 1, 1992

Effect of Forced-Running Stress on ,8-Adrenergic Receptors in Rat Brain Regions and Liver Toyonori Nakamura, M.D. Department of Psychiatry, Mie University School of Medicine, Tsu

Abstract: Rats were exposed to forced-running stress for 1 day, 3 days or a long term (approximately 2 weeks), and P-adrenergic receptor binding was then assayed using [~H]dihydroalprenoloI(DHA) in six brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in Fadrenergic receptor density was first evident on day 1. In contrast, a decrease in Fadrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [3H]DHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased ['HI DHA binding subsequently returned to control levels despite the exposure to the forced-running stress. In the cerebellum and the liver, ['HIDHA binding did not change significantly throughout the stress. These results indicate that the forced-running stress induces both the time- and region-specific changes in Fadrenergic receptors. Moreover, the rats showed either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term stress. Thus, we also examined the relationship of P-adrenergic receptors to these behavioral differences. ['HIDHA binding for the behavioral depression group was lower in the hippocampus and higher in the liver than for the spontaneous recovery group. Key Words:

stress, P-adrenergic receptors, behavioral depression, rat

Jpn J Psychialr Neurol 46: 187-195, 1992

INTRODUCTION Most mental disorders are considered as the results of a combination of internal conditioning factors (e.g. genetical predisposition) and the effects of external stress. The investigation of stress-induced changes in neuronal functioning will thus have important implications for our understanding Received for publication on Aug. 19, 1991. Mailing address: Toyonori Nakamura, M.D., Department of Psychiatry, Mie University School of Medicine, 174, Edobashi 2-chome, Tsu-shi, Mie 514, Japan.

of the pathophysiology of various psychoses. Acute and chronic stresses produce a number of changes in the noradrenergic system in the brain.' Acute stress increases the noradrenaline turnover, and this change is reflected as a decreased concentration of noradrenaline and/or an increased concentration of metabolites. Unlike acute stress, chronic stress does not decrease a noradrenaline concentration. Following chronic stress, the rats show a behavioral depression in which the concentration of noradrenaline in the locus coeruleus is significantly depleted.30 These results suggest that the noradrenergic

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function and behavioral activity vary according to the time course of stress. I n addition to the effects on noradrenaline, various forms of stress are known to modify P-adrenergic receptor binding.2n However, it has never been reported whether the forced-running stress affects P-adrenergic receptor binding. In the present study, we investigated changes in P-adrenergic receptors related to the time course of the forcedrunning stress in 6 brain regions and the liver. Moreover, since the rats show either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term forcedrunning stress as previously reported' * I IL', we also examined the relationship of /3adrenergic receptors to these behavioral differences.

MATERIALS AND METHODS

Animuls

Female Wistar rats (Shizuoka Animal Center, Japan), weighing 200 to 250 g, were housed in revolving drum cages with a I m circumference in a room maintained at 23OC under a 12-h light-dark cycle (light on at 07 :00 h ) . Food and water were freely available throughout all experiments. The female rats were chosen because they are more vulnerable than males to repeated stress'", and because women have a higher risk of depressive disorder' ;''. Furthermore, there was no effect of the estrous cycle on padrenergic receptor number.:i5 :Ix Forced-Running Stress

Spontaneous running activity was recorded as the number of drum revolutions per day for 4 weeks (Fig. 1 ) . We selected rats having a spontaneous running activity

3

SPONTANEOUS RECOVERY BEHAVIORAL DEPRESSION

RUNNING STRESS

2

1

3

4

I

5

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TIME (WEEKS)

Fig. 1 : Spontaneous running activity before and after forced-running stress, Spontaneous running activity was recorded as the number of drum revolutions per day. We selected rats having a spontaneous running activity of greater than 4,000 revolutions per day for subsequent exposure to forced-running stress. During the 2 weeks following the end of long-term forced-running stress, rats were divided into hehavioral depression and spontaneous recovery groups on the basis of their leve!s of spontaneous running activity as described in materials and methods.

Effect of Stress on P-Adrenergic Receptors of greater than 4,000 revolutions per day for this study. They were transferred into automatically revolving drums ( 1 m circumference, 5 revolutions/min) for subsequent exposure to the forced-running stress. The rat rectal temperature was measured every 3 hours throughout the exposure to the forced-running stress. The rat was given a 24-hour rest when the rectal temperature decreased to 34OC. However, the I-day and 3-day stress groups had no rest because the rectal temperature of these groups did not decrease to 34OC. For the long-term stress group, a series of stress and rest cycles was repeated 3 times. The duration of the forcedrunning stress tolerated by individual rats varied, with an average of 6.1 days for the initial exposure, 4.6 days for the second, and 3.3 days for the third. Following the end of the long-term forcedrunning stress, the rats were returned to the previously used revolving cages, and their spontaneous running activities were recorded every day for 2 weeks. As previously reported from our laboratory' l 1 12, the rats were divided into 2 behavioral groups on the basis of their levels of spontaneous running activity 2 weeks following the end of the long-term forced-running stress (Fig. 1 ). In the behavioral depression group, spontaneous running activity was under 2,000 revolutions per day for 2 weeks following the end of the long-term stress. In the spontaneous recovery group, however, spontaneous running activity increased gradually, and was greater than 4,000 revolutions per day at least for the last week before decapitation. The rats not meeting these criteria were excluded from the experiments. Binding Experiments

Following either 1-day or 3-day exposure to the forced-running stress, the rats were immediately sacrificed by decapitation. The long-term stress rats were sacrificed immediately after the third stress, without the third rest. The behavioral depression and spontaneous recovery rats were sacrificed 2

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weeks after the end of the long-term stress. The brains and right liver lobes were rapidly removed, and the brains were dissected into 6 regions (cerebral cortex, hippocampus, cerebellum, hypothalamus, pons + med.obl. and midbrain) according to the method of Glowinski and Iversen..' Tissues were homogenized in a 50 mM Tris-HCI buffer (pH 7.6 at 23OC) using an Ultra-Turrax for 25 sec. In the midbrain and pons + med.obl., the homogenate was centrifuged for 10 min at 1,OOO X g and then the supernatant was recentrifuged for 15 min at 43,000 X g. In other brain regions and the liver, the homogenates were centrifuged for 15 min at 43,000 X g. The respective pellets were stored at - 8OoC until assayed. Frozen tissues were suspended in the 50 mM Tris-HC1 buffer, maintained at 23OC for 10 min, homogenized for 2 0 sec, and then centrifuged for 15 min at 43,000 X g. The resulting pellets were resuspended in the same buffer and used for the binding assays. A ["HIDHA binding assay was done by a modification of a previously described method.") Membrane suspensions were incubated in triplicate for 20 min at 23OC with [3H]dihydroalprenolo1 ( ["HIDHA) (New England Nuclear, 90.3 Ci/mmole) in a total volume of 500 pl containing the 50 mM Tris-HCI buffer (pH 7.6 at 23OC). The binding assays were terminated by the addition of 7 ml of the same ice-cold buffer followed by a rapid filtration under a reduced pressure through Whatman G F / B filters. The filters were rinsed twice with 7 ml of the same buffer and transferred to counting vials containing 10 ml of UniverGel (Nakarai Chemicals). After 24 hours samples were counted by liquid scintillation spectrometry at an efficiency of 45-5070. The protein concentrations were determined by the method of Lowry et a1.I' using bovine serum albumin as the standard. In the cerebral cortex, hippocampus and cerebellum, the maximum number of [3H]DHA binding sites (Bmax)and the disso-

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ciation constant (K,) were obtained from Scatchard analyses.?; In other brain regions and the liver, p-adrenergic receptor density was assayed using 0.7 nM ["IDHA. Nonspecific binding was determined in parallel samples containing 10 pM I-alprenolol. Specific binding represented approximately 80% of the total binding in the cerebral cortex and the liver, 70% in the cerebellum and midbrain, and 60% in the pons + med.obl., hypothalamus and hippocampus at 0.7 nM ['HIDHA. Stutisticul Anulysis

All data are presented as means and standard deviations. Differences between the groups were tested using a one-way analysis of variance (ANOVA) followed by TurkeyKramer's multiple comparison test. RESULTS

Fig. 2 shows the effect of the forced-running stress on ["HIDHA binding in the 6 brain regions and the liver. The forced-running stress produced a significant reduction in /3-adrenergic receptor binding in the pons + med.ob1. [F(3,19) = 14.02, P < O.OOl], hypothalamus [F(3,19) = 9.46,

PONS t MED.OBL.

HYPOTHALAMUS

< 0.0011, midbrain [F(3,19) = 4.75, P < 0.051, cerebral cortex [F(3,19) = 19.06, P < 0.001] and hippocampus [F(3,19) = 15.86, P < 0.0011.

P

Moreover, the multiple comparison tests revealed that changes in the p-adrenergic receptor number related to the time course of the forced-running stress differed among the 6 brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in p-adrenergic receptor density was first evident on day 1 . In contrast, a decrease in p-adrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [:'HIDHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased ["HIDHA binding subsequently returned to the control levels despite the exposure to the forcedrunning stress. In the cerebellum and the liver, [:'HIDHA binding did not change significantly throughout the stress [cerebellum, F(3,19) = 3.10, N.S.; liver, F(3,19) = 0.93, N.S.]. Since the rats showed either the behavioral depression or the spontaneous recovery of

LIVER

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a, a

3.DAY STRESS

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Fig. 2: Effect of forcedrunning stress on p-adrenergic receptors in 6 brain regions and the liver. Columns and vertical bars are means and standard deviations from 4-8 experiments done in triplicate. a P < 0.01, compared to control. b P < 0.05, compared to control. P < 0.01. compared to control and long-term stress. d P < 0.01, compared to control and 1-day stress.

Effect of Stress on p-Adrenergic Receptors

PONS+ MEO.OBL.

30r

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'Z

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; -. =.

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0 CONTROL

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running activity during the 2 weeks following the end of the long-term stress (Fig. 11, we also investigated the relationship of p-adrenergic receptor density to these 2 types of behavior (Fig. 3 ) . Comparing padrenergic receptor bindings among the control, behavioral depression and spontaneous recovery groups, significant differences were observed in the cerebral cortex [F(2,14) = 18.97, P < 0.001], hippocampus [F(2,14) = 21.48, P < 0.001] and liver [F(2,14) =41.18, P < 0.0011. However, in the other regions no significant differences were observed [cerebellum, F( 2,14) = 0.18, N.S.; midbrain, F(2,14) = 3.39, N.S.; hypothalamus, F(2,14) = 0.99, N.S.; pons + med. obl., F(2,14) = 0.02, N.S.]. In the cerebral cortex, the P-adrenergic receptor number decreased in both the behavioral depression and spontaneous recovery groups, but there was no significant difference between these 2 groups. In contrast, [3H]DHA binding for the bchavioral depression group was lower in thc hippocampus and higher in the liver than binding for the spontaneous recovery group. The Scatchard analyses for the cerebral cortex, hippocampus and cerebellum revealed that the K, for ["HIDHA did not

Fig. 3: [:'HIDHA binding in control, behavioral depression and spontaneous recovery groups. Columns and vertical bars are means and standard deviations from 5-7 experiments done in triplicate. a P < 0.01, compared to control and spontaneous recovery. b P < 0.0 1, compared to control.

change significantly in any of these brain regions throughout the stress. The mean values (2SD) for K, (nM) were 1.96 A 0.26 in the cxebral cortex, 3.20 k 0.51 in hippocampus, and 0.70 f 0.18 in cerebellum. DISCUSSION

Previous reports have shown that various forms of stress including tail shocki3, immobilization3', i~olation'~, food deprivation"" and REM sleep deprivationl0 decrease the number of P-adrenergic receptors. In the present study, the forced-running stress also reduced p-adrenergic receptor density. Since a wide range of stressors induce a decrease in the P-adrenergic receptor number, this change may be a general response to most forms of stressful stimulation. Moreover, the present study demonstrated that changes in P-adrenergic receptor binding related to the time course of stress differed among the 6 brain regions and the liver. These regional characteristics can be classified into the following 4 types (Fig. 4): ( 1 ) region where P-adrenergic receptor binding begins to decrease after 1-day stress, and then plateaus for the duration of the forced-

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1) PONS t MED.OBL.

2) HYPOTHALAMUS

MIDBRAIN

3 ) CEREBRAL CORTEX HIPPOCAMPUS

1-DAY

3-DAY

LONG-TERM

Fig. 4: A schematic presentation of regional characteristics of ['HIDHA binding

4) CEREBELLUM LIVER

1-DAY

running stress (pons + medmbl.), ( 2 ) regions where the reduction in p-adrenergic receptor density becomes apparent on day I , and subsequently [{HIDHA binding returns to the control levels despite the exposure to the forced-running stress (hypothalamus and midbrain), ( 3 ) regions where the decrease in the p-adrenergic receptor number becomes most apparent on day 3, and then [{HIDHA binding plateaus for the duration of the forced-running stress (cerebral cortex and hippocampus), and ( 4 ) regions where padrenergic receptor density does not change significantly (liver and cerebellum). These regional characteristics may be attributed to regional variations in stress-induced noradrenaline release.:" :I2 In the hypothalamus, pons + med.obl. and midbrain, I day of the forced-running stress was required to elicit the decrease in p-adrenergic receptor density, whereas 3 days were necessary for this effect in the cerebral cortex and hippocampus (Fig. 2 ) . In accordance with the results of Stone and Platt", these results suggest that p-adrenergic receptors in the former regions are more sensitive to stress, while P-adrenergic receptors in the latter regions are more resistant. In previous studies, a decrease in the p-adrenergic receptor number in the cerebral

3-DAY

LONG-TERM

during forced-running stress. The ordinate indicates [:'HIDHA binding expressed as a percentage of the control value, and the abscissa indicates the time course of forced-running stress.

cortex was observed following 14 days of 2.5 hr/day immobilization stress'j4, or after 5 days of 90 min/day tail shock stress":'. In

the present study, [{HIDHA binding in the cerebral cortex showed a marked decrease after 3 days of the forced-running stress (Fig. 2 ) . This discrepancy may be due to differences in the duration and intensity of stress. In the midbrain and hypothalamus, [{HIDHA binding first decreased, but subsequently returned to the control levels despite the exposure to the forced-running stress reported similar (Fig. 2 ) . Torda er results for spleen tissue following a 40-daily exposure to a 2.5 hr/day immobilization stress. Recovery of ['HIDHA binding may represent an adaptation to stress in the midbrain and hypothalamus. In the liver and cerebellum, where the PLIadrenergic receptor subtype is dominant I' I N , the forced-running stress did not induce a reduction in p-adrenergic receptor binding (Fig. 2 ) . However, in the cerebral cortex and hippocampus, where the p,-adrenergic receptor subtype is dominant" IN, ['HIDHA binding decreased (Fig. 2 ) . The &adrenergic receptor subtype may thus exhibit a response to stress which differs from the p,-adrenergic receptor subtype. Interestingly,

Effect of Stress on P-Adrenergic Receptors a down-regulation of P-adrenergic receptors induced by antidepressants is restricted to the PI-adrenergic receptor subtype.n I s Comparison of rH]DHA binding between the behavioral depression and spontaneous recovery groups revealed that the hippocampal P-adrenergic receptor number in the behavioral depression group was lower than in the spontaneous recovery group (Fig. 3). Sherman and Petty" reported that infusion of noradrenaline into the hippocampus prior to exposure to uncontrollable shock prevents an avoidance-escape deficit from occurring. Sapolsky and M c E w e P have demonstrated that the hippocampus is associated with stress-induced cortisol secretion in the rat. Clinical studies of depressive disorder have shown the elevated plasma cortisol levels and an abnormality in the dexamethasone suppression test.' l5 2o Moreover, a high density of ['Hldesmethylimipramine binding sites has been reported for human hippocampus." Taken together, the hippocampus is most likely involved not only in behavioral depression of rats, but also in human depression. In the liver, P-adrenergic receptor density did not change significantly throughout the exposure to the forced-running stress, but nevertheless the p-adrenergic receptor number increased 2 weeks after the end of the long-term stress only in the behavioral depression group (Fig. 3). Although it is unclear what mechanisms are involved in an elevation of the liver P-adrenergic receptor number in the behavioral depression group, this results may be similar to an increase in serum transaminase induced by liver injury during stress." Furthermore, there are reports that electric stimulation of the ventromedial hypothalamic nucleus in the rat brain and acute human psychoses induce liver dysfunction (e.g. disturbance of androgen metabolism and of enzymatic activities in the urea cycle) .21 This dysfunction has been attributed to a breakdown in cerebro-hepatic homeostasis. Thus, the elevation of liver p-adrenergic receptor binding in the behav-

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ioral depression group may also be associated with the breakdown in cerebro-hepatic homeostasis. The present study demonstrates that the forced-running stress induced time- and region-specific changes in p-adrenergic receptors, and these regional characteristics were classified into 4 types. In addition, hippocampal and liver P-adrenergic receptors were associated with the behavioral depression. The precise role of P-adrenergic receptors in the pathophysiology of the behavioral depression, however, remains to be clarified in the future. ACKNOWLEDGMENTS The author wishes to thank Professor J. Nomura for his encouragement and support, and Drs. R. Tsujimura and I. Kitayama for helpful suggestions on the manuscript. This research was supported by Grant-in-Aid (#63770814) from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Anisman,

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