Epinephrine, norepinephrine, dopamine and serotonin: Differential effects of acute and chronic stress on regional brain amines

Brain Research, 239 (1982) 417424

417

Elsevier Biomedical Press

EPINEPHRINE, NOREPINEPHR1NE, DOPAMINE AND SEROTONIN: D I F F E R E N T I A L EFFECTS OF A C U T E A N D C H R O N I C STRESS O N REGIONAL BRAIN AMINES

KEVIN A. ROTH, IVAN M. MEFEORD and JACK D. BARCHAS Nancy Pritzker Laboratory of Behavioral Neuroehemistry, Department of P~ychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.)

(Accepted October 8th, 1981) Key word~: epinephrine - - norepinephrine - - dopamine - - serotonin stress

brain amines

SUMMARY Following acute cold swim stress, hypothalamic epinephrine concentrations were markedly lowered and remained decreased for 24 h, while norepinephrine concentrations were decreased, but returned to baseline within 14 h. With oscillation stress repeated daily for 21 days, hypothalamic norepinephrine, hypothalamic epinephrine, and hippocampal norepinephrine turnover were decreased and absolute concentrations were increased. Repeated stress had little effect on serotonin, dopamine or their metabolites. These results suggest that hypothalamic epinephrine concentration and turnover are particularly responsive to acute and chronic stress. The decreased epinephrine and norepinephrine turnover under chronic stress may be responsible in part for the behavioral and endocrine changes observed in chronically stressed rats.

INTRODUCTION Acute and chronic stress produces a number of changes in catecholaminergic and serotinergic systems in the central nervous system. Acutely, stress increase the turnover of catecholamines and with relatively severe stress serotonin turnover1,2, H. These changes are reflected as decreased concentration of transmitter and/or increased concentration of metabolites. Unlike acute stress which decreases norepinephrine concentration in brain, chronic stress may not alter, or may increase norepinephrine concentrations 1. Other work has indicated changes in serotinergicZ, dopaminergic 3 and epinergic 12 systems with chronic stress. Several lines of evidence suggest that brain epinephrine is particularly responsive to stress. Hypothalamic epinephrine concentrations decrease by a greater percentage 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

418 than dopamine, norepinephrine and serotonin concentrations after acute stress t3 and changes in phenylethanolamine-N-methyltransferase (PNMT) activity occur under acute and repeated stress12,15. We have undertaken a series of studies on the effects of acute and chronic stress on brain catecholamines and serotonin with particular emphasis on the epinergic system in hypothalamus. Initially we examined the time course of recovery from acute stress in hypothalamic catecholamine and serotonin concentrations to ascertain if epinephrine concentrations rebound to control levels with a similar time course as other monoamines. It has been reported that after inescapable shock norepinephrine concentrations were still decreased at considerable time periods thereafter17 1.% Secondly, we examined the effects of chronic and repeated stressors on catecholamine and serotonin concentrations in several brain regions. The turnover of norepinephrine and epinephrine after repeated stress was assessed using a dopamine-/3-hydroxylase inhibitor. MATERIALS AND METHODS Adult male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA j 180-200 g were used throughout. Animals were housed in groups of 4 with food and water available ad libitum and 12 h day/night cycles. After appropriate treatment rats were sacrificed by decapitation, and the brains removed and dissected on a bed of crushed ice. In individual experiments, one or more of the following regions were removed (approximate tissue weights given in parentheses): hypothalamus (30 mg), brain stem (130 mg), basal ganglia including caudate and putamen (70 mg), hippocampus (60 mg) and frontal cortex (30 mg). Samples were placed in 1.5 ml polypropylene tubes and stored on dry-ice until assayed. The tissue concentration of dopamine, its metabolites dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), norepinephrine (NE), epinephrine (EPI), and serotonin and its metabolite, 5-hydroxyindoleacetic acid (5-H1AA), were determined by ion pairing reverse phase HPLC with electrochemical detection 8,9. Statistical analysis was performed with analysis of variance and Student's t-test. All data is presented as X ~ S.E.M. In the first experiment, rats were exposed to either a 30-min oscillation stress or a 3-min cold swim (4 °C) and sacrificed at various time points after the stress. Hypothalami were removed and catecholamine and serotonin concentrations measured. For the oscillation stress, rats were placed in groups of four on a 12 ~ 18 inch platform which oscillated horizontally at approximately 3 Hz. Rats were unrestrained during this procedure. In experiment two, the effect of repeated oscillation stress on hypothalamic catecholamines and serotonin was examined over time. Rats were given either 1, 2, 3, 7, 14 or 21 days of 30 min oscillation stress/day, and sacrificed 24 h after the last stress and hypothalamic amines measured. After 21 days, catecholamine and serotonin concentrations were measured in all the regions mentioned above. In the final experiment, the turnover of norepinephrine and epinephrine after 7 days of 0.5 h oscillation stress/day were assessed after dopamine-fl-hydroxylase inhibition with

74 5- 03 57 ~ 02* 57 i 05*

EPI

7.56; df = 2, 19; P <~ 0.01 4.12; df = 2, 19; P -< 0.05 11.51;df = 2, 1 7 ; P <." 0.01 11.85; df = 2, 19 ;P < 0.01

* P -< 0.05 vs. control.

F = F = F~ F --

(12) (5) (5)

Control 0 h poststress 3 h poststress

EPI: NE: DOPAC: HVA:

(n)

Group 2673 ~ 102 2225 ± 81" 2606 ± 87

NE 415 ! 31 5005- 44 437 ± 53

Dopamine 60 ± 03 91 ± 10" 62±0.3

DOPAC

Effect o f O.5 h oscillation stress on hypothalamic dopamine, serotonin and metabolites (ng/g tissue)

TABLE 1

52 5- 04 95 5- 13" 55 ~ 05

HVA

1604 + 46 1654 + 95 1565 i 8 1

5-HT

939 5- 49 1119 ~ 53 942 ~ 5 l

5-HIAA

4~

420 80

i

70

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i

~

.~"

NOREPINEPHRINE ~.,,.

60

W

3086 2700 '~ z 2314

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1829 ~. I IN EPHRIN'N~E. ' N ~

I

-~ 40

1543

c~ -~

20

771

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10

386

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I BASAL

I 0

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I 48 HRS POST COLD SWIM

Fig. 1. Hypothalamic epinephrine and norepinephrine recovery from acute cold swim stress. Basal group, n == 10; poststress groups, n 4-5. Epinephrine: F =: 13.18; df = 5,28; P <" 0.01. Norepinephrine: F =- 2.87, df ..... 5,29; P --~ 0.05. *P < 0.05 vs basal. fusaric acid. Twenty-four hours after the last stressor experimental and control rats were injected intraperitoneally with either saline or 80 mg/kg fusaric acid and sacrificed 60 min later. The degree of depletion after synthesis inhibition gives an indication of turnover, a marked depletion indicating high turnover, a slight depletion indicating low turnover. Epinephrine concentrations were measured in hypothatamus and brain stem, norepinephrine concentrations were measured in the regions mentioned earlier. RESULTS Both acute stressors, cold swim and oscillation, produced similar effects on catecholamines. Immediately after acute stress, hypothalamic D O P A C and HVA were markedly increased, and returned to basal levels within 3 h (Table 1). Dopamine concentrations were unaffected. In the hypothalamus, norpinephrine and epinephrine concentrations were decreased immediately after stress and maximally decreased one hour after stress (Fig. 1 and Table I); 1 h after cold swim norepinephrine concentrations decreased about 20 ~,,, and epinephrine concentrations about 35 o/. After cold swim stress, norepinephrine concentrations returned to basal levels within 14 h; however, epinephrine concentrations were still decreased after 24 h and returned to baseline within 48 h (Fig. 1). With repeated oscillation stress for 0-21 days and measuring hypothalamic catecholamines and serotonin 24 h after the last stress, no change in dopamine, DOPAC, serotonin and 5-HIAA was seen. Hypothalamic norepinephrine and epinephrine concentrations were approximately equal to basal concentrations after 1--3

(5) (5)

Basal Stressed

* P < 0.05 versus basal ** n.d, = n o t determined.

Cortex

Basal Stressed

Hippocampus

(5) (5)

(5) (5)

(5) (5) (5) (5) (5)

Basal Stressed

Basal ganglia

Basal Stressed

Brain stem

Basal Stressed

Hypothalamus

(n)

319 ± 28 350 ± 13

224 ± 16 308 =k 32*

318 i 40 382 ± 70

768 :k 63 814 ± 46

2431 ± 78 2874 a~ lOl*

NE

A n i m a l s were sacrified 24 h after the last stress.

n.d. n.d.

n.d. n.d.

n.d.** n.d.

08 i 01 09 i 01

57 ~ 03 71 ~ 04*

EPI

n.d. n,d.

n.d. n.d.

7666 :k 554 7714 ± 428

71 ± 04 74 ± 06

572 i 19 574 ± 16

Dopamine

n.d, n.d.

n.d. n.d.

914 ± 70 930 ± 86

23 :k 03 19 ± 02

90 :k 08 98 ~ 06

DOPA C

Effect o f 21 clays ~?fO.5 h oscillation stress~day on brain catecholamines, serotonin and metabolites (ng/g tissue)

T A B L E II

653 ± 65 613 ± 27

530 ± 55 620 ! 25

914 ± 39 953 ± 64

924 ± 37 865 ± 31

1261 ± 69 1240 ± 29

5-HT

294 £ 34 283 ± 16

357 i 30 368 ± 20

615 _-k 32 605 -k 46

443 ± 15 400 ± 17

594 =k 41 661 i 29

5-HIAA

4~

422 i i i i

z ~E
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i

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, ~ . . . . ~ _

EPINEPHRINE

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llO

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80

,~ "r

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2> I

O

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i

i

i

0 1 2 3

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1

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14

21

,

DAYS

Fig. 2. Effect of repeated oscillation stress on hypothalamic epinephrine, norepinephrine and dopamine. Subjects were sacrificed 24 h after the last stress. Unstressed group, n ~:- t 6 ; stressed groups, n -~ 5. E p i n e p h r i n e : F = 3 . 0 7 ; d f ~ 6 , 3 9 ; P ~-: 0.05. Norepinephrine: F ~:- 3 . 6 3 ; d f : : 6 , 3 9 ; P < 0 . 0 1 , * P < 0.05 vs 0 day.

days of oscillation stress and were significantly increased from 7 to 21 days (Fig. 2). In animals exposed to 21 days of oscillation stress, no changes in dopamine, DOPAC, serotonin or 5-HIAA were observed in any region. Hypothatamic epinephrine and norepinephrine were significantly increased; also, hippocampal norepinephrine was increased (Table !I). Turnover studies with fusaric acid indicated that 7 days of oscillation stress produced an apparent decrease in turnover of norepinephrine in both hypothalamus and hippocampus, and epinephrine turnover was decreased in hypothalamus and brain stem, (Fig. 3). 140

F - ' l NAIVE 120

I~

REPEATEDSTRESS

.~ 1 0 0

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0 (..)

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±

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40 20

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NOREPINEPHRINE

/.i

// // //

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z~ // // // / /

//

i// /1" // l/ // // /-/ ,r/

EPINEPHRINE

Fig. 3. Effect of the D B H inhibitor fusaric acid, 80 mg/kg i.p., on brain norepinephrine and epinephrine concentrations in naive and repeatedly stressed (7 days of 0.5 h oscillation stress/day) rats. Injection of fusaric acid was 24 h after the last stress and rats were sacrificed I h postinjection n ~ 6-8/ group. Naive saline values were (ng/g tissue): norepinephrine; hypothalamus, 2488 ± 81 ; brain stem, 885 _-E 42; hippocampus, 380 5: 40; cortex, 4t5 ~ 32; basal ganglia, 270 :t: 47. Epinephrine: hypothalamus, 55 3_ 01 ; brain stem, 10 ± 01. * P < 0.05, ** P < 0.01 naive vs repeated stress.

423 DISCUSSION Our results suggest several interesting findings on the effects of acute and chronic stress on brain catecholamines and serotonin. [n the hypothalamus, an area critically involved in stress responding, it was shown that acute cold swim stress produced a prolonged decrease in norepinephrine (1-14 h) and epinephrine concentrations (24-48 h). With repeated stress both these hypothalamic transmitters showed a marked decline in turnover and an increase in absolute level. Chronic repeated stress also resulted in elevations in hippocampal norepinephrine concentrations and a decreased turnover rate. The findings that chronic stress results in changes in hippocampal norepinephrine and hypothalamic norepinephrine and epinephrine is particularly interesting. These amines have been implicated in the control of ACTH release~,a°; it is known that P N M T inhibition as well as DBH inhibition results in elevated hypothalamicpituitary-adrenal activity. We have previously reported that chronic intermittent stress elevates the corticosterone concentration in plasmaV; this may be a result of the decreased norepinephrine and epinephrine turnover. Acute stress was shown to elevate the dopamine metabolites, HVA and DOPAC, in hypothalamus and brain stem, dopamine concentrations being relatively unaffected. This result indicates that acute stress can increase dopamine turnover; chronic stress failed to affect the levels of dopamine or its metabolites suggesting little effect of chronic stress on dopamine turnover. Hypothalamic serotonin and 5-HIAA were unaffected by acute oscillation stress; in chronically stressed rats no changes in serotonin or 5-HIAA were observed in the regions tested. In preliminary studies of the effects of chronic intermittent stress on brain catecholamines, we did not find elevated epinephrine concentrations in hypothalamus 11. Recently, we have found decreased hypothalamic epinephrine turnover in these animals (unpublished observations). The differential effects of repeated stress versus chronic intermittent stress on hypothalamic epinephrine concentrations needs to be examined; however, more importantly, both stress procedures resulted in decreased epinephrine turnover. A possible relationship between stressful life events and depression has been noted by numerous authors 5, and there had been work suggesting that epinephrinergic neurons may be involved in human depression4,~6; thus, it is interesting that animals exposed to repeated stress have decreased epinephrine turnover. It will be relevant to determine if the changes produced by chronic stress can be prevented or reversed by antidepressants. Regardless of the relationship between chronic stress and human depression, our results suggest that several reproducible changes occur in rats exposed to chronic stress. Particularly responsive to stress, either chronic or acute, were hypothalamic epinephrine and norepinephrine; decreased turnover of norepinephrine and epinephrine may be responsible, in part, for the behavioral and endocrine disturbances seen in chronically stressed rats and this possibility requires further investigation.

424 ACKNOWLEDGEMENTS This work supported by a Selected Research O p p o r t u n i t y A w a r d from the Office of N a v a l Research, SR0-001 : N00014-79-C-0796. We t h a n k Sue Poage for her assistance in preparing this manuscript. REFERENCES 1 Anisman, H., Neurochemicat changes elicited by stress: behavioral correlates. In H. Anisman and G. Bignami (Eds.), Psychopharmacology of Aversively Motivated Behavior, Plenum Press, New York, 1978, pp. 119-171. 2 Barchas, J. D. and Freedman, D. X., Brain amines: response to physiological stress, Biochem. Pharmacol., 12 (1963) 1232-1235. 3 Bliss, E. L., Effects of behavioral manipulations upon brain serotonin and dopamine. In J. D. Barchas and E. Usdin (Eds.), Serotonin and Behavior, Academic Press, New York~ 1973, pp. 315-324. 4 Christensen, N. J., Vestergaard, P., Sorenson, T. and Rafaelsen, O. J., Cerebrospinal fluid adrenaline and noradrenaline in depressed patients, Acta psychiat, scand., 6t (1980) I78-182. 5 Depue, R. A. (Ed.), The Psychobiology of the Depressive Disorders- Implications for the Effects of Stress, Academic Press, New York, 1979. 6 Ganong, W. F., Neurotransmitters and pituitary function: regulation of ACTH secretion, bed. Proc., 39 (1980) 2923-2930. 7 Katz, R. J., Roth, K. A. and Carroll, B. J., Acute and chronic stress effects on open field activity in the rat - - implications for a model of depression, Neurosci. Biobeha~. Rev., 5 (1981) 247-251. 8 Mefford, I. N., Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neuorsci. Meth., 3 (1981) 207-224. 9 Mefford, I. N., Gilberg, M. and Barchas, J. D., Simultaneous determination of catecholamines and 3,4-dihydroxyphenylacetic acid (DOPAC) in rat brain by HPLC with electrochemical detection, Analyt. Biochem., 104 (1980) 469-472. 10 Roth, K. A., Katz, R. J., Sibel, M., Mefford, I. N., Barchas, J. D. and Carroll, B. J., Central epinerglc inhibition of corticosterone release in rat, Life Sci., 28 (1981) 2389-2394. 11 Roth, K. A., Mefford, I. N., Barchas, J. D. and Katz, R. J., Central monoamine changes in chronically stressed rats, Neurosci. Abstr., 6 (1980) 452. 12 Saavedra, J. M. and Torda, T., Increased brainstem and decreased hypothalamic adrenalineforming enzyme after acute and repeated immobilizationstress in the rat, Neuroendocrinology, 31 (1980) 142-146. 13 Sauter, A. M., Baba, Y., Stone, E. A. and Goldstein, M., Effect of stress and of phenylethanolamine-N-methyltransferase inhibition on central norepinephrine and epinephrine levels, Brain Research, 144 (1978) 415--419. 14 Stone, E. A., Stress and catecholamines. In A. J. Friedhoff (Ed.), Catecholamines and Behaviour, Vol. 2, Plenum Press, New York, 1975, pp. 31-72. 15 Turner, B. B., Katz, R. J., Roth, K. A. and Carroll, B. J., Central elevation of phenylethanolamine N-methyltransferase activity following stress, Brain Research, 153 (1978) 419-422. 16 VonVoigtlander, P. F., Triezenberg, H. J. and Losey, E. O,, Interactions between clonidine and antidepressant drugs: A method for identifyingantidepressant-like agents, Neuropharmacology, 17 (1978) 375-381. 17 Stolk, J. M., Barchas, J. D., Goldstein, M,, Boggan, W. and Freedman, D. X., A comparison of psychotomimetic drug effects on rat brain norepinephrine metabolism, J. PharmacoL exp. Ther., 189 (1974) 42-50. 18 Stolk, J. M., Conner, R. L., Levine, S. and Barchas, J. D., Brain norepinephrine metabolism and shock-induced fighting behavior in rats: differential effects of shock and fighting on the neurochemical response to a common footshock stimulus, J. Pharmacol. exp, Ther., 190(1974) 193-209. 19 Weiss, J. M., Glazer, H. I., Pohorecky, L. A., Bailey, W. H. and Schneider, L. H., Coping behavior and stress-induced depression: studies of the role of brain catecholamines. In R. A. Depue (Ed.), The Psychobiology of the Depressive Disorders - - Implications for the Effects of Stress, Academic Press, New York, 1979, pp. 125-160.