- Email: [email protected]
Differential hormonal and physiological responses to stress in Roman high- and low-avoidance rats
Physiology & Behavior, Vol. 28, pp. 259-263. Pergamon Press and Brain Research Publ., 1982. Printed in the U.S.A.
Differential Hormonal and Physiological Responses to Stress in Roman Highand Low-Avoidance Rats C. G E N T S C H , * M. L I C H T S T E I N E R , *
P. D R I S C O L L t
A N D H. F E E R *
*Psychiatric University Clinic, Basel Switzerland and tBehavioural Science Institute, ETHZ, Ziirich, Switzerland R e c e i v e d 10 A u g u s t 1981 GENTSCH, C., M. LICHTSTEINER, P. DRISCOLL AND H. FEER. Differential hormonal and physiological responses to stress in Roman high- and low-avoidance rats. PHYSIOL. BEHAV. 28(2) 259-263, 1982.--In the present experiments, plasma concentrations of corticosterone, ACTH, prolactin, blood glucose, serum free fatty acids, core temperature and fecal boli were measured immediately following the exposure of Roman high- and low-avoidance rats (RHANerh, R L A N e r h ) to various stressful situations. As compared to R L A N e r h rats, the RHA/Verh animals showed an attenuated emotional response in "non-self-menacing" (novel environment) situations. Differences between the 2 selected lines were not generally found in unstressed rats or in "high-stress" (ether, immobilisation, footshock) situations.
Stress-parameters
Emotional response
Selected rat lines
ROMAN high- and Roman low-avoidance rats (RHA/Verh and RLA/Verh, respectively) are selected for rapid learning of the conditioned avoidance response in a two-way shuttle box versus failing to learn that response. Comparisons of these two psychogenetically selected lines in further behavioural tests (e.g., openfield, complex labyrinth, HebbWilliams maze) have revealed additional differences between RHA/Verh and RLA/Verh animals, which have been primarily interpreted in terms of differences in activity levels and in emotional behaviour [1, 5, 7]. During a recent comparison on several genetically selected rat lines with individually and group-housed rats, we have described RHA/Verh rats as being more active, defecating less and showing an attenuated corticosterone secretion following exposure to a novel environment, than their RLA/Verh counterparts [9]. The present study was conducted in order to investigate more closely whether such differences between the two Roman lines may be generalized to other, more severe, stress situations. In addition to corticosterone levels [4, 8, 22, 23] and defecation score [3, 8, 9, 13], other hormonal and physiological parameters which have been proposed to be influenced by stress were also measured, such as plasma ACTH [4,15], prolactin [22] and blood glucose [17] levels, serum free fatty acids (FFA) [12,19] and body- (core) temperature [2,24]. METHOD
Naive RHA/Verh and R L A N e r h male rats, bred at the Behavioural Science Institute E T H and averaging 6 months of age, were used throughout all experiments.
After a 3 weeks acclimatisation period to the new environmental conditions (3 animals per cage, cage size 42x26x 14 cm; 12 hr light/12 hr dark cycle; 25°C room temperature; food and water ad lib), animals were individually housed (macrolon cages 26x20× 14 cm) for 24 hr prior to being tested. Such pre-isolation has previously been shown not to affect behavioural measures or basal hormone concentrations and ensured that each rat could be removed from its homecage without disturbing other experimental animals. All experiments were carded out in the middle of the dark phase with behavioural testing being followed immediately by sacrifice through decapitation and collection of trunk blood. For each test-situation six RHA/Verh and six RLA/Verh rats were used.
Test-Situations Tests were made under the following conditions:
(a) Unstressed controls. Animals were killed immediately after removal from homecage (6 rats of each line were used in the three replications). (b) Injection. Animals were injected intraperitoneally with 500/zl of saline and were killed following a further 10 rain in the homecage. (c) New cage. Rats were placed for 10 min in a new cage, identical to the homecage (furnished with fresh sawdust). (d) Openfield. Animals were placed for 10 rain in a wooden box (56x66 cm). The field was bordered by 12 cm side walls and illuminated by a 100 W red lamp suspended 80 cm above the centre. (e) Shuttle box as novel environment. Animals were
C o p y r i g h t © 1982 Brain R e s e a r c h Publications Inc.--0031-9384/82/020259-05503.00/1
260
(i~NTS('H t:t 4i.
placed in a shuttle box for I0 min. (Model 7502, U. Basile. Comerio, Italy), with no current applied. (J') Ether stress, Animals were placed in a glass cylinder (20 cm diameter, 26 cm high) which had been saturated with ether vapour. After 1 min of exposure, animals were transfered again to the homeeage, where they were allowed to recover for 9 min. (g) lmmobilisation. Animals were forced to remain for 10 min in a black coloured plastic tube (10 cm diameter), immobilizing them through the pressure of a metallic slide. (h) Inescapable footshock. The above-mentioned shuttle box was used. However, during these 10 min periods, inescapable footshocks at 30 sec intervals (5 sec, 100 V) were delivered by a static scrambler through the stainless steel grid floor.
Determinations (a/c) Plasma corticosterone and ACTH concentrations were determined by RIA [11], as were plasma prolactin concentrations [14]. (d) Blood glucose was determined by the hexokinase-method (Glucoquant, Boehringer Mannheim GmbH) using deproteinized plasma. (e) Serum free fatty acids (FFA) were determined according to the method of Lorch [16]. (f) Core temperature was measured immediately following sacrifice by a metallic probe (Ellab Instruments, Elekrolaboratoriet, Kopenhagen) to a depth of 6 cm from the anus. (g) Fecal boli were counted for each animal at the end of all 10 rain test periods. RESULTS
The results for all determinations, for all experimental situations, are summarised in Fig. 1. In regard to baseline secretion levels, only plasma corticosterone concentrations were significantly different (twoway analysis of variance F(1/30=4.6, p<0.05) between RHA/Verh and RLA/Verh animals. However, several differences between RHA/Verh and RLA/Verh rats were shown to reach a significant level (Student's t-test) in the stress situations. The most marked differences between the two genetically selected lines were found after exposures to novel environments (new cage, openfield and shuttle box as novel environment). During these three situations RHA/Verh rats showed a significantly attenuated response when compared with their RLA/Verh counterparts in respect to corticosterone-, ACTH- and prolactin-secretion as well as in defecation scores. A similar difference in hormone secretion was found after an IP injection of saline. During the more serious (physical) stress-situations, such as ether-stress, immobilisation and inescapable footshock, differences between RHA/Verh and RLA/Verh rats were not normally observed, seldom reaching a significant level. Concerning blood glucose and F F A concentrations, RHA/Verh and RLA/Verh animals differed neither under unstressed conditions nor after any of the seven stressexposures. Core temperatures were also not significantly different between the two lines, either in unstressed animals or after having been exposed to the various stressful situations, except after IP injection of saline. Further analysis of the present data (Spearman's rank correlation) revealed highly significant correlations between corticosterone, A C T H , prolactin, glucose and defecation score in both selected lines. On the other hand, no significant correlations between F F A or core temperature existed to the other stress parameters, with only two exceptions (Table 1).
I)ISCUSS|ON
In the present study, hormonal and physiological "stress-parameters" have been measured in unstressed male RHA/Verh and RLA/Verh rats, after exposure to se,: eral experimental situations. In unstressed rats the only >ignificant difference between the two Roman lines was l\mnd in plasma corticosterone concentration. As all other parameters showed equal values in both rat lines, it has to be assumed that RHA/Verh and RLA/Verh animals do not differ drastically in their basal secretion levels. Different stress-induced secretion levels, however, were found between RHA/Verh and RLA/Verh rats (Fig. I), par-ticularly in "novel environment" situations. These differences appear to be situation-dependent, as in "high-stress" situations (e.g., inescapable footshock, ether and immobilisation) no significant differences between the two selected lines existed. These results may be either due to a ceiling effect, or they may indicate that both lines react similarly to a strong stress whereas they differ in their ability to interpret certain "non-self-menacing" situations te.g., novel environments/. To help indicate which of the two Roman lines shows the " a b e r r a n t " reactivity in response to stress-exposure, similar experiments to those described above were carried out in a group of unselected rats derived from a local Wistar strain (unpublished results). Marked parallels between RLA/Verh rats and the unselected animals were observed, e.g., open-field exposure was already a strong stressor and IP injection of saline induced a significant elevation in stress hormone concentrations. We therefore tend to interpret the hormonal and physiological differences between RHA/Verh and RLA/Verh rats to indicate that RHA/Verh animals represent a rat line characterized by an unusually attenuated emotional responsiveness and stress reactivity. Some experimental conditions could have influenced the present findings and should be mentioned here. The differences between RHA/Verh and RLA/Verh rats could be due to a temporal dissimilarity in hormonal secretion or, although quite improbable [4,22], the differences in plasma concentrations between the two selected lines could be caused, if maximal secretion would have been already passed after only 10 min, by a temporal difference in returning to baseline secretion levels. Additionally, possible differences due to an unequal circadian rhythm in RHA/Verh and RLA/Verh animals, which would induce not only a different baseline secretion level but also different stress-induced hormone secretions [4, 22, 23], could have influenced the present findings. Although an interpretation on the grounds of such kinetic or circadian differences between the two Roman lines cannot ultimately be excluded by our data, such an explanation for the dissimilarities between RHA/Verh and RLA/Verh rats seems to us rather improbable. The present data would, in that case, indicate that such effects would be situation-dependent, only occuring under "'low-stress" conditions. We would rather believe that the different stressreactivity in RHA/Verh and RLA/Verh rats is caused by a difference in informational input and/or central processing, ultimately depending upon neurochemical differences. Although few neurochemical analyses have been performed on the two selected rat lines to date, several studies have been recently completed or are in progress. Whereas Overstreet ct al. [18] found no significant differences in muscarinic cholinergic receptor binding in several brain regions, differ-
RESPONSES TO STRESS
ug%
i ,o®
261
PRO" ACTH LACTIN pg Iml nglml
,L
1,50:.
~,I.UCOSE
FFA FEqlIt
mgllOOml
~
BOLl
,k
60..
200..
60. ~
20Q.
3o(3
38
6_
,b,
}
=I
I=OQ~
0
!
1 ~ . ~
=i _®
~'.i=©
60..
00 ,o.1
151X
3O(
,L
20Q 100.
d8 I,--
,o
6~~~ 2~i] --1
10(1
FIG. 1. Responses of RHA/Verh and RLA/Verh rats after exposure to seven experimental situations as measured by changes in different "stress-parameters." Each column represents the mean_+S.E.M, of six animals, with the exception of unstressed animals, (N= 18 for those columns). A: p<0.05; B: p<0.005; C: p<0.0005. I~RLA/Verh, IIRHANerh.
262
GENTSCH f 7 Ai TABLE I SPEARMAN'S RANK CORRELATION COEFFICIENT qo) BETWEEN HORMONAL AND PHYSIOLOGICAI "STRESS-PARAMETERS," INCLUDING ALL SEVEN EXPERIMENTAl_,SITUATIONS (ALWAYS42 DETERMINATIONSPER RAT LINE)
Corticosterone ACTH Prolactin Glucose FFA Temperature Defecation
Corticosterone
ACTH
Prolactin
Glucose
FFA
Temperature
+0.36* +0.60:~ +0.55¢ +0.48t +0.62:~ +0.69~ +0.13 +0.17 +0.25 +0.00 +0.44~ +0.73:~
+0.49~ +0.43t +0.55:~ +0.61~t +0.10 +0.03 + 0.22 -0.02 +0.61 $ +0.68~
+0.47t +0.65~: +0.01 -0.02 +0.29* +0.06 +0.51:~ +0.63z~
+0.26 +0.16 +0.22 +0.31" +0.61:~ +0.74:~
+0.21 -0.17 +0.29* +0.20
+0.36* +0.13
Defecation
*p<0.05, fp<0.01, ~p<0.001. Upper value: RLA/Verh. Lower value: RHA/Verh.
ences in 5-HT metabolism have been described [6], and Krfiuchi (personal communication) has observed a reduced binding of WB-4101 to hypothalamic a-adrenerglc receptors in R L A N e r h rats when compared to their RHA/Verh counterparts. Such findings may be of special interest for the understanding o f differences in release-mechanisms for pituitary-hormones. We have recently determined aH-diazepam binding in various central regions of RHA/Verh and RLA/Verh rats [10]. R H A N e r h animals showed a higher binding (4-12%, depending on subregion) than their RLA/Verh counterparts. These results are quantitatively and qualitatively consistent with data obtained with Maudsley non reactive (MNR) and Maudsley reactive (MR) rats [20]. Robertson [20] postulated that a lowered emotionality is connected with an enhanced 3H-diazepam binding in the central nervous system, and he was able to confirm this hypothesis in several mouse strains differing in emotionality [21]. These findings are of particular interest, as it has been shown that the behavioural responses of R H A N e r h and R L A N e r h rats in stress situations are also generally comparable to those of the MNR and MR strains, respectively [5]. Looking briefly at the different hormonal and physiological parameters which have been proposed and used here for the quantification o f stress, we have found significant correlations between plasma corticosterone, ACTH, prolactin and blood glucose concentrations. Such findings support the measurement of these parameters as reliable indicators of stress. Defecation score has once again been shown as another valuable parameter for the determination of stress experienced by an animal, reflecting very consistently differences observed in stress-hormone plasma concentrations. No correlation between F F A concentration and any other parameter (stress hormones) could be detected, but since plasma catecholamines were not measured, it would be premature to pass judgement on the reliability of F F A as an indicator of stress. Plasma catecholamines are not only known to be instantly and drastically affected by the decapi-
tation process, but the momentary reaction of plasma catecholamines to stress plays an important role in the activation of lipase-caused increases in F F A . Correlations between core temperature and the other parameters were not consistent. N o significant difference between the two Roman lines was observed, except for after IP injection of saline. This latter finding has been subsequently replicated (unpublished results), but is for the moment not clearly understood, being unique within all determinations in that RHA/Verh rats showed a significantly stronger reaction than did their RLA/Verh counterparts. In summary, comparison of hormonal and physiological responses to experimental stress-situations in RHA/Verh and R L A N e r h male rats revealed an attenuated reactivity in R H A N e r h animals following exposure to "non-selfmenacing" situations, as compared to control (baseline) and "high-stress" conditions, where few differences were found between the two selected rat lines. Plasma concentrations of corticosterone, ACTH, prolactin, blood glucose, and defecation score were supported as reliable indicators of stress experienced by an animal, whereas core temperature was not. In order to fully evaluate the value of F F A , further studies would have to be conducted incorporating measurements of plasma catecholamines as well.
ACKNOWLEDGEMENTS We would like to thank Dr. J. Baumann (Endokrinologische Abteilung, Universitfits-Kinderspital, Basel) for corticosterone and ACTH determinations, Dr. P. Marbach (Sandoz AG, Basel) for prolactin determinations and Mr. P. Kerker (Hoffmann La Roche AG, Basel) for FFA determinations. We thank Miss M. L. Sfoggia for her help in taking care of the animals and her technical assistance. One of the authors (C. G.) was supported by "Schweizerischer Nationalfonds zur F6rderung der wissenschaftlichen Forschung" (Grant No. 3.855.0.79).
RESPONSES
TO STRESS
263
REFERENCES
1. Bhttig, K., P. Driscoll, J. Schlatter and H. J. Uster. Effects of nicotine on the exploratory locomotion patterns of female Roman high- and low-avoidance rats. Pharrnac. Biochem. Behav. 4: 435-439, 1976. 2. Bl~sig, J., V. H611t, U. B~iuede and A. Herz. Involvement of endorphins in emotional hyperthermia of rats. Life Sci. 23: 2525-2532, 1978. 3. Broadhurst, P. L. Determinants of emotionality in the rat. I. Situational Factors. Br. J. Psychol. 48: 1-12, 1957. 4. Dallman M. F. Circadian patterns of stress-induced ACTH secretion are modified by corticosterone responses. Endocrinology 100: 138--147, 1977. 5. Driscoll, P. and K. B~ittig. Behavioral and physiological correlates of psychogenetic selection (RHA/Verh vs RLA/Verh rats). In: L'animal de laboratoire au service de l'homme. Lyon: Coll. Found. M6rieux, 1979, pp. 477-487. 6. Driscoll, P., J. Dedek, J. R. Martin and K. B~ttig. Regional 5-HT analysis in Roman high- and Roman low-avoidance rats following MAO inhibition. Eur. J. Pharmac. 68: 373-376, 1980. 7. Diitsch, H. R. and K. B~ittig. Psychogenetische Unterschiede (RHA- vs RLA-Ratten) im Vermeidungslernen, Offenfeldverhalten, Hebb-Williams Intelligenztest und bei der Labyrinthexploration. Z. exp. angew. Psychol 24: 230-243, 1977. 8. File, S. E. and S. V. Vellucci. Behavioural and biochemical measures of stress in hooded rats from different sources. Physiol. Behav. 22: 31-35, 1979. 9. Gentsch, C., M. Lichtsteiner and H. Feer. Locomotor activity, defecation score and corticosterone levels during an openfleld exposure: A comparison among individually and group-housed rats, and genetically selected rat lines. Physiol. Behav. 27" 183186, 1981. 10. Gentsch, C. M. Lichtsteiner and H. Feer. 3H-diazepam binding sites in Roman high- and Roman low-avoidance rats. Experientia, in press, 1982. 11. Girard, J., J. B. Baumann, U. Biihler, K. Zuppiger, H. G. Haas, J. J. Staub and H. I. Wyss. Cyproteroneacetate and ACTH adrenal function. J. clin. Endocr. Metab. 47: 581-586, 1978. 12. Gottschalk, L. A., J. M. Cleghorn, G. C. Gleser and J. M. Ianoco. Studies of relationships of emotions to plasma lipids. Psychosom. Med. 27: 102-111, 1965.
13. Hall, C. S. Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. J. comp. Physiol. 18: 385-403, 1934. 14. H~usler, A., H. P. Rohr, P. Marbach and E. FI0ckiger. Changes in prolactin secretion in lactating rats assessed by correlative morphometric and biochemical methods. J. Ultrastruct. Res. 64: 74--84, 1978. 15. Kokka, N., J. F. Garcia, R. George and H. W. Elliott. Growth hormone and ACTH secretion: evidence for an inverse relationship in rats. Endocrinology 90: 735-743, 1972. 16. Lorch, E. Extraction of free fatty acids from plasma or serum in a continuous-flow system. Analyt. Biochem. 2,11:307-312, 1969. 17. McMurthy, J. P. and B. C. Wexler. Hypersensitivity of spontaneously hypertensive rats (SHR) to heat, ether, and immobilisation. Endocrinology 108: 1730-1736, 1981. 18. Overstreet, D. H., P. Driscoll, J. R. Martin and H. I. Yamamura. Brain muscarinergic cholinergic receptor binding in Roman high- and low-avoidance rats. Psychopharmacology 72: 143-145, 1981. 19. Pavle, P. and W. L. Holmes. Free fatty acid metabolism during stress: exercise, acute cold exposure and anaphylactic shock. Lipids 8: 142-150, 1973. 20. Robertson, H. A. Differences in benzodiazepine receptor binding in Maudsley reactive and Maudsley non-reactive rats. Eur. J. Pharmac. 50: 455---457, 1978. 21. Robertson, H. A. Benzodiazepine receptors in "emotional" and "non-emotional" mice: comparison of four strains. Eur. J. Pharmac. 56: 163-166, 1979. 22. Seggie, J. A. and G. M. Brown. Stress response patterns of plasma corticosterone, prolactin, and growth hormone in the rat, following handling or exposure to novel environments. Can. J. Physiol. Pharmac. 53: 629-637, 1975. 23. Torrellas, A., C. Guaza, J. Borrell and S. Borreli. Adrenal hormones and brain catecholamines responses to morning and afternoon immobilization stress in rats. Physiol. Behav. 26: 129133, 1981. 24. Yokoi, Y. Effect of ambient temperature upon emotional hypertermia and hypothermia in rabbits. J. appl. Physiol. 21: 17951798, 1965.
Differential Hormonal and Physiological Responses to Stress in Roman Highand Low-Avoidance Rats C. G E N T S C H , * M. L I C H T S T E I N E R , *
P. D R I S C O L L t
A N D H. F E E R *
*Psychiatric University Clinic, Basel Switzerland and tBehavioural Science Institute, ETHZ, Ziirich, Switzerland R e c e i v e d 10 A u g u s t 1981 GENTSCH, C., M. LICHTSTEINER, P. DRISCOLL AND H. FEER. Differential hormonal and physiological responses to stress in Roman high- and low-avoidance rats. PHYSIOL. BEHAV. 28(2) 259-263, 1982.--In the present experiments, plasma concentrations of corticosterone, ACTH, prolactin, blood glucose, serum free fatty acids, core temperature and fecal boli were measured immediately following the exposure of Roman high- and low-avoidance rats (RHANerh, R L A N e r h ) to various stressful situations. As compared to R L A N e r h rats, the RHA/Verh animals showed an attenuated emotional response in "non-self-menacing" (novel environment) situations. Differences between the 2 selected lines were not generally found in unstressed rats or in "high-stress" (ether, immobilisation, footshock) situations.
Stress-parameters
Emotional response
Selected rat lines
ROMAN high- and Roman low-avoidance rats (RHA/Verh and RLA/Verh, respectively) are selected for rapid learning of the conditioned avoidance response in a two-way shuttle box versus failing to learn that response. Comparisons of these two psychogenetically selected lines in further behavioural tests (e.g., openfield, complex labyrinth, HebbWilliams maze) have revealed additional differences between RHA/Verh and RLA/Verh animals, which have been primarily interpreted in terms of differences in activity levels and in emotional behaviour [1, 5, 7]. During a recent comparison on several genetically selected rat lines with individually and group-housed rats, we have described RHA/Verh rats as being more active, defecating less and showing an attenuated corticosterone secretion following exposure to a novel environment, than their RLA/Verh counterparts [9]. The present study was conducted in order to investigate more closely whether such differences between the two Roman lines may be generalized to other, more severe, stress situations. In addition to corticosterone levels [4, 8, 22, 23] and defecation score [3, 8, 9, 13], other hormonal and physiological parameters which have been proposed to be influenced by stress were also measured, such as plasma ACTH [4,15], prolactin [22] and blood glucose [17] levels, serum free fatty acids (FFA) [12,19] and body- (core) temperature [2,24]. METHOD
Naive RHA/Verh and R L A N e r h male rats, bred at the Behavioural Science Institute E T H and averaging 6 months of age, were used throughout all experiments.
After a 3 weeks acclimatisation period to the new environmental conditions (3 animals per cage, cage size 42x26x 14 cm; 12 hr light/12 hr dark cycle; 25°C room temperature; food and water ad lib), animals were individually housed (macrolon cages 26x20× 14 cm) for 24 hr prior to being tested. Such pre-isolation has previously been shown not to affect behavioural measures or basal hormone concentrations and ensured that each rat could be removed from its homecage without disturbing other experimental animals. All experiments were carded out in the middle of the dark phase with behavioural testing being followed immediately by sacrifice through decapitation and collection of trunk blood. For each test-situation six RHA/Verh and six RLA/Verh rats were used.
Test-Situations Tests were made under the following conditions:
(a) Unstressed controls. Animals were killed immediately after removal from homecage (6 rats of each line were used in the three replications). (b) Injection. Animals were injected intraperitoneally with 500/zl of saline and were killed following a further 10 rain in the homecage. (c) New cage. Rats were placed for 10 min in a new cage, identical to the homecage (furnished with fresh sawdust). (d) Openfield. Animals were placed for 10 rain in a wooden box (56x66 cm). The field was bordered by 12 cm side walls and illuminated by a 100 W red lamp suspended 80 cm above the centre. (e) Shuttle box as novel environment. Animals were
C o p y r i g h t © 1982 Brain R e s e a r c h Publications Inc.--0031-9384/82/020259-05503.00/1
260
(i~NTS('H t:t 4i.
placed in a shuttle box for I0 min. (Model 7502, U. Basile. Comerio, Italy), with no current applied. (J') Ether stress, Animals were placed in a glass cylinder (20 cm diameter, 26 cm high) which had been saturated with ether vapour. After 1 min of exposure, animals were transfered again to the homeeage, where they were allowed to recover for 9 min. (g) lmmobilisation. Animals were forced to remain for 10 min in a black coloured plastic tube (10 cm diameter), immobilizing them through the pressure of a metallic slide. (h) Inescapable footshock. The above-mentioned shuttle box was used. However, during these 10 min periods, inescapable footshocks at 30 sec intervals (5 sec, 100 V) were delivered by a static scrambler through the stainless steel grid floor.
Determinations (a/c) Plasma corticosterone and ACTH concentrations were determined by RIA [11], as were plasma prolactin concentrations [14]. (d) Blood glucose was determined by the hexokinase-method (Glucoquant, Boehringer Mannheim GmbH) using deproteinized plasma. (e) Serum free fatty acids (FFA) were determined according to the method of Lorch [16]. (f) Core temperature was measured immediately following sacrifice by a metallic probe (Ellab Instruments, Elekrolaboratoriet, Kopenhagen) to a depth of 6 cm from the anus. (g) Fecal boli were counted for each animal at the end of all 10 rain test periods. RESULTS
The results for all determinations, for all experimental situations, are summarised in Fig. 1. In regard to baseline secretion levels, only plasma corticosterone concentrations were significantly different (twoway analysis of variance F(1/30=4.6, p<0.05) between RHA/Verh and RLA/Verh animals. However, several differences between RHA/Verh and RLA/Verh rats were shown to reach a significant level (Student's t-test) in the stress situations. The most marked differences between the two genetically selected lines were found after exposures to novel environments (new cage, openfield and shuttle box as novel environment). During these three situations RHA/Verh rats showed a significantly attenuated response when compared with their RLA/Verh counterparts in respect to corticosterone-, ACTH- and prolactin-secretion as well as in defecation scores. A similar difference in hormone secretion was found after an IP injection of saline. During the more serious (physical) stress-situations, such as ether-stress, immobilisation and inescapable footshock, differences between RHA/Verh and RLA/Verh rats were not normally observed, seldom reaching a significant level. Concerning blood glucose and F F A concentrations, RHA/Verh and RLA/Verh animals differed neither under unstressed conditions nor after any of the seven stressexposures. Core temperatures were also not significantly different between the two lines, either in unstressed animals or after having been exposed to the various stressful situations, except after IP injection of saline. Further analysis of the present data (Spearman's rank correlation) revealed highly significant correlations between corticosterone, A C T H , prolactin, glucose and defecation score in both selected lines. On the other hand, no significant correlations between F F A or core temperature existed to the other stress parameters, with only two exceptions (Table 1).
I)ISCUSS|ON
In the present study, hormonal and physiological "stress-parameters" have been measured in unstressed male RHA/Verh and RLA/Verh rats, after exposure to se,: eral experimental situations. In unstressed rats the only >ignificant difference between the two Roman lines was l\mnd in plasma corticosterone concentration. As all other parameters showed equal values in both rat lines, it has to be assumed that RHA/Verh and RLA/Verh animals do not differ drastically in their basal secretion levels. Different stress-induced secretion levels, however, were found between RHA/Verh and RLA/Verh rats (Fig. I), par-ticularly in "novel environment" situations. These differences appear to be situation-dependent, as in "high-stress" situations (e.g., inescapable footshock, ether and immobilisation) no significant differences between the two selected lines existed. These results may be either due to a ceiling effect, or they may indicate that both lines react similarly to a strong stress whereas they differ in their ability to interpret certain "non-self-menacing" situations te.g., novel environments/. To help indicate which of the two Roman lines shows the " a b e r r a n t " reactivity in response to stress-exposure, similar experiments to those described above were carried out in a group of unselected rats derived from a local Wistar strain (unpublished results). Marked parallels between RLA/Verh rats and the unselected animals were observed, e.g., open-field exposure was already a strong stressor and IP injection of saline induced a significant elevation in stress hormone concentrations. We therefore tend to interpret the hormonal and physiological differences between RHA/Verh and RLA/Verh rats to indicate that RHA/Verh animals represent a rat line characterized by an unusually attenuated emotional responsiveness and stress reactivity. Some experimental conditions could have influenced the present findings and should be mentioned here. The differences between RHA/Verh and RLA/Verh rats could be due to a temporal dissimilarity in hormonal secretion or, although quite improbable [4,22], the differences in plasma concentrations between the two selected lines could be caused, if maximal secretion would have been already passed after only 10 min, by a temporal difference in returning to baseline secretion levels. Additionally, possible differences due to an unequal circadian rhythm in RHA/Verh and RLA/Verh animals, which would induce not only a different baseline secretion level but also different stress-induced hormone secretions [4, 22, 23], could have influenced the present findings. Although an interpretation on the grounds of such kinetic or circadian differences between the two Roman lines cannot ultimately be excluded by our data, such an explanation for the dissimilarities between RHA/Verh and RLA/Verh rats seems to us rather improbable. The present data would, in that case, indicate that such effects would be situation-dependent, only occuring under "'low-stress" conditions. We would rather believe that the different stressreactivity in RHA/Verh and RLA/Verh rats is caused by a difference in informational input and/or central processing, ultimately depending upon neurochemical differences. Although few neurochemical analyses have been performed on the two selected rat lines to date, several studies have been recently completed or are in progress. Whereas Overstreet ct al. [18] found no significant differences in muscarinic cholinergic receptor binding in several brain regions, differ-
RESPONSES TO STRESS
ug%
i ,o®
261
PRO" ACTH LACTIN pg Iml nglml
,L
1,50:.
~,I.UCOSE
FFA FEqlIt
mgllOOml
~
BOLl
,k
60..
200..
60. ~
20Q.
3o(3
38
6_
,b,
}
=I
I=OQ~
0
!
1 ~ . ~
=i _®
~'.i=©
60..
00 ,o.1
151X
3O(
,L
20Q 100.
d8 I,--
,o
6~~~ 2~i] --1
10(1
FIG. 1. Responses of RHA/Verh and RLA/Verh rats after exposure to seven experimental situations as measured by changes in different "stress-parameters." Each column represents the mean_+S.E.M, of six animals, with the exception of unstressed animals, (N= 18 for those columns). A: p<0.05; B: p<0.005; C: p<0.0005. I~RLA/Verh, IIRHANerh.
262
GENTSCH f 7 Ai TABLE I SPEARMAN'S RANK CORRELATION COEFFICIENT qo) BETWEEN HORMONAL AND PHYSIOLOGICAI "STRESS-PARAMETERS," INCLUDING ALL SEVEN EXPERIMENTAl_,SITUATIONS (ALWAYS42 DETERMINATIONSPER RAT LINE)
Corticosterone ACTH Prolactin Glucose FFA Temperature Defecation
Corticosterone
ACTH
Prolactin
Glucose
FFA
Temperature
+0.36* +0.60:~ +0.55¢ +0.48t +0.62:~ +0.69~ +0.13 +0.17 +0.25 +0.00 +0.44~ +0.73:~
+0.49~ +0.43t +0.55:~ +0.61~t +0.10 +0.03 + 0.22 -0.02 +0.61 $ +0.68~
+0.47t +0.65~: +0.01 -0.02 +0.29* +0.06 +0.51:~ +0.63z~
+0.26 +0.16 +0.22 +0.31" +0.61:~ +0.74:~
+0.21 -0.17 +0.29* +0.20
+0.36* +0.13
Defecation
*p<0.05, fp<0.01, ~p<0.001. Upper value: RLA/Verh. Lower value: RHA/Verh.
ences in 5-HT metabolism have been described [6], and Krfiuchi (personal communication) has observed a reduced binding of WB-4101 to hypothalamic a-adrenerglc receptors in R L A N e r h rats when compared to their RHA/Verh counterparts. Such findings may be of special interest for the understanding o f differences in release-mechanisms for pituitary-hormones. We have recently determined aH-diazepam binding in various central regions of RHA/Verh and RLA/Verh rats [10]. R H A N e r h animals showed a higher binding (4-12%, depending on subregion) than their RLA/Verh counterparts. These results are quantitatively and qualitatively consistent with data obtained with Maudsley non reactive (MNR) and Maudsley reactive (MR) rats [20]. Robertson [20] postulated that a lowered emotionality is connected with an enhanced 3H-diazepam binding in the central nervous system, and he was able to confirm this hypothesis in several mouse strains differing in emotionality [21]. These findings are of particular interest, as it has been shown that the behavioural responses of R H A N e r h and R L A N e r h rats in stress situations are also generally comparable to those of the MNR and MR strains, respectively [5]. Looking briefly at the different hormonal and physiological parameters which have been proposed and used here for the quantification o f stress, we have found significant correlations between plasma corticosterone, ACTH, prolactin and blood glucose concentrations. Such findings support the measurement of these parameters as reliable indicators of stress. Defecation score has once again been shown as another valuable parameter for the determination of stress experienced by an animal, reflecting very consistently differences observed in stress-hormone plasma concentrations. No correlation between F F A concentration and any other parameter (stress hormones) could be detected, but since plasma catecholamines were not measured, it would be premature to pass judgement on the reliability of F F A as an indicator of stress. Plasma catecholamines are not only known to be instantly and drastically affected by the decapi-
tation process, but the momentary reaction of plasma catecholamines to stress plays an important role in the activation of lipase-caused increases in F F A . Correlations between core temperature and the other parameters were not consistent. N o significant difference between the two Roman lines was observed, except for after IP injection of saline. This latter finding has been subsequently replicated (unpublished results), but is for the moment not clearly understood, being unique within all determinations in that RHA/Verh rats showed a significantly stronger reaction than did their RLA/Verh counterparts. In summary, comparison of hormonal and physiological responses to experimental stress-situations in RHA/Verh and R L A N e r h male rats revealed an attenuated reactivity in R H A N e r h animals following exposure to "non-selfmenacing" situations, as compared to control (baseline) and "high-stress" conditions, where few differences were found between the two selected rat lines. Plasma concentrations of corticosterone, ACTH, prolactin, blood glucose, and defecation score were supported as reliable indicators of stress experienced by an animal, whereas core temperature was not. In order to fully evaluate the value of F F A , further studies would have to be conducted incorporating measurements of plasma catecholamines as well.
ACKNOWLEDGEMENTS We would like to thank Dr. J. Baumann (Endokrinologische Abteilung, Universitfits-Kinderspital, Basel) for corticosterone and ACTH determinations, Dr. P. Marbach (Sandoz AG, Basel) for prolactin determinations and Mr. P. Kerker (Hoffmann La Roche AG, Basel) for FFA determinations. We thank Miss M. L. Sfoggia for her help in taking care of the animals and her technical assistance. One of the authors (C. G.) was supported by "Schweizerischer Nationalfonds zur F6rderung der wissenschaftlichen Forschung" (Grant No. 3.855.0.79).
RESPONSES
TO STRESS
263
REFERENCES
1. Bhttig, K., P. Driscoll, J. Schlatter and H. J. Uster. Effects of nicotine on the exploratory locomotion patterns of female Roman high- and low-avoidance rats. Pharrnac. Biochem. Behav. 4: 435-439, 1976. 2. Bl~sig, J., V. H611t, U. B~iuede and A. Herz. Involvement of endorphins in emotional hyperthermia of rats. Life Sci. 23: 2525-2532, 1978. 3. Broadhurst, P. L. Determinants of emotionality in the rat. I. Situational Factors. Br. J. Psychol. 48: 1-12, 1957. 4. Dallman M. F. Circadian patterns of stress-induced ACTH secretion are modified by corticosterone responses. Endocrinology 100: 138--147, 1977. 5. Driscoll, P. and K. B~ittig. Behavioral and physiological correlates of psychogenetic selection (RHA/Verh vs RLA/Verh rats). In: L'animal de laboratoire au service de l'homme. Lyon: Coll. Found. M6rieux, 1979, pp. 477-487. 6. Driscoll, P., J. Dedek, J. R. Martin and K. B~ttig. Regional 5-HT analysis in Roman high- and Roman low-avoidance rats following MAO inhibition. Eur. J. Pharmac. 68: 373-376, 1980. 7. Diitsch, H. R. and K. B~ittig. Psychogenetische Unterschiede (RHA- vs RLA-Ratten) im Vermeidungslernen, Offenfeldverhalten, Hebb-Williams Intelligenztest und bei der Labyrinthexploration. Z. exp. angew. Psychol 24: 230-243, 1977. 8. File, S. E. and S. V. Vellucci. Behavioural and biochemical measures of stress in hooded rats from different sources. Physiol. Behav. 22: 31-35, 1979. 9. Gentsch, C., M. Lichtsteiner and H. Feer. Locomotor activity, defecation score and corticosterone levels during an openfleld exposure: A comparison among individually and group-housed rats, and genetically selected rat lines. Physiol. Behav. 27" 183186, 1981. 10. Gentsch, C. M. Lichtsteiner and H. Feer. 3H-diazepam binding sites in Roman high- and Roman low-avoidance rats. Experientia, in press, 1982. 11. Girard, J., J. B. Baumann, U. Biihler, K. Zuppiger, H. G. Haas, J. J. Staub and H. I. Wyss. Cyproteroneacetate and ACTH adrenal function. J. clin. Endocr. Metab. 47: 581-586, 1978. 12. Gottschalk, L. A., J. M. Cleghorn, G. C. Gleser and J. M. Ianoco. Studies of relationships of emotions to plasma lipids. Psychosom. Med. 27: 102-111, 1965.
13. Hall, C. S. Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. J. comp. Physiol. 18: 385-403, 1934. 14. H~usler, A., H. P. Rohr, P. Marbach and E. FI0ckiger. Changes in prolactin secretion in lactating rats assessed by correlative morphometric and biochemical methods. J. Ultrastruct. Res. 64: 74--84, 1978. 15. Kokka, N., J. F. Garcia, R. George and H. W. Elliott. Growth hormone and ACTH secretion: evidence for an inverse relationship in rats. Endocrinology 90: 735-743, 1972. 16. Lorch, E. Extraction of free fatty acids from plasma or serum in a continuous-flow system. Analyt. Biochem. 2,11:307-312, 1969. 17. McMurthy, J. P. and B. C. Wexler. Hypersensitivity of spontaneously hypertensive rats (SHR) to heat, ether, and immobilisation. Endocrinology 108: 1730-1736, 1981. 18. Overstreet, D. H., P. Driscoll, J. R. Martin and H. I. Yamamura. Brain muscarinergic cholinergic receptor binding in Roman high- and low-avoidance rats. Psychopharmacology 72: 143-145, 1981. 19. Pavle, P. and W. L. Holmes. Free fatty acid metabolism during stress: exercise, acute cold exposure and anaphylactic shock. Lipids 8: 142-150, 1973. 20. Robertson, H. A. Differences in benzodiazepine receptor binding in Maudsley reactive and Maudsley non-reactive rats. Eur. J. Pharmac. 50: 455---457, 1978. 21. Robertson, H. A. Benzodiazepine receptors in "emotional" and "non-emotional" mice: comparison of four strains. Eur. J. Pharmac. 56: 163-166, 1979. 22. Seggie, J. A. and G. M. Brown. Stress response patterns of plasma corticosterone, prolactin, and growth hormone in the rat, following handling or exposure to novel environments. Can. J. Physiol. Pharmac. 53: 629-637, 1975. 23. Torrellas, A., C. Guaza, J. Borrell and S. Borreli. Adrenal hormones and brain catecholamines responses to morning and afternoon immobilization stress in rats. Physiol. Behav. 26: 129133, 1981. 24. Yokoi, Y. Effect of ambient temperature upon emotional hypertermia and hypothermia in rabbits. J. appl. Physiol. 21: 17951798, 1965.