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The peripheral sympathetic nervous system in rat strains selectively bred for differences in response to stress
BEHAVIORAL AND NEURAL BIOLOGY
34, 319-325 (1982)
BRIEF REPORT The Peripheral Sympathetic Nervous System in Rat Strains Selectively Bred for Differences in Response to Stress 1
DAVID A . BLIZARD, HARVEY J. ALTMAN, 2 AND LEWIS S. FREEDMAN 2
Department of Physiology & Pharmacology, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, North Carolina 27103 Male rats of two Maudsley Nonreactive sublines, the MNRA/Har and MNPU Har strains, both selected for low levels of open-field defecation (OFD), exhibit higher concentrations of norepinephrine (NE) in descending and transverse colon than Maudsley Reactive (MR/Har) males, selected for high levels of OFD. These differences in NE concentration in regions of the gastrointestinal system involved in defecation support the hypothesis that genetic selection for differences in OFD in the Maudsley strains has been mediated by alterations in their peripheral sympathetic nervous system (SNS). Rats of one Nonreactive subline (MNRA/ Har) also had higher NE concentrations than MR/Hat animals in small intestine and submaxillary gland; thus, previous data on other organs (heart, spleen, adrenal) and the present findings show that the differences in NE concentration between MR/Har and the MNRA/Har subline have been found in most peripheral tissues studied and may reflect a widespread influence of genes on the SNS. On the other hand, differences in tissue NE concentration between MR/Hat and the other Nonreactive subline (MNR/Har) were restricted to descending and transverse colon in the present study. Thus, when considering strain variation in the SNS across all three Maudsley strains the present results suggest that both general and organ-specific factors contribute to their determination.
Selection for differences in open-field defecation (OFD) in the Maudsley strains (Broadhurst, 1960) has been accompanied by alterations in the concentration of neurotransmitter in their central and peripheral noradrenergic systems. Maudsley Reactive (MR/Har) rats exhibit lower concentrations of norepinephrine (NE) in heart, spleen, and hypothalamus and lower total catecholamines (CA) in the adrenal gland than rats of both Maudsley Nonreactive sublines (MNR/Har and MNRA/Har) (Slater, This research was supported by NIH Grant GM 26962. 2 Division of Behavioral Neurology, N.Y. University Medical Center, 341 E 25th Street, New York, N.Y. 10010. 319 0163-1047/82/030319-07 $02.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
320
BLIZARD, ALTMAN, AND FREEDMAN
Blizard, & Pohorecky, 1977; Liang & Blizard, 1978). MR/Har rats also exhibit lower concentrations of NE in plasma under resting conditions, possibly reflecting a lower basal release of NE in Reactive vs Nonreactive rats (Blizard, Liang, & Emmel, 1980). On the basis of these observations, it has been suggested that differences in emotionality are mediated by alterations in the sympathetic nervous system (SNS) (Blizard, 1981), an assertion first made by C. S. Hall nearly 50 years ago (Hall, 1934). In the present study, it was hypothesized that if differences in the peripheral SNS mediate differences in OFD in the Maudsley strains they should be seen in parts of the gastrointestinal (GI) tract that play a direct role in the act of defecation. Therefore, NE concentrations were compared in the descending and transverse colon; other parts of the GI tract and other tissues were also assayed to assess the specificity of any strain differences found. Two batches of male rats from the MR/Har, MNR/Har, and MNRA/ Har strains were used in these experiments. They were kindly supplied by Dr. Gordon M. Harrington, Department of Psychology, University of Northern Iowa. The first group (MR/Har, N = 5; MNRA/Har, N = 4; MNRA/Har, N = 5) was approximately 200 days old at the time of sacrifice, and the second group, comprising similar numbers from each strain, was 230 days old. Both groups were maintained in the laboratory for 2 to 3 weeks before use in this study on a 15-hr light/9-hr dark diurnal cycle with food and water available ad lib. All three strains are albino; the two Nonreactive strains have been separated since at least the 8th generation of inbreeding; MNR/Har rats are AA and MNRA/Har rats are at the agouti locus (Harrington, 1981). All rats were decapitated during the light segment of the daily diurnal cycle. The order of killing was balanced across strains. Submaxillary glands, descending colons, (Groups I and 2); transverse colon (Group 1), small intestine (from the duodenum adjacent to the stomach), ascending colon, and vas deferens, (Group 2); were dissected out and stored temporarily on dry ice and subsequently at -70°C in a freezer until assay. The sections of the gastrointestinal tract, approximately 6 cm in length, were trimmed of fat and their contents expunged with a large tweezer. For the norepinephrine assay a modification of the procedure of Roth and Stone (1971) was followed. This is fully described in Liang and Blizard (1978). The results were analyzed by one-way analysis of variance and t tests and are shown in Table 1. Variations in degrees of freedom are due to occasional loss of tissue or faulty assay procedure. The MNRA/Har strain exhibited higher concentrations of NE than both MR/Har and MNR/Har strains in descending and transverse colon,
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
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BLIZARD, ALTMAN, AND FREEDMAN
small intestine, and submaxillary gland, but did not differ in the ascending colon. The other Nonreactive strain, the MNR/Har, had higher NE concentrations than MR/Har rats in the descending and transverse colon, but did not differ in other parts of the GI system. In contrast, in the vas deferens, the only extragastrointestinal tissue assayed, MR/Hat rats had higher NE concentrations than both Nonreactive strains; the two Nonreactive strains did not differ from each other. MR/Hat rats had heavier submaxillary glands than both Nonreactive strains. When the customary strain differences in body weight were taken into account, however, MR/Har and MNRA/Har rats exhibited significantly heavier submaxillary glands per gram of body weight than MNR/ Har rats (Table 2). With the strain differences in gland weight in mind, comparisons were made of the absolute amount of NE in the submaxillary glands of the three strains. In this analysis, MR/Har (M = 0.73 ~g _ 0.03) and MNRA/Har (M = 0.67 ~g _+ 0.01) rats were found to have significantly higher amounts of NE than MNR/Har (M = 0.47 ~g ___ 0.01) rats (F(2, 21) = 48.4, p < .001; MR/Har vs MNR/Har, t(16) -8.68, p < .001; MNRA/Har vs MNR/Har, t(12) = 12.3, < .001) but did not differ from each other. It was hypothesized that alterations would be seen in the peripheral SNS in regions of the GI tract that play a role in open-field defecation, the criterion used to differentiate the Maudsley strains. The present findings support this hypothesis; both Nonreactive strains had higher NE concentrations than Reactive rats in the descending and transverse colon (Table 1). The lack of strain variation in NE levels of ascending colon is also consistent with the above hypothesis because this part of the colon is not intimately involved in the act of defecation. It is well established that the noradrenergic system exercises an inhibitory influence on colonic activity; therefore, it is tempting to speculate that the lower open-field defecation and higher NE concentrations in descending colon reflect a higher sympathetic tone in Nonreactive vs Reactive rats. Previous findings in other tissues support the belief that the higher tissue concentrations of NE in Nonreactive rats are correlated with higher neuronal activity; compared with MR/Har rats, MNRA/Har animals exhibited an increased rate of NE metabolism in heart (Slater et al., 1977); in another study, compared with the MR/Har strain, basal plasma NE levels were elevated in both Nonreactive strains, possibly indicating elevated NE release (Blizard et al., 1980). Thus, it would be consistent with previous findings in other tissues if the higher NE concentrations in descending and transverse colon of Nonreactive rats were also correlated with increased neuronal activity. Nevertheless, to conclusively establish the correlation, direct estimates of NE turnover should be carried out in descending colon.
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
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BLIZARD, ALTMAN, AND FREEDMAN
MNRA/Har animals may be distinguished from their Nonreactive cousins (MNR/Har) in that they exhibited significantly higher concentrations of NE in descending and transverse colon, small intestine, and submaxillary gland. In this respect, the present results parallel previous findings on the heart (Liang & Blizard, 1978). It is possible that MNRA/Har rats inherited more alleles with the net effect of decreasing emotionality than did MNR/Har animals and that these have exerted a correspondingly greater or more pervasive influence on the SNS. If so, the effect was probably fortuitous because the two Nonreactive strains do not differ in their mean levels of open-field defecation, the criterion used to differentiate the strains during genetic selection. The results of previous research were consistent with the idea that genetic selection for OFD produced a generalized or widespread difference in the SNS of the Maudsley strains: both Nonreactive strains had higher concentrations of NE or total CAs in all peripheral tissues studied (heart, spleen, and adrenal gland). The present experiment provides more data with which to evaluate this assertion and emphasizes important differences between the two Nonreactive strains. Providing evidence of a generalized but not all-pervasive difference in their SNS, MNRA/Har rats exhibited significantly higher concentrations of NE than MR/Har animals in four of five comparisons involving the GI tract and submaxillary gland. On the other hand, indicative of more restricted differences in their SNS, MNR/Har rats had higher NE levels than did MR/Har rats in only two out of five comparisons involving the GI system. Thus, the hypothesis that there is a generalized difference between the sympathetic nervous systems of the Maudsley Reactive and Maudsley Nonreactive strains accounts quite well for comparisons of MR/Har with one Nonreactive subline (MNRA/Har), but somewhat less successfully for comparisons of MR/Har with the other subline (MNR/Har). In both comparisons, however, the hypothesis must be modified to take into consideration organ-specific factors that produce opposite strain differences in NE concentrations of vas deferens (MR/Har greater than both Nonreactive sublines) to those seen in most other tissues. REFERENCES Blizard, D. A. (1981). The Maudsley Reactive and Non-Reactive strains---A North American perspective. Behavior Genetics, 11, 469-489. Blizard, D. A., & Liang, B. (1979). Central serotonergic function and behavior in the Maudsley Reactive and Non-Reactive strains: A reevaluation. Behavior Genetics, 9, 413-418. Blizard, D. A., Liang, B., & Emmel, D. K. (1980). Blood-pressure, heart-rate, and plasma catecholamines under resting conditions in rat strains selectively bred for differences in response to stress. Behavioral and Neural Biology, 29, 487-497. Broadhurst, P. L. (1960). Experiments in psychogenetics. In H. J. Eysenck (Ed.), Experiments in Personality, Psychogenetics and Psychopharmacology, Vol. 1, pp. 3-102. London: Routledge & Kegan Paul.
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
325
Hall, C. S. (1934). Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. Journal of Comparative Psychology, 18, 385-403. Harrington, G. M. (1981). Genetics and behavior of the rat: Characteristics and origins of defined strains and stocks. Behavior Genetics, 11, 445-468. Liang, B., & Blizard, D. A. (1978). Central and peripheral norepinephrine concentrations in rat strains selectively bred for differences in response to stress: Confirmation and extension. Pharmacology Biochemistry and Behavior, 8, 75-80. Roth, R. H., & Stone, E. A. (1971). The action of reserpine on noradrenaline biosynthesis in sympathetic nerve tissue. Biochemical Pharmacology, 17, 1581-1590. Slater, J., Blizard, D. A., & Pohorecky, L. A. (1977). Central and peripheral norepinephrine metabolism in rat strains selectively bred for differences in response to stress. Pharmacology Biochemistry and Behavior, 6, 511-520.
34, 319-325 (1982)
BRIEF REPORT The Peripheral Sympathetic Nervous System in Rat Strains Selectively Bred for Differences in Response to Stress 1
DAVID A . BLIZARD, HARVEY J. ALTMAN, 2 AND LEWIS S. FREEDMAN 2
Department of Physiology & Pharmacology, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, North Carolina 27103 Male rats of two Maudsley Nonreactive sublines, the MNRA/Har and MNPU Har strains, both selected for low levels of open-field defecation (OFD), exhibit higher concentrations of norepinephrine (NE) in descending and transverse colon than Maudsley Reactive (MR/Har) males, selected for high levels of OFD. These differences in NE concentration in regions of the gastrointestinal system involved in defecation support the hypothesis that genetic selection for differences in OFD in the Maudsley strains has been mediated by alterations in their peripheral sympathetic nervous system (SNS). Rats of one Nonreactive subline (MNRA/ Har) also had higher NE concentrations than MR/Hat animals in small intestine and submaxillary gland; thus, previous data on other organs (heart, spleen, adrenal) and the present findings show that the differences in NE concentration between MR/Har and the MNRA/Har subline have been found in most peripheral tissues studied and may reflect a widespread influence of genes on the SNS. On the other hand, differences in tissue NE concentration between MR/Hat and the other Nonreactive subline (MNR/Har) were restricted to descending and transverse colon in the present study. Thus, when considering strain variation in the SNS across all three Maudsley strains the present results suggest that both general and organ-specific factors contribute to their determination.
Selection for differences in open-field defecation (OFD) in the Maudsley strains (Broadhurst, 1960) has been accompanied by alterations in the concentration of neurotransmitter in their central and peripheral noradrenergic systems. Maudsley Reactive (MR/Har) rats exhibit lower concentrations of norepinephrine (NE) in heart, spleen, and hypothalamus and lower total catecholamines (CA) in the adrenal gland than rats of both Maudsley Nonreactive sublines (MNR/Har and MNRA/Har) (Slater, This research was supported by NIH Grant GM 26962. 2 Division of Behavioral Neurology, N.Y. University Medical Center, 341 E 25th Street, New York, N.Y. 10010. 319 0163-1047/82/030319-07 $02.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
320
BLIZARD, ALTMAN, AND FREEDMAN
Blizard, & Pohorecky, 1977; Liang & Blizard, 1978). MR/Har rats also exhibit lower concentrations of NE in plasma under resting conditions, possibly reflecting a lower basal release of NE in Reactive vs Nonreactive rats (Blizard, Liang, & Emmel, 1980). On the basis of these observations, it has been suggested that differences in emotionality are mediated by alterations in the sympathetic nervous system (SNS) (Blizard, 1981), an assertion first made by C. S. Hall nearly 50 years ago (Hall, 1934). In the present study, it was hypothesized that if differences in the peripheral SNS mediate differences in OFD in the Maudsley strains they should be seen in parts of the gastrointestinal (GI) tract that play a direct role in the act of defecation. Therefore, NE concentrations were compared in the descending and transverse colon; other parts of the GI tract and other tissues were also assayed to assess the specificity of any strain differences found. Two batches of male rats from the MR/Har, MNR/Har, and MNRA/ Har strains were used in these experiments. They were kindly supplied by Dr. Gordon M. Harrington, Department of Psychology, University of Northern Iowa. The first group (MR/Har, N = 5; MNRA/Har, N = 4; MNRA/Har, N = 5) was approximately 200 days old at the time of sacrifice, and the second group, comprising similar numbers from each strain, was 230 days old. Both groups were maintained in the laboratory for 2 to 3 weeks before use in this study on a 15-hr light/9-hr dark diurnal cycle with food and water available ad lib. All three strains are albino; the two Nonreactive strains have been separated since at least the 8th generation of inbreeding; MNR/Har rats are AA and MNRA/Har rats are at the agouti locus (Harrington, 1981). All rats were decapitated during the light segment of the daily diurnal cycle. The order of killing was balanced across strains. Submaxillary glands, descending colons, (Groups I and 2); transverse colon (Group 1), small intestine (from the duodenum adjacent to the stomach), ascending colon, and vas deferens, (Group 2); were dissected out and stored temporarily on dry ice and subsequently at -70°C in a freezer until assay. The sections of the gastrointestinal tract, approximately 6 cm in length, were trimmed of fat and their contents expunged with a large tweezer. For the norepinephrine assay a modification of the procedure of Roth and Stone (1971) was followed. This is fully described in Liang and Blizard (1978). The results were analyzed by one-way analysis of variance and t tests and are shown in Table 1. Variations in degrees of freedom are due to occasional loss of tissue or faulty assay procedure. The MNRA/Har strain exhibited higher concentrations of NE than both MR/Har and MNR/Har strains in descending and transverse colon,
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
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BLIZARD, ALTMAN, AND FREEDMAN
small intestine, and submaxillary gland, but did not differ in the ascending colon. The other Nonreactive strain, the MNR/Har, had higher NE concentrations than MR/Har rats in the descending and transverse colon, but did not differ in other parts of the GI system. In contrast, in the vas deferens, the only extragastrointestinal tissue assayed, MR/Hat rats had higher NE concentrations than both Nonreactive strains; the two Nonreactive strains did not differ from each other. MR/Hat rats had heavier submaxillary glands than both Nonreactive strains. When the customary strain differences in body weight were taken into account, however, MR/Har and MNRA/Har rats exhibited significantly heavier submaxillary glands per gram of body weight than MNR/ Har rats (Table 2). With the strain differences in gland weight in mind, comparisons were made of the absolute amount of NE in the submaxillary glands of the three strains. In this analysis, MR/Har (M = 0.73 ~g _ 0.03) and MNRA/Har (M = 0.67 ~g _+ 0.01) rats were found to have significantly higher amounts of NE than MNR/Har (M = 0.47 ~g ___ 0.01) rats (F(2, 21) = 48.4, p < .001; MR/Har vs MNR/Har, t(16) -8.68, p < .001; MNRA/Har vs MNR/Har, t(12) = 12.3, < .001) but did not differ from each other. It was hypothesized that alterations would be seen in the peripheral SNS in regions of the GI tract that play a role in open-field defecation, the criterion used to differentiate the Maudsley strains. The present findings support this hypothesis; both Nonreactive strains had higher NE concentrations than Reactive rats in the descending and transverse colon (Table 1). The lack of strain variation in NE levels of ascending colon is also consistent with the above hypothesis because this part of the colon is not intimately involved in the act of defecation. It is well established that the noradrenergic system exercises an inhibitory influence on colonic activity; therefore, it is tempting to speculate that the lower open-field defecation and higher NE concentrations in descending colon reflect a higher sympathetic tone in Nonreactive vs Reactive rats. Previous findings in other tissues support the belief that the higher tissue concentrations of NE in Nonreactive rats are correlated with higher neuronal activity; compared with MR/Har rats, MNRA/Har animals exhibited an increased rate of NE metabolism in heart (Slater et al., 1977); in another study, compared with the MR/Har strain, basal plasma NE levels were elevated in both Nonreactive strains, possibly indicating elevated NE release (Blizard et al., 1980). Thus, it would be consistent with previous findings in other tissues if the higher NE concentrations in descending and transverse colon of Nonreactive rats were also correlated with increased neuronal activity. Nevertheless, to conclusively establish the correlation, direct estimates of NE turnover should be carried out in descending colon.
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
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BLIZARD, ALTMAN, AND FREEDMAN
MNRA/Har animals may be distinguished from their Nonreactive cousins (MNR/Har) in that they exhibited significantly higher concentrations of NE in descending and transverse colon, small intestine, and submaxillary gland. In this respect, the present results parallel previous findings on the heart (Liang & Blizard, 1978). It is possible that MNRA/Har rats inherited more alleles with the net effect of decreasing emotionality than did MNR/Har animals and that these have exerted a correspondingly greater or more pervasive influence on the SNS. If so, the effect was probably fortuitous because the two Nonreactive strains do not differ in their mean levels of open-field defecation, the criterion used to differentiate the strains during genetic selection. The results of previous research were consistent with the idea that genetic selection for OFD produced a generalized or widespread difference in the SNS of the Maudsley strains: both Nonreactive strains had higher concentrations of NE or total CAs in all peripheral tissues studied (heart, spleen, and adrenal gland). The present experiment provides more data with which to evaluate this assertion and emphasizes important differences between the two Nonreactive strains. Providing evidence of a generalized but not all-pervasive difference in their SNS, MNRA/Har rats exhibited significantly higher concentrations of NE than MR/Har animals in four of five comparisons involving the GI tract and submaxillary gland. On the other hand, indicative of more restricted differences in their SNS, MNR/Har rats had higher NE levels than did MR/Har rats in only two out of five comparisons involving the GI system. Thus, the hypothesis that there is a generalized difference between the sympathetic nervous systems of the Maudsley Reactive and Maudsley Nonreactive strains accounts quite well for comparisons of MR/Har with one Nonreactive subline (MNRA/Har), but somewhat less successfully for comparisons of MR/Har with the other subline (MNR/Har). In both comparisons, however, the hypothesis must be modified to take into consideration organ-specific factors that produce opposite strain differences in NE concentrations of vas deferens (MR/Har greater than both Nonreactive sublines) to those seen in most other tissues. REFERENCES Blizard, D. A. (1981). The Maudsley Reactive and Non-Reactive strains---A North American perspective. Behavior Genetics, 11, 469-489. Blizard, D. A., & Liang, B. (1979). Central serotonergic function and behavior in the Maudsley Reactive and Non-Reactive strains: A reevaluation. Behavior Genetics, 9, 413-418. Blizard, D. A., Liang, B., & Emmel, D. K. (1980). Blood-pressure, heart-rate, and plasma catecholamines under resting conditions in rat strains selectively bred for differences in response to stress. Behavioral and Neural Biology, 29, 487-497. Broadhurst, P. L. (1960). Experiments in psychogenetics. In H. J. Eysenck (Ed.), Experiments in Personality, Psychogenetics and Psychopharmacology, Vol. 1, pp. 3-102. London: Routledge & Kegan Paul.
SYMPATHETIC NERVOUS SYSTEM AND STRESS-RESPONSE
325
Hall, C. S. (1934). Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. Journal of Comparative Psychology, 18, 385-403. Harrington, G. M. (1981). Genetics and behavior of the rat: Characteristics and origins of defined strains and stocks. Behavior Genetics, 11, 445-468. Liang, B., & Blizard, D. A. (1978). Central and peripheral norepinephrine concentrations in rat strains selectively bred for differences in response to stress: Confirmation and extension. Pharmacology Biochemistry and Behavior, 8, 75-80. Roth, R. H., & Stone, E. A. (1971). The action of reserpine on noradrenaline biosynthesis in sympathetic nerve tissue. Biochemical Pharmacology, 17, 1581-1590. Slater, J., Blizard, D. A., & Pohorecky, L. A. (1977). Central and peripheral norepinephrine metabolism in rat strains selectively bred for differences in response to stress. Pharmacology Biochemistry and Behavior, 6, 511-520.