Life Sciences Vol . 17, pp . 143-150 Printed in the U .S .A .
Pergamon Press
EFFECT OF IMMOBILIZATION STRESS ON SEROTONIN CONTENT AND TURNOVER IN REGIONS OF THE RAT BRAIN` William W . Morgen, P . Kevin Rudeen and Karla A . Pfeil Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, T axas
78284
(Received in final form May 30, 1975) Summary Sprague-Dawley rata were stressed by immobilization from 30 to 300 minutes and the effects on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cerebral cortex, diencephalon, striatum, hippocampus and the brainatem . In a subsequent study 5-HT turnover rate in these brain areas was estimated by measuring 5-HIAA accumulation 0, 30, 60 and 90 minutes after probenecid. The content of 5-HIAA and the turnover rate of 5-HT were significantly increased in the cerebral cortex shortly after the onset of immobilization . The content of 5-HIAA in the brainstem was increased by immobilization although 5-HT turnover rate was not increased . Short term increases in 5-HIAA content were observed in the striatum and hippocampus . However, no significant changes in 5-HT turnover rate were observed in either of these 2 brain areas . immobilization did not affect 5-HIAA content or 5-HT turnover in the diencephalon. The sensitivity of the serotonergic system in the cerebral cortex to immobilization stress suggests that this brain region could be used in future studies of the interrelationships between stress and the brain serotonergic system . Acute atreae has been shown to affect the serotonergic system in the whole brain particularly by increasing the turnover of serotonin (5-HT) (1, 2, 3). It is well established that 5-HT containing cell bodies and their nerve end ings are not homogenously distributed in the brain but are localized in only very specific brain regions (4 ) . Therefore, studies which have concentrated on the effects of stress on the serotonergic system in the whole brain have ignored the distribution of the serotonergic neurone and have precluded the demonstration of regional differences in the degree or direction of response of the serotonergic system to stress . If regional differences do exist, it would be more profitable in future studies of the role of the brain serotonergic system in the response to atreae to concentrate on those brain areas in which the serotonergic system Is particularly sensitive to stress . To determine if Supported by NASA Grant NSG 9012
144
Stress and Serotonin Turnover
Vol. 17, No . 1
there are regional differences in the response of the aerotonergic system to stress, 5-HT content, 5-hydroxyindoleacetic acid (5-HIAA) content and 5-HT turnover were analyzed in 5 discrete brain areas of rate which were acutely stressed by immobilization. Methode Adult male Sprague-Dawley rate (150 grams) were housed for 1 week at near constant temperature (22 f 1 °C) and were exposed to a 14 :10 lightdark lighting regimen (lights on 0600-2000 Central Standard Time) . Food (Wayne Lab Blox) and water were provided ad libitum . Two separate studies were performed. In the first study, rate were stressed by immobilization (5) for periods of 0, 30, 60, 120, 180 or 300 minutes and were then sacrificed by decapitation . The contents of 5-HT and 5-HIAA were compared is brain areas of control versus stressed rate . In the second study, 5-HT turnover rate was measured. Rate were sacrificed 0, 30, 60 and 90 minutes after probenecid (Merck, Sharp and Dohme) administration (6 ). The stressed animals were immobilized for 1 hour before probenecid administration . Thus the rate in this second study had been atresaed for periods of 60, 90, 120 and 150 minutes at sacrifice. The slope and y intercept of the beet fit line for the accumulation of 5-HIAA after probenecid administration was determined by the least squares method for the control and for the stressed rats (7). Immediately after sacrifice, the brain was removed and rapidly dieaected by one investigator Into 5 discrete brain areas : the brainetem, diencephalon (thalamus, hypothalamus), hippocampus, striatum and cerebral cortex. The brainetem included that portion of the brain from the gracile nuclei to the superior colliculus . The diencephalon included that portion from the anterior commissure anteriorly to the internal capsule laterally . The striatum included that portion between the internal capsule medially and the external capsule laterally. The hippocampal region included the fornix, hippocampus and the dentate gyrus . The remaining telencephalon after dissection of the striatum and hlppocampal formation was taken collectively ae the cerebral tortes. The cerebellum was discarded. After dissection, these brain areas were frozen on dry ice, weighed and stored at -55 ° C for 24 to 48 hours . In the first study, blood was collected from the cervical vessels immediately after decapitation. Each brain sample was homogenized in acidified butanol and 5-HT and 5-HIAA were extracted by a modification of the procedure of Cox and Perhach (8). In the modified procedure 5-HIAA was extracted from the organic phase with 0. 6 ml 0. 5M phosphate buffer (pH 7. O) . Serotonin (5-HT) was extracted from the same organic phase with 2. 3 ml 0. O1N HCl. Serotonin (5-HT) and 5-HIAA were quantltated by the methods of Maickel_et al . (9) and Curzon and Green (10), respectively. The blood was allowed to clot at room temperature. Following centrifugation, serum was collected and frozen (-25 ° C) for subsequent corticosterone analysis (11). The statistical significance of differences in the parameters studied in control sad atresaed animals was determined initially by the analysis of
Vol.
17,
No .
Stress and Serotonin Turnover
1
14 5
variance (12) and then by the Student-Newman-Keuls test (13) . The statistical significance of differences in the slopes and in the y intercepts of the best fit lines for 5-HIAA accumulation with time after probenecid in the control versus stressed animals was determined by a t teat (14) . Results Immobilization stress produced a significant increase in the content of 5-HIAA ín different areas of the rat brain (Figure 1) . In the cerebral cortex 5-HIAA content was significantly elevated (17%, p< 0.05) within 30 minutes after immobilization (Figure 1) . The peak elevation of 5-HTAA content in the cerebral cortex occurred after 120 minutes (38%, p< 0 .001). By 300 minutes, 5-HTAA levels had decreased slightly although they remained significantly OiENCEPHALON
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FIG. 1 The Effect of Immobilization Stress on 5-Hydroxyindoleacetic Acid Content is Discrete Areas of the Rat Brain . Each bar represents the meanfstandard error of a group of animals. The number is parenthesis indicates the number of values averaged to obtain the mean. The symbol above the bar indicates the statistical significance from control ae determined by the Student-Neuman-Keuls teat (13) . ~` p< 0.05 $p
Pergamon Press
EFFECT OF IMMOBILIZATION STRESS ON SEROTONIN CONTENT AND TURNOVER IN REGIONS OF THE RAT BRAIN` William W . Morgen, P . Kevin Rudeen and Karla A . Pfeil Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, T axas
78284
(Received in final form May 30, 1975) Summary Sprague-Dawley rata were stressed by immobilization from 30 to 300 minutes and the effects on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cerebral cortex, diencephalon, striatum, hippocampus and the brainatem . In a subsequent study 5-HT turnover rate in these brain areas was estimated by measuring 5-HIAA accumulation 0, 30, 60 and 90 minutes after probenecid. The content of 5-HIAA and the turnover rate of 5-HT were significantly increased in the cerebral cortex shortly after the onset of immobilization . The content of 5-HIAA in the brainstem was increased by immobilization although 5-HT turnover rate was not increased . Short term increases in 5-HIAA content were observed in the striatum and hippocampus . However, no significant changes in 5-HT turnover rate were observed in either of these 2 brain areas . immobilization did not affect 5-HIAA content or 5-HT turnover in the diencephalon. The sensitivity of the serotonergic system in the cerebral cortex to immobilization stress suggests that this brain region could be used in future studies of the interrelationships between stress and the brain serotonergic system . Acute atreae has been shown to affect the serotonergic system in the whole brain particularly by increasing the turnover of serotonin (5-HT) (1, 2, 3). It is well established that 5-HT containing cell bodies and their nerve end ings are not homogenously distributed in the brain but are localized in only very specific brain regions (4 ) . Therefore, studies which have concentrated on the effects of stress on the serotonergic system in the whole brain have ignored the distribution of the serotonergic neurone and have precluded the demonstration of regional differences in the degree or direction of response of the serotonergic system to stress . If regional differences do exist, it would be more profitable in future studies of the role of the brain serotonergic system in the response to atreae to concentrate on those brain areas in which the serotonergic system Is particularly sensitive to stress . To determine if Supported by NASA Grant NSG 9012
144
Stress and Serotonin Turnover
Vol. 17, No . 1
there are regional differences in the response of the aerotonergic system to stress, 5-HT content, 5-hydroxyindoleacetic acid (5-HIAA) content and 5-HT turnover were analyzed in 5 discrete brain areas of rate which were acutely stressed by immobilization. Methode Adult male Sprague-Dawley rate (150 grams) were housed for 1 week at near constant temperature (22 f 1 °C) and were exposed to a 14 :10 lightdark lighting regimen (lights on 0600-2000 Central Standard Time) . Food (Wayne Lab Blox) and water were provided ad libitum . Two separate studies were performed. In the first study, rate were stressed by immobilization (5) for periods of 0, 30, 60, 120, 180 or 300 minutes and were then sacrificed by decapitation . The contents of 5-HT and 5-HIAA were compared is brain areas of control versus stressed rate . In the second study, 5-HT turnover rate was measured. Rate were sacrificed 0, 30, 60 and 90 minutes after probenecid (Merck, Sharp and Dohme) administration (6 ). The stressed animals were immobilized for 1 hour before probenecid administration . Thus the rate in this second study had been atresaed for periods of 60, 90, 120 and 150 minutes at sacrifice. The slope and y intercept of the beet fit line for the accumulation of 5-HIAA after probenecid administration was determined by the least squares method for the control and for the stressed rats (7). Immediately after sacrifice, the brain was removed and rapidly dieaected by one investigator Into 5 discrete brain areas : the brainetem, diencephalon (thalamus, hypothalamus), hippocampus, striatum and cerebral cortex. The brainetem included that portion of the brain from the gracile nuclei to the superior colliculus . The diencephalon included that portion from the anterior commissure anteriorly to the internal capsule laterally . The striatum included that portion between the internal capsule medially and the external capsule laterally. The hippocampal region included the fornix, hippocampus and the dentate gyrus . The remaining telencephalon after dissection of the striatum and hlppocampal formation was taken collectively ae the cerebral tortes. The cerebellum was discarded. After dissection, these brain areas were frozen on dry ice, weighed and stored at -55 ° C for 24 to 48 hours . In the first study, blood was collected from the cervical vessels immediately after decapitation. Each brain sample was homogenized in acidified butanol and 5-HT and 5-HIAA were extracted by a modification of the procedure of Cox and Perhach (8). In the modified procedure 5-HIAA was extracted from the organic phase with 0. 6 ml 0. 5M phosphate buffer (pH 7. O) . Serotonin (5-HT) was extracted from the same organic phase with 2. 3 ml 0. O1N HCl. Serotonin (5-HT) and 5-HIAA were quantltated by the methods of Maickel_et al . (9) and Curzon and Green (10), respectively. The blood was allowed to clot at room temperature. Following centrifugation, serum was collected and frozen (-25 ° C) for subsequent corticosterone analysis (11). The statistical significance of differences in the parameters studied in control sad atresaed animals was determined initially by the analysis of
Vol.
17,
No .
Stress and Serotonin Turnover
1
14 5
variance (12) and then by the Student-Newman-Keuls test (13) . The statistical significance of differences in the slopes and in the y intercepts of the best fit lines for 5-HIAA accumulation with time after probenecid in the control versus stressed animals was determined by a t teat (14) . Results Immobilization stress produced a significant increase in the content of 5-HIAA ín different areas of the rat brain (Figure 1) . In the cerebral cortex 5-HIAA content was significantly elevated (17%, p< 0.05) within 30 minutes after immobilization (Figure 1) . The peak elevation of 5-HTAA content in the cerebral cortex occurred after 120 minutes (38%, p< 0 .001). By 300 minutes, 5-HTAA levels had decreased slightly although they remained significantly OiENCEPHALON
(16 1B)(Bl
1 .4
(811e1 iT1
4
06
0
HIPPOCAYPUS
a
(81 18) ~ 17)(e)
I.
.
caNrna
® STRESS-301m
I
S~SS-60rm
" BTRE55-I20rm S7RE56 -IBOIm
® S7HESS-300nin
ì""
â v STRIATUM
181
1.4
IB) ~ Iel
I
BRAINSTEM ~l ~ i 1~ ~
o
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~ (-~ i . f71
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CEREBRAL CORTEX
1i61 o.
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FIG. 1 The Effect of Immobilization Stress on 5-Hydroxyindoleacetic Acid Content is Discrete Areas of the Rat Brain . Each bar represents the meanfstandard error of a group of animals. The number is parenthesis indicates the number of values averaged to obtain the mean. The symbol above the bar indicates the statistical significance from control ae determined by the Student-Neuman-Keuls teat (13) . ~` p< 0.05 $p
