- Email: [email protected]
Differential effects of inescapable footshocks and of stimuli previously paired with inescapable footshocks on dopamine turnover in cortical and limbic areas of the rat
Life Sciences, Vol. 30, pp. 2207-2214 Printed in the U.S.A.
Pergamon Press
DIFFERENTIAL EFFECTS OF INESCAPABLE FOOTSHOCKS AND OF STIMULI PREVIOUSLY PAIRED WITH INESCAPABLE FOOTSHOCKS ON DOPAMINE TURNOVER IN CORTICAL AND LIMBIC AREAS OF THE RAT J.P. Herman, D. Guillonneau, R. Dantzer, B. Scatton ~, L. Semerdjian-Rouquier ~ and M. Le Moal Lab. Neurobiologie des Comportements, Universit~ de Bordeaux II, 146 Rue L~o Saignat, 33076 BORDEAUX CEDEX and ~ Synthelabo-LERS, 31 Avenue P.V. Couturier, BAGNEUX FRANCE (Received in final form April 12, 1982) SUMMARY The effect of electric footshocks and of exposure to environmental stimuli paired with electrical shocks upon the dopaminergic activity in various cortical and limbic areas of the rat were evaluated by measuring dihydroxyphenylacetic acid (DOPAC) levels in these areas. In animals exposed to a 20 min electric footshock session DOPAC concentrations were significantly increased in the anteromedial and sulcal frontal cortices, olfactory tubercle, nucleus accumbens and amygdaloid complex (by 66, 37, 28, 55 and 90% respectively). Re-exposure of rats to an environment where they had been shocked 24 h earlier induced an elevation of DOPAC content only in the anteromedial frontal cortex (by 47%). Plasma corticosterone levels were elevated in both situations. No change in serotonin or 5-hydroxyindolacetic acid content of these areas could he detected in either situation. The results show that electric footshocks and environmental stimuli associated to previous shocks both activate central dopaminergic systems, altough the patterns of activation are different. Recent studies suggest that the mesocorticofrontal and the mesollmbic dopaminergic (DA) neurons originating from the cell group located within the ventral tegmental area (1) are implicated in the control of cognitive processes and emotional behavior. Lesions with 6-hydroxydopamine (6-OHDA) of the mesocortical DA system at the terminal level induce learning and retention impairments in a delayed alternation task (2, 3). Lesions of the limbic DA terminals within the nucleus accumbens lead to perseveration, impairment of ongoing behavior and of investigatory exploration and more generally to impairment in the functional processes leading from motivation to action (4). These two DA systems are also activated during stressful situations. Thus, an increase in DA turnover in mesocortical and mesolimbic neurons has been observed following electric shocks (5-7), prolonged immobilization (8) or exposure to a new environment (9). These biochemical changes have been related to anxiety or fear induced by the experimental situation. However, various performance factors such as level of motor activity and reactivity to electric shocks could also account for the observed changes in the latter studies. A m a i n characteristic of emotional states is that they are subject to conditioning, i.e. they can he elicited by the presentation of previously neutral stimuli which have been paired with aversive stimuli. As a matter of fact it has been recently reported that changes in whole brain or hypothalamic noradrenergic activity induced by a stressor could be reinstated by a re0024-3205/82/252207-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2208
Conditioned
DOPAC Changes After Stress
exposure to stimuli associated with the stressor
Vol. 30, No. 25, 1982
(I0).
The aim of the present study was to determine whether a similar phenomenon could be observed in dopaminergic neurons following electric footshocks. The activity of DA neurons was assessed by measuring changes in the concentrations of DA, and one of its metabolite, DOPAC in various projection areas of the DA systems. Since noradrenergic and serotoninergic systems have been shown to be affected by stressful situations (11-13), we have also measured levels of noradrenaline (NA), serotonin (5-HT) and its metabolite, 5-hydroxyindolacetic acid (5 HIAA) in the same structures. METHODS Animals and housin$ conditions Male Sprague-Dawley rats weighing 300-350 g were used. The animals were housed in plastic cages in groups of five with food and water ad libitum. Temperature (23°C) and relative humidity (60%) of the animal house were kept constant. Light was on between 08.00 and 20.00 h. Experiments began after a 14-day period of acclimatization. Handlin$ and habituation
to environmental
stimuli
In order to minimize reactions to the experimental routine and to habituate the animals to the new environment represented by the experimental apparatus rats were handled for I0 days. Each day the animals were brought first to a room located nearby the experimental room and in which a white noise was maintained. Each rat was then brought to the experimental room and put into a conditioning chamber for 20 min. The chambers (Campden Instr.) were housed in a sound attenuating cubicle and were equipped with fan, light and a speaker connected to a white noise generator. After 20 min the animal was taken back to the first room and placed into a cage different from its home cage in order to habituate the remaining animals to the removal of one member of the group. The time schedule was kept constant throughout the period of handling and was identical to that fixed for the experiment itself. Handling as well as the experiment was conducted between 12.00 and 18.00 h. First experiment
: Inescapable
footshock session
The procedure described above was followed for the footshock session. Scrambled electric shocks were delivered through the stainless steel grid of the conditioning chamber by a constant current shock generator (Campden Instr.) Trains of 6 shocks (1.5 mA, 180 msec on, 180 msec off) were delivered at 8 sec intervals for 20 min. Control animals were placed in separate chambers in which no electric shocks were delivered. Second experiment ' : Repeated shocks and exposure to stimuli previously paired to shocks One group of rats submitted to electric shocks as described above were divided into subgroups 24 h later. Rats of the first subgroup received again the same shock treatment (Shock-shock group) while the rats of the second subgroup were placed into the conditioning chamber but not shocked (Shock-no shock group). Control animals were placed on both days in the chambers for 20 min, without receiving any electric shock.
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2209
Biochemical analysis Inmediately following the last experimental session, the animals were sacrificed by decapitation in an adjacent room. Trunk blood was collected for corticosterone determination. The brain was quickly removed and cut into l or 2 am thick coronal slices, perpendicular to the dorsal surface of the brain. Slices were placed on a refrigerated stage, brain structures corresponding to limbic, striatal and cortical DA projection areas were dissected out (see Fig.l for details) and put onto dry ice. The plasma and brain samples were stored at - 76°C until the biochemical analysis. 1lolo-losoop
FIG.
I
Schematic representation of sampling from rat brain slices. Approximate coordinates are given according to the atlas of Kbnig and Klippel (22). Abbreviations used are : FCm : anteromedial frontal cortex, FCs : sulcal frontal cortex ; CC : cingular cortex ; OT : olfactory tubercle ; ACC : nucleus accumbens; NC : caudate-putamen ; A : amygdaloid complex ; S : septum. Plasma corticosterone was measured by a competitive protein binding assay (]4), catecholamine and DOPAC by a radioenzymatic method (15). Indole compounds were measured by high performance liquid chromatography with electrochemlcal detection (16). Statistical analysis was performed using either the Student's t-test or analysis of variance followed by a Newman-Keuls' test for multiple comparisons. RESULTS Effests of inescapable footshocks After a 20 min shock session plasma corticosterone level increased
2210
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
markedly : from 206 ± 56 ng/ml in control rats to 528 ± 20 ng/ml in shocked rats (t = 5.3532, d.f. = 10, p < O.001). Under these experimental conditions DOPAC levels were significantly increased in the anteromedial and sulcal frontal corti ces, the nucleus accumbens, the olfactory tubercle and the amygdaloid complex but remained unaffected in the cingular cortex, caudate nucleus and septum (Fig 2). The electric shock session left DA levels unchanged in the structures examined but was accompanied by a significant decrease o~ NA levels in the anteromedial frontal cortex, amygdaloid complex and septum (Pig.2). No change in the concentrations of 5 HT or 5-HIAA could be detected in any of the regiorsexamined under these conditions (results not shown).
.~ l i e o
E 100
i 1 .2_ u
FCne
FCI
CC
OT
ACC
IIC
4
S
FIG. 2 Effects of electric shocks upon catecholamine and DOPAC content of different brain areas. Results are means with SEM of data obtained on 6 rats and are expressed as percentage of respective control values. Control values for DA, NA and DOPAC were in pg/mg tissue. Control values (]00%) DA NA DOPAC FCm (anteromedial frontal cortex) 187 ± 13 550 ± 28 53 ± 3 FCs (sulcal frontal cortex) 93 ± ]3 361 ± 35 60 ± 5 CC (cingulate cortex) 123 ± 7 838 ± 137 83 ± ]3 OT (olfactory tubercle) ]0100 ± 1]70 232 ± 40 ] ] 9 0 ± ]22 ACC (nucleus accumbens) I0700 ± 1000 390 ± 33 1350 ± 150 NC (caudate putamen) 22300 ± 1740 Not m e a s u r e d 2460 ± I ] 2 A (amygdaloid complex) 798 ± 65 1960 ± ]55 124 ± 18 S (septum) 562 ± 69 985 ± 10] 107 ± 69 .: p < 0.05
; , .:p < 0.01
; , . .:p < 0.005
Effects of stimuli previously paired with electric shocks Placement of the animals in the conditioning chamber in which they had been previously shocked (Shock-no shock group) resulted in plasma corticosterone levels of 398 ± 45 ng/ml (n = 6). This value differs significantly
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2211
(Newman-Keuls'test) from that of the control group (212 ± 33 ng/ml ; n = 6) and was not different from that found in the group (Shock-shock group) which received the shocks twice (533 ± 36 ng/ml ; n = 6). The pattern of DOPAC level changes was the same in the Shock-shock group as in rats exposed to a single shock session (Fig. 3 vs Fig. 2). In contrast, rats of the Shock-no shock group showed a significant elevation of DOPAC levels only in the anteromedial frontal cortex. This effect was similar in magnitude to that observed in the Shock-shock situation (Fig.3). The levels of DA, NA, 5-HT and 5-HIAA in the Shock-no shock group was unchanged compared to the levels found in the control group in all the structures examined. The reactivity of the anteromedial frontal cortex in the Shock-no shock group could have been due to a long term effect of the electric shocks received the day before. Therefore, DOPAC levels in this structure were measured in rats sacrificed inEaediately after removal from their home-cage 24 h after having been submitted to an inescapable footshock session. DOPAC levels at this time have returned to normal ; while control level was 58 ± 7 p g/mg tissue (n = 6) that of the experimental group was 59 ± 14 pg/mg tissue (n = 6).
F~.|beck-shosk • greup
~.8beck-ee sheck |ro|p "
ZOO $
15e
1 Ill
se
N
I FCn
FC|
ACC
OT
A
FIG. 3 Effects of repeated electric shocks and exposure to an environment previously paired with shocks upon DOPAC content of different brain areas. Results are means with SEM of data obtained on 6 rats and are expressed as percentage of respective ~ontrol values. For abbreviations see F!g.l.@: p < 0.05 ; ~,e :p < 0.01.
DISCUSSION The present study indicates that central DA systems are activated not only by electric footshocks but also by environmental stimuli previously paired with inescapable electric shocks. The patterns of activation observed in the
2212
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
two situations are qualitatively different. Placing the animals back into the environment in which they have been previously shocked results in an increase of DA turnover limited to the anteromedial frontal cortex whereas exposure to electric footshocks activates DA metabolism not only in this area but also in various cortical and limbic structures i.e. the sulcal frontal cortex, the nucleus accumbens, the amygdaloid complex and the olfactory tubercle. Endogenous NA levels were decreased in the anteromedial frontal cortex, the amygdaloid region and the septum only following electric shocks, probably as a result of an activation of the noradrenergic system in these regions (17). In contrast, endogenous serotonin and 5-HIAA were not affected in the different brain regions in either experimental situation. The increase in DA metabolism found in the anteromedial frontal cortex and the nucleus accumbens after electric shocks it in agreement with previous reports (5-7). However our results demonstrate that changes in DA metabolism were occuring also in other DA-rich brain areas (see Fig.2). The largest increase in DOPAC level has been found in the amygdala, where Lavielle et al. (6) did not notice significant differences compared to controls. The possible reasons of this discrepancy are not yet understood but may lie in the different dissection procedure used. Exposure to the environmental stimuli previously paired with electric footshocks is clearly stressful as evidenced by the plasma corticosterone level increase. However DA activation in this experimental condition is confined to the anteromedial frontal cortex. The lack of DA turnover changes in the other brain regions where DA metabolism is augmented following acute electric shocks does not appear to be connected to a tolerance to the repeated stress. Thus animals of the shock-shock group exhibit a similar increase in DOPAC levels as the animals receiving a single shock. Likewise the reaction of the anteromedial frontal cortex does not reflect an after-effect of the first exposure to electric shocks since the animals shocked 24 h before and not re-exposed to shocks did not show an elevation of DOPAC level in this region. Previous studies have shown that neurochemical changes observed following inescapable shocks were subject to sensitization or conditioning and could be induced again by the presence in small quantity of the unconditioned stimulus (18) or by the presence of conditioned stimuli, i.e. the environmental stimuli previously paired with the unconditioned stimulus (I0). These studies were concerned essentially with changes in whole brain or hypothalamic NA activity. It is interesting to note that no change in NA levels could be detected in the present experiment in the shock-no shock animals. It is possible that the effects described by Cassens et al. (10) for the whole brain reflect an activation localized in structures other than those examined here. As far as central DA systems are concerned it appears that only the anteromedial frontal cortex is subject to a conditioning process in the case of the strong stressor used here. As no control for sensitization was inclued in the present experiment a non-speclfic increase of reactivity to environmental stimuli cannot be entirely ruled out. The functional interpretation of the changes of DA metabolism is complicated by the multiple reactions elicited by the stressors like pain, hormonal changes, emotional and motor reactions, etc. However the elevation of DOPA£ levels do not seem to be directly linked to the neuroendocrinological reaction as no significant correlation could be detected between DOPAC increase in any structure and plasma corticosterone levels. This lack of correlation may be due however to a ceiling effect of the stressor used on the pituitary-adrenal response. Conversely a possible causal relationship between DA activation and motor activity cannot be excluded. Stimulation of the DA receptors in the nuleus accumbens increase motor activity (19) while DA terminals of the frontal
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2213
cortex have been hypothetized to have an inhibitory action in locomotor regulation (20). In fact under the effect of electric shocks rats show intense motor activity while in the presence of environmental cues associated with electric shocks their motor activity is greatly reduced. On the other hand the structures concerned are also known to be involved in emotional reactions (21) and the activation of their DA terminals could be an indication of this role. This interpretation is supported by the inhibitory activity of anxiolytics on the shock-induced DA activation (6, 7). The mesocortical DA system has also been implicated in the cognitive representational processes which are part of the learning experiment (2, 3); animals re-exposed to stimuli previously paired with electric shocks assess the significance of the stimuli according to their previous experience and develop anticipatory fear reactions. The increase of DOPAC levels in the anteromedial frontal cortex in the shock-no shock group could represent a correlate of this cognitive process. Our results offer further support for the implication of the mesocortical system in cognitive processes and/or in emotional behavior. Further work is, however, clearly needed to better understand the functional significance of the specific neurochemical changes observed in the mesocortical DA system of rats exposed to stimuli previously paired with electric shocks.
ACKNOWLEDGEMENTS The authors are grateful to Ms R.M. Bluthe for her expert technical assistance. This study was supported by the grant DGRST n ° 80.E.0883.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
14. 15.
A. DAHLSTROM and K. FUXE, A. Physiol. Stand., Suppl. 232, 1-55 (1965). H. SIMON, B. SCATTON and M. LE MOAL, Nature, 286 , 150-151 (1979). H. SIMON, J. Physiol. (Paris) 77 , 81-95 (1980)'. A. LOUILLOT, H. SIMON, J.P. HERMAN and M. LE MOAL, Abstracts of the 5th Europ. Neurosei. Congr., Naurosci. Lett., Suppl.7, p. 47 (1981). A.M. THIERRY, J.P. TASSlN, G. BLANC and J. GLOWINSKI, Nature, 263 , 242244 (1976). S. LAVIELLE, J.P. TASSlN, A.M. THIERRY, G. BLANC, D. HERVE, C. BARTHELEMY and J. GLOWINSKI, Brain Res., 168 , 585-594 (1978). F. FADDA, A. ARGIOLAS, M.L. MELIS, A.M. TISSARI, P.L. ONALI and G.L. GESSA, Life Sci., 2 3 , 2219-2224 (1978). L.T. KENNEDY and M.J. ZIGMOND, Soc. Neurosci., Abstr. 6 , p. 44 (1980). J.P. TASSlN, D. HERVE, G. BLANC and J. GLOWINSKI, Neurosci. Lett., 17 , 67-71 (]980). G. CASSENS, A. KURUC, M. ROFFMAN, P.J. ORSULAK and J.J. SCHILDKRAUT, Behav. Brain Res., ~ , 387-407 (1980). E.L. BLISS, J. AILON and J. ZWANZINGER, J. Pharm. Exp. Ther., 164 , 122-134 (1968). A.M. THIERRY, F. JAVOY, J. GLOWINSKI and S. KETY, J. Pharm. Exp. Ther., 163 , 163-171 (1968). A.M. THIERRY, M. FEKETE and J. GLOWINSKI, Eur. J. Pharmacol., 4 , 384-389 (1968). D . E . P . MURPHY, J . C l i n . E n d o c r i n o l . , 2_7 , 973-990 ( 1 9 6 7 ) . M.I.K. FEKETE, B. KANYICSKA and J.P. HERMAN, Life Sci., 2 3 , 1549-1556 (1978)
o
16. L. SEMERDJIAN-ROUQUIER, L. BOSSI and B. SCATTON, J. Chromatogr., in press.
2214
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
17. J. KORF, R.H. ROTH and G.K. AGHAJANIAN, Europ. J. Pharmacol., 23 , 276-282 ( 1973). 18. H. ANISHAN and L . S . SKLAR, J . Comp. P h y s i o l . P s y c h o l . , 93 , 610-625 (1979). 19. A.J.J. PIJNENBURG, W.M.M. HONIG, J.A.M. Van Der HEYDEN and J.M. Van ROSSUM, Eur. J. Pharmacol., 35 , 45-58 (1976). 20. J.P. TASSIN, L. STINUS, H. SIMON, G. BLANC, A.M. THIERRY, M. LE MOAL, B. CARDO and J. GLOWINSKI, Brain Res., 141 , 267-28! (1978). 21. R.L. ISAACSON : The Limbic System, Plenum Press, New York (1974). 22. J.F.R. KONIG and R.A. KLIPPEL : The Rat Brain, Williams and Wilkins Comp., Baltimore (1967).
Pergamon Press
DIFFERENTIAL EFFECTS OF INESCAPABLE FOOTSHOCKS AND OF STIMULI PREVIOUSLY PAIRED WITH INESCAPABLE FOOTSHOCKS ON DOPAMINE TURNOVER IN CORTICAL AND LIMBIC AREAS OF THE RAT J.P. Herman, D. Guillonneau, R. Dantzer, B. Scatton ~, L. Semerdjian-Rouquier ~ and M. Le Moal Lab. Neurobiologie des Comportements, Universit~ de Bordeaux II, 146 Rue L~o Saignat, 33076 BORDEAUX CEDEX and ~ Synthelabo-LERS, 31 Avenue P.V. Couturier, BAGNEUX FRANCE (Received in final form April 12, 1982) SUMMARY The effect of electric footshocks and of exposure to environmental stimuli paired with electrical shocks upon the dopaminergic activity in various cortical and limbic areas of the rat were evaluated by measuring dihydroxyphenylacetic acid (DOPAC) levels in these areas. In animals exposed to a 20 min electric footshock session DOPAC concentrations were significantly increased in the anteromedial and sulcal frontal cortices, olfactory tubercle, nucleus accumbens and amygdaloid complex (by 66, 37, 28, 55 and 90% respectively). Re-exposure of rats to an environment where they had been shocked 24 h earlier induced an elevation of DOPAC content only in the anteromedial frontal cortex (by 47%). Plasma corticosterone levels were elevated in both situations. No change in serotonin or 5-hydroxyindolacetic acid content of these areas could he detected in either situation. The results show that electric footshocks and environmental stimuli associated to previous shocks both activate central dopaminergic systems, altough the patterns of activation are different. Recent studies suggest that the mesocorticofrontal and the mesollmbic dopaminergic (DA) neurons originating from the cell group located within the ventral tegmental area (1) are implicated in the control of cognitive processes and emotional behavior. Lesions with 6-hydroxydopamine (6-OHDA) of the mesocortical DA system at the terminal level induce learning and retention impairments in a delayed alternation task (2, 3). Lesions of the limbic DA terminals within the nucleus accumbens lead to perseveration, impairment of ongoing behavior and of investigatory exploration and more generally to impairment in the functional processes leading from motivation to action (4). These two DA systems are also activated during stressful situations. Thus, an increase in DA turnover in mesocortical and mesolimbic neurons has been observed following electric shocks (5-7), prolonged immobilization (8) or exposure to a new environment (9). These biochemical changes have been related to anxiety or fear induced by the experimental situation. However, various performance factors such as level of motor activity and reactivity to electric shocks could also account for the observed changes in the latter studies. A m a i n characteristic of emotional states is that they are subject to conditioning, i.e. they can he elicited by the presentation of previously neutral stimuli which have been paired with aversive stimuli. As a matter of fact it has been recently reported that changes in whole brain or hypothalamic noradrenergic activity induced by a stressor could be reinstated by a re0024-3205/82/252207-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2208
Conditioned
DOPAC Changes After Stress
exposure to stimuli associated with the stressor
Vol. 30, No. 25, 1982
(I0).
The aim of the present study was to determine whether a similar phenomenon could be observed in dopaminergic neurons following electric footshocks. The activity of DA neurons was assessed by measuring changes in the concentrations of DA, and one of its metabolite, DOPAC in various projection areas of the DA systems. Since noradrenergic and serotoninergic systems have been shown to be affected by stressful situations (11-13), we have also measured levels of noradrenaline (NA), serotonin (5-HT) and its metabolite, 5-hydroxyindolacetic acid (5 HIAA) in the same structures. METHODS Animals and housin$ conditions Male Sprague-Dawley rats weighing 300-350 g were used. The animals were housed in plastic cages in groups of five with food and water ad libitum. Temperature (23°C) and relative humidity (60%) of the animal house were kept constant. Light was on between 08.00 and 20.00 h. Experiments began after a 14-day period of acclimatization. Handlin$ and habituation
to environmental
stimuli
In order to minimize reactions to the experimental routine and to habituate the animals to the new environment represented by the experimental apparatus rats were handled for I0 days. Each day the animals were brought first to a room located nearby the experimental room and in which a white noise was maintained. Each rat was then brought to the experimental room and put into a conditioning chamber for 20 min. The chambers (Campden Instr.) were housed in a sound attenuating cubicle and were equipped with fan, light and a speaker connected to a white noise generator. After 20 min the animal was taken back to the first room and placed into a cage different from its home cage in order to habituate the remaining animals to the removal of one member of the group. The time schedule was kept constant throughout the period of handling and was identical to that fixed for the experiment itself. Handling as well as the experiment was conducted between 12.00 and 18.00 h. First experiment
: Inescapable
footshock session
The procedure described above was followed for the footshock session. Scrambled electric shocks were delivered through the stainless steel grid of the conditioning chamber by a constant current shock generator (Campden Instr.) Trains of 6 shocks (1.5 mA, 180 msec on, 180 msec off) were delivered at 8 sec intervals for 20 min. Control animals were placed in separate chambers in which no electric shocks were delivered. Second experiment ' : Repeated shocks and exposure to stimuli previously paired to shocks One group of rats submitted to electric shocks as described above were divided into subgroups 24 h later. Rats of the first subgroup received again the same shock treatment (Shock-shock group) while the rats of the second subgroup were placed into the conditioning chamber but not shocked (Shock-no shock group). Control animals were placed on both days in the chambers for 20 min, without receiving any electric shock.
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2209
Biochemical analysis Inmediately following the last experimental session, the animals were sacrificed by decapitation in an adjacent room. Trunk blood was collected for corticosterone determination. The brain was quickly removed and cut into l or 2 am thick coronal slices, perpendicular to the dorsal surface of the brain. Slices were placed on a refrigerated stage, brain structures corresponding to limbic, striatal and cortical DA projection areas were dissected out (see Fig.l for details) and put onto dry ice. The plasma and brain samples were stored at - 76°C until the biochemical analysis. 1lolo-losoop
FIG.
I
Schematic representation of sampling from rat brain slices. Approximate coordinates are given according to the atlas of Kbnig and Klippel (22). Abbreviations used are : FCm : anteromedial frontal cortex, FCs : sulcal frontal cortex ; CC : cingular cortex ; OT : olfactory tubercle ; ACC : nucleus accumbens; NC : caudate-putamen ; A : amygdaloid complex ; S : septum. Plasma corticosterone was measured by a competitive protein binding assay (]4), catecholamine and DOPAC by a radioenzymatic method (15). Indole compounds were measured by high performance liquid chromatography with electrochemlcal detection (16). Statistical analysis was performed using either the Student's t-test or analysis of variance followed by a Newman-Keuls' test for multiple comparisons. RESULTS Effests of inescapable footshocks After a 20 min shock session plasma corticosterone level increased
2210
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
markedly : from 206 ± 56 ng/ml in control rats to 528 ± 20 ng/ml in shocked rats (t = 5.3532, d.f. = 10, p < O.001). Under these experimental conditions DOPAC levels were significantly increased in the anteromedial and sulcal frontal corti ces, the nucleus accumbens, the olfactory tubercle and the amygdaloid complex but remained unaffected in the cingular cortex, caudate nucleus and septum (Fig 2). The electric shock session left DA levels unchanged in the structures examined but was accompanied by a significant decrease o~ NA levels in the anteromedial frontal cortex, amygdaloid complex and septum (Pig.2). No change in the concentrations of 5 HT or 5-HIAA could be detected in any of the regiorsexamined under these conditions (results not shown).
.~ l i e o
E 100
i 1 .2_ u
FCne
FCI
CC
OT
ACC
IIC
4
S
FIG. 2 Effects of electric shocks upon catecholamine and DOPAC content of different brain areas. Results are means with SEM of data obtained on 6 rats and are expressed as percentage of respective control values. Control values for DA, NA and DOPAC were in pg/mg tissue. Control values (]00%) DA NA DOPAC FCm (anteromedial frontal cortex) 187 ± 13 550 ± 28 53 ± 3 FCs (sulcal frontal cortex) 93 ± ]3 361 ± 35 60 ± 5 CC (cingulate cortex) 123 ± 7 838 ± 137 83 ± ]3 OT (olfactory tubercle) ]0100 ± 1]70 232 ± 40 ] ] 9 0 ± ]22 ACC (nucleus accumbens) I0700 ± 1000 390 ± 33 1350 ± 150 NC (caudate putamen) 22300 ± 1740 Not m e a s u r e d 2460 ± I ] 2 A (amygdaloid complex) 798 ± 65 1960 ± ]55 124 ± 18 S (septum) 562 ± 69 985 ± 10] 107 ± 69 .: p < 0.05
; , .:p < 0.01
; , . .:p < 0.005
Effects of stimuli previously paired with electric shocks Placement of the animals in the conditioning chamber in which they had been previously shocked (Shock-no shock group) resulted in plasma corticosterone levels of 398 ± 45 ng/ml (n = 6). This value differs significantly
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2211
(Newman-Keuls'test) from that of the control group (212 ± 33 ng/ml ; n = 6) and was not different from that found in the group (Shock-shock group) which received the shocks twice (533 ± 36 ng/ml ; n = 6). The pattern of DOPAC level changes was the same in the Shock-shock group as in rats exposed to a single shock session (Fig. 3 vs Fig. 2). In contrast, rats of the Shock-no shock group showed a significant elevation of DOPAC levels only in the anteromedial frontal cortex. This effect was similar in magnitude to that observed in the Shock-shock situation (Fig.3). The levels of DA, NA, 5-HT and 5-HIAA in the Shock-no shock group was unchanged compared to the levels found in the control group in all the structures examined. The reactivity of the anteromedial frontal cortex in the Shock-no shock group could have been due to a long term effect of the electric shocks received the day before. Therefore, DOPAC levels in this structure were measured in rats sacrificed inEaediately after removal from their home-cage 24 h after having been submitted to an inescapable footshock session. DOPAC levels at this time have returned to normal ; while control level was 58 ± 7 p g/mg tissue (n = 6) that of the experimental group was 59 ± 14 pg/mg tissue (n = 6).
F~.|beck-shosk • greup
~.8beck-ee sheck |ro|p "
ZOO $
15e
1 Ill
se
N
I FCn
FC|
ACC
OT
A
FIG. 3 Effects of repeated electric shocks and exposure to an environment previously paired with shocks upon DOPAC content of different brain areas. Results are means with SEM of data obtained on 6 rats and are expressed as percentage of respective ~ontrol values. For abbreviations see F!g.l.@: p < 0.05 ; ~,e :p < 0.01.
DISCUSSION The present study indicates that central DA systems are activated not only by electric footshocks but also by environmental stimuli previously paired with inescapable electric shocks. The patterns of activation observed in the
2212
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
two situations are qualitatively different. Placing the animals back into the environment in which they have been previously shocked results in an increase of DA turnover limited to the anteromedial frontal cortex whereas exposure to electric footshocks activates DA metabolism not only in this area but also in various cortical and limbic structures i.e. the sulcal frontal cortex, the nucleus accumbens, the amygdaloid complex and the olfactory tubercle. Endogenous NA levels were decreased in the anteromedial frontal cortex, the amygdaloid region and the septum only following electric shocks, probably as a result of an activation of the noradrenergic system in these regions (17). In contrast, endogenous serotonin and 5-HIAA were not affected in the different brain regions in either experimental situation. The increase in DA metabolism found in the anteromedial frontal cortex and the nucleus accumbens after electric shocks it in agreement with previous reports (5-7). However our results demonstrate that changes in DA metabolism were occuring also in other DA-rich brain areas (see Fig.2). The largest increase in DOPAC level has been found in the amygdala, where Lavielle et al. (6) did not notice significant differences compared to controls. The possible reasons of this discrepancy are not yet understood but may lie in the different dissection procedure used. Exposure to the environmental stimuli previously paired with electric footshocks is clearly stressful as evidenced by the plasma corticosterone level increase. However DA activation in this experimental condition is confined to the anteromedial frontal cortex. The lack of DA turnover changes in the other brain regions where DA metabolism is augmented following acute electric shocks does not appear to be connected to a tolerance to the repeated stress. Thus animals of the shock-shock group exhibit a similar increase in DOPAC levels as the animals receiving a single shock. Likewise the reaction of the anteromedial frontal cortex does not reflect an after-effect of the first exposure to electric shocks since the animals shocked 24 h before and not re-exposed to shocks did not show an elevation of DOPAC level in this region. Previous studies have shown that neurochemical changes observed following inescapable shocks were subject to sensitization or conditioning and could be induced again by the presence in small quantity of the unconditioned stimulus (18) or by the presence of conditioned stimuli, i.e. the environmental stimuli previously paired with the unconditioned stimulus (I0). These studies were concerned essentially with changes in whole brain or hypothalamic NA activity. It is interesting to note that no change in NA levels could be detected in the present experiment in the shock-no shock animals. It is possible that the effects described by Cassens et al. (10) for the whole brain reflect an activation localized in structures other than those examined here. As far as central DA systems are concerned it appears that only the anteromedial frontal cortex is subject to a conditioning process in the case of the strong stressor used here. As no control for sensitization was inclued in the present experiment a non-speclfic increase of reactivity to environmental stimuli cannot be entirely ruled out. The functional interpretation of the changes of DA metabolism is complicated by the multiple reactions elicited by the stressors like pain, hormonal changes, emotional and motor reactions, etc. However the elevation of DOPA£ levels do not seem to be directly linked to the neuroendocrinological reaction as no significant correlation could be detected between DOPAC increase in any structure and plasma corticosterone levels. This lack of correlation may be due however to a ceiling effect of the stressor used on the pituitary-adrenal response. Conversely a possible causal relationship between DA activation and motor activity cannot be excluded. Stimulation of the DA receptors in the nuleus accumbens increase motor activity (19) while DA terminals of the frontal
Vol. 30, No. 25, 1982
Conditioned DOPAC Changes After Stress
2213
cortex have been hypothetized to have an inhibitory action in locomotor regulation (20). In fact under the effect of electric shocks rats show intense motor activity while in the presence of environmental cues associated with electric shocks their motor activity is greatly reduced. On the other hand the structures concerned are also known to be involved in emotional reactions (21) and the activation of their DA terminals could be an indication of this role. This interpretation is supported by the inhibitory activity of anxiolytics on the shock-induced DA activation (6, 7). The mesocortical DA system has also been implicated in the cognitive representational processes which are part of the learning experiment (2, 3); animals re-exposed to stimuli previously paired with electric shocks assess the significance of the stimuli according to their previous experience and develop anticipatory fear reactions. The increase of DOPAC levels in the anteromedial frontal cortex in the shock-no shock group could represent a correlate of this cognitive process. Our results offer further support for the implication of the mesocortical system in cognitive processes and/or in emotional behavior. Further work is, however, clearly needed to better understand the functional significance of the specific neurochemical changes observed in the mesocortical DA system of rats exposed to stimuli previously paired with electric shocks.
ACKNOWLEDGEMENTS The authors are grateful to Ms R.M. Bluthe for her expert technical assistance. This study was supported by the grant DGRST n ° 80.E.0883.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
14. 15.
A. DAHLSTROM and K. FUXE, A. Physiol. Stand., Suppl. 232, 1-55 (1965). H. SIMON, B. SCATTON and M. LE MOAL, Nature, 286 , 150-151 (1979). H. SIMON, J. Physiol. (Paris) 77 , 81-95 (1980)'. A. LOUILLOT, H. SIMON, J.P. HERMAN and M. LE MOAL, Abstracts of the 5th Europ. Neurosei. Congr., Naurosci. Lett., Suppl.7, p. 47 (1981). A.M. THIERRY, J.P. TASSlN, G. BLANC and J. GLOWINSKI, Nature, 263 , 242244 (1976). S. LAVIELLE, J.P. TASSlN, A.M. THIERRY, G. BLANC, D. HERVE, C. BARTHELEMY and J. GLOWINSKI, Brain Res., 168 , 585-594 (1978). F. FADDA, A. ARGIOLAS, M.L. MELIS, A.M. TISSARI, P.L. ONALI and G.L. GESSA, Life Sci., 2 3 , 2219-2224 (1978). L.T. KENNEDY and M.J. ZIGMOND, Soc. Neurosci., Abstr. 6 , p. 44 (1980). J.P. TASSlN, D. HERVE, G. BLANC and J. GLOWINSKI, Neurosci. Lett., 17 , 67-71 (]980). G. CASSENS, A. KURUC, M. ROFFMAN, P.J. ORSULAK and J.J. SCHILDKRAUT, Behav. Brain Res., ~ , 387-407 (1980). E.L. BLISS, J. AILON and J. ZWANZINGER, J. Pharm. Exp. Ther., 164 , 122-134 (1968). A.M. THIERRY, F. JAVOY, J. GLOWINSKI and S. KETY, J. Pharm. Exp. Ther., 163 , 163-171 (1968). A.M. THIERRY, M. FEKETE and J. GLOWINSKI, Eur. J. Pharmacol., 4 , 384-389 (1968). D . E . P . MURPHY, J . C l i n . E n d o c r i n o l . , 2_7 , 973-990 ( 1 9 6 7 ) . M.I.K. FEKETE, B. KANYICSKA and J.P. HERMAN, Life Sci., 2 3 , 1549-1556 (1978)
o
16. L. SEMERDJIAN-ROUQUIER, L. BOSSI and B. SCATTON, J. Chromatogr., in press.
2214
Conditioned DOPAC Changes After Stress
Vol. 30, No. 25, 1982
17. J. KORF, R.H. ROTH and G.K. AGHAJANIAN, Europ. J. Pharmacol., 23 , 276-282 ( 1973). 18. H. ANISHAN and L . S . SKLAR, J . Comp. P h y s i o l . P s y c h o l . , 93 , 610-625 (1979). 19. A.J.J. PIJNENBURG, W.M.M. HONIG, J.A.M. Van Der HEYDEN and J.M. Van ROSSUM, Eur. J. Pharmacol., 35 , 45-58 (1976). 20. J.P. TASSIN, L. STINUS, H. SIMON, G. BLANC, A.M. THIERRY, M. LE MOAL, B. CARDO and J. GLOWINSKI, Brain Res., 141 , 267-28! (1978). 21. R.L. ISAACSON : The Limbic System, Plenum Press, New York (1974). 22. J.F.R. KONIG and R.A. KLIPPEL : The Rat Brain, Williams and Wilkins Comp., Baltimore (1967).