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Effect of electroconvulsive shock on the content of thyrotropin-releasing hormone in rat brain
Life Sciences, Volume 34, pp. 1149-1152 Printed in the U.S.A.
Pergamon Press
EFFECT OF ELECTROCONVULSIVE SHOCK ON THE CONTENT OF THYROTROPIN-RELEASING HORMONE IN RAT BRAIN Michael J. Kubek and Albert Sattin Departments of Anatomy and Psychiatry Indiana University School of Medicine, and V.A. Medical Center Indianapolis, Indiana 46223 (Received in final form January 13, 1984) Summary Five grand-mal seizures were electrically induced in rats on alternate days. Forty-eight hours following the last seizure, TRH was quantitated in extracts of anterior cortex, hippocampus, striatum, thalamus plus midbrain, and hypothalamus. When compared to sham treated controls, TRH was found to be elevated 5-fold in the hippocampus and 2-fold in the striatum with no changes observed in the remaining regions. Since the time chosen for analysis excludes acute post-ictal effects, these results draw attention to a prolonged alteration of TRH levels in specific brain regions in an animal model of electroconvulsive treatment. Thyrotropin-Releasing Hormone (TRH) has been implicated in the regulation of several mammalian behaviors. Although the results of clinical testing are equivocal, it has been suggested that TRH also might play a role in affective illness (1,2). In the biological classification of depressive disorders, the TSH response to intravenous TRH has been useful since certain subgroups of depressed patients show a blunted response to TRH (1,3). Moreover, the reversal of this blunted TSH response following antidepressant therapy can be used to predict the prognosis in certain endogenous depressions (3). Immunoreactive TRH is found widely distributed throughout the neuroaxis of rats and humans (4-6). Since affective disorders are thought to represent limbic system dysfunctions in the CNS, we undertook an examination of five limbic and associated regions with a view toward possible changes in the regional content of im~unoreactive TRH following application of electroconvulsive shock (ECS) using a paradigm that mimics the most effective clinical antidepressant treatment, electroconvulsive therapy (ECT) (7). A preliminary account of these findings has been presented (8). Methods Male Sprague-Dawley rats (150-175 gm), obtained from Harlan Industries (Indianapolis), were used for all experiments. They were housed in pairs in a facility employing NIH animal care standards with unrestricted access to standard feed and water and in a 12-hour lighting cycle. For ECS, rats were hand-held while smooth disc electrodes were applied to the ears with spring-held clips. Electrode paste was used to increase conductance, and the electrodes were cleaned with fine sandpaper before each ECS.
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
1150
Electroconvulsive Shock on TRH Content
Vol. 34, No. 12, 1984
To approximate constant current output, 1300 volts were passed for 1 second through an internal resistance of 40,000 ohms giving 30 m A output (a.c.) through a standard 5,000 ohm external resistance. When the external resistance was replaced by the animal, this current produced, sequentially, tonic extension of forelimbs, hindlimbs, then clonus, and transiently altered behavior for several minutes. Sham animals were treated identically except current was shunted through the external resistance. All rats were first adapted to handling. This avoids fear-induced aggression. Individual members of each caged pair of rats always received the same treatment. Sham ECS and ECS was given beginning at 1600 hrs. on alternate days for a total of 5 treatments. All rats continued to grow during the treatment, but ECS rats grew at 50% of the control rate. As we were not interested in acute post-ictal effects, the time chosen for sacrifice was 48 + 3 hrs. after the last treatment. Following decapitation, the brains were removed immediately following midcollicular transection and dissected on the surface of an ice-chilled Petri plate. The hypothalamus was dissected with a circular incision from the ventral surface. The hippocampi were reflected from each hemi-cortex and separated by blunt dissection. Striata were dissected with a special knife made by curving and sharpening the flat end of a weighing spatula. Cortical tissue anterior to a coronal cut at the mid-point of the ventricular aspect of the striatum was defined as anterior cortex. The remainder of the subcortical forebrain tissue was designated as thalamus + midbrain. Designated regions were quickly weighed, then frozen on solid CO 2 and stored at -70 ° until further processed. All tissues were extracted with acetic acid, and TRH was measured by radioimmunoassay (RIA) as previously described, using the original antibody at a final dilution of 1:100,400 (5,9). The assay, highly stereospecific for TRH, has a lower limit of sensitivity between 2-4 pg per tube. Assay analysis was performed using the NIH log-logit RIA program (i0) through Indiana University Computing Services. The interassay coefficient of variation was 13.2% (n=59). Values, uncorrected for recovery, are expressed as pg/mg wet weight, and statistical comparisons were performed using a 2-tailed Student t-test. Results and Discussion Two days after the last of 5 alternate-day ECS, tissue content of TRH was significantly elevated over the control level in two of the five brain regions examined (Table I). Tissue weights did not differ between the two treatment groups. The magnitude of the ECS effect was nearly 5-fold in hippocampus and 2-fold in striatum. As no effect was seen in the other regions examined, it TABLE I Effect of ECS on TRH Content in Five Regions of Rat Brain
Region Anterior Cortex Hippocampus Striatum Thalamus + Midbrain Hypothalamus
TRH (pg/mg wet weight) Sham ECS ECS 3.57 6.18 9.26 28.53 292.1
+ 0.50 + 1.01 + 1.37 ~ 2.14 + 26.3
(16) (15) (16) (16) (13)
4.39 *30.37 +19.01 35.50 299.7
+ 0.50 + 2.75 + 2.35 ~ 3.83 + 19.2
(15) (15) (15) (13) (17)
Results are means + SEM (number of rats) pooled from three separate experiments. Differences from sham ECS were: *p <0.0005 and +p <0.005.
Vol. 34, No. 12, 1984
Electroconvulsive Shock on TRH Content
1151
would appear that the effect of ECS on brain TRH is selective. Furthermore, the effect is prolonged for at least 2 days after completion of the treatment. Further work is planned to characterize the time course of the increase and possible decline in TRH content of these and other relevant brain regions. The regional selectivity of the ECS effect has been further characterized by preliminary data in which ECS-induced increases of TRH in hippocampus and striatum were extended to include amygdala-pyriform cortex and the remainder of cerebral cortex, but no effect was observed in n. accumbens. Importantly, subconvulsive shock, although stressful to the rats, had no effect on TRH content in any of these regions except for striatum where the increase was as great or greater than that following ECS (II). Prolonged and large increases in an endogenous brain peptide of the magnitude reported here are unusual. Assaying immunoreactive met-enkephalin in rat brain, Hong et al. found almost a 2-fold increase 24 hours after I0 daily ECS in hypothalamus, and smaller increases in the accumbens, septum, caudate, and amygdala. Subconvulsive shock had no effect in these regions (12). Although their ECS paradigm differs from ours, it seems likely that the regional effects of ECS on TRH differ from the effects on met-enkephalin. In contrast with the latter, we have not observed any ECS-induced increase in TRH in n. accumbens or in whole hypothalamus. However, because of the very high initial content of TRH in whole hypothalamus, it is possible that a change in TRH content was concealed in one or more hypothalamic nuclei. The mechanism responsible for accumulation of TRH in specific CNS loci following ECS is not presently understood. Possible neurochemical explanations include increased rate of TRH synthesis, or decreased rate of metabolism or release. The neurophysiological meaning of such increased TRH content is unknown. Spindel et al. have reported greater than a 2-fold increase in rat striatal TRH 5 days following hemisection of the nigrostriatal tract. This increased tissue content of TRH was hypothesized to be due to loss of GABAergic inhibition of TRH release (13). Additionally, the acute release of biogenic amines and possibly other transmitters during each seizure might initiate neuromodulatory effects that result, over time, in the observed accumulation of TRH. Functionally-induced long-term modulation of transmitter activity has been described in simple nervous systems (14). Alternatively, seizures may have a more direct effect on TRH itself. TRH has been shown to interact with and modulate catecholamine, cholinergic, and possibly indoleamine systems in brain (1,2,9). Since these transmitter systems are believed to be involved in affective disorders, it will be important to obtain further data concerning the interactions of TRH with these systems. In summary, the present results provide the first evidence that ECS, given in a temporal paradigm used clinically, can induce significant and prolonged elevations of TRH in specific brain loci. This initial finding suggests that alterations of TRH content might contribute to our understanding of the antidepressant effect of ECT, and further implicates TRH as a neurotransmitter and/or neuromodulator that is relevant to affective function. Acknowledsements Supported by MH 29126, Indiana University Biomedical Research Grant, and the Mental Health Research and Education Foundation, Indianapolis. We thank Donald Etchison and Marianna Zaphiriou for technical assistance, and James Norton, Ph.D. for statistical advice.
1152
Electroconvulsive Shock on TRH Content
Vol. 34, No. 12, 1984
References I. 2. 3. 4.
C.B. NEMEROFF, P.T. LOOSEN, G. BISSETTE, P.J. MANBERG, I.C. WILSON, M.A. LIPTON and A.J. PRANGE, Jr., Psychoneuroendocrinol. 3 279-310 (1979). G.G. YARBROUGH, Progr. in Neurobiol. 12 291-312 (1979). C. KIRKEGAARD, Psychoneuroendocrinol. 6 189-212 (1981). M.J. BROWNSTEIN, M. PALKOVITS, J.M. SAAVEDRA, R.M. BASSIRI and R.D. UTIGER, Science 185 167-169 (1974). M.J. KUBEK, M.A. LORINCZ and J.F. WlLBER, Brain Reso 126 196-200 (1977). M.J. KUBEK, J.F. WILBER and J.M. GEORGE, Endocrinol. 105 537-540 (1979). F.H. FRANKEL, Electroconvulsive Therapy: Report of the Task Force on Electroconvulsive Therapy of the American Psychiatric Association, The American Psychiatric Association, Washington, DC (1978). M.J. KUBEK, D. ETCHISON and A. SATTIN, Abstr. Soc. Neurosci. 7 379 (1981). J.F. WILBER, E. MONTOYA, N.P. PLONTNIKOFF, W.F. WHITE, R. GENDRICH, L. RENAUD and J.B. MARTIN, Rec. Prog. Horm. Res. 32 117-159 (1976). D. RODBARD, Clin. Chem. 20 1255-1270 (1974). M.Jo KUBEK, J.L. MEYERHOFF and A. SATTIN, Abstr. Soc. Neurosci. 8 982 (1982). J.S. HONG, J.C. GILLIN, Y.-Y. YANG and E. COSTA, Brain Res. 177 273-278 (1979). EoR. SPINDEL, D.J. PETTIBONE and R.J. WURTMAN, Brain Res. 216 323-331 (1981). E.R. KANDEL and J.H. SCHWARTZ, Science 218 433-443 (1982). J.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14.
Pergamon Press
EFFECT OF ELECTROCONVULSIVE SHOCK ON THE CONTENT OF THYROTROPIN-RELEASING HORMONE IN RAT BRAIN Michael J. Kubek and Albert Sattin Departments of Anatomy and Psychiatry Indiana University School of Medicine, and V.A. Medical Center Indianapolis, Indiana 46223 (Received in final form January 13, 1984) Summary Five grand-mal seizures were electrically induced in rats on alternate days. Forty-eight hours following the last seizure, TRH was quantitated in extracts of anterior cortex, hippocampus, striatum, thalamus plus midbrain, and hypothalamus. When compared to sham treated controls, TRH was found to be elevated 5-fold in the hippocampus and 2-fold in the striatum with no changes observed in the remaining regions. Since the time chosen for analysis excludes acute post-ictal effects, these results draw attention to a prolonged alteration of TRH levels in specific brain regions in an animal model of electroconvulsive treatment. Thyrotropin-Releasing Hormone (TRH) has been implicated in the regulation of several mammalian behaviors. Although the results of clinical testing are equivocal, it has been suggested that TRH also might play a role in affective illness (1,2). In the biological classification of depressive disorders, the TSH response to intravenous TRH has been useful since certain subgroups of depressed patients show a blunted response to TRH (1,3). Moreover, the reversal of this blunted TSH response following antidepressant therapy can be used to predict the prognosis in certain endogenous depressions (3). Immunoreactive TRH is found widely distributed throughout the neuroaxis of rats and humans (4-6). Since affective disorders are thought to represent limbic system dysfunctions in the CNS, we undertook an examination of five limbic and associated regions with a view toward possible changes in the regional content of im~unoreactive TRH following application of electroconvulsive shock (ECS) using a paradigm that mimics the most effective clinical antidepressant treatment, electroconvulsive therapy (ECT) (7). A preliminary account of these findings has been presented (8). Methods Male Sprague-Dawley rats (150-175 gm), obtained from Harlan Industries (Indianapolis), were used for all experiments. They were housed in pairs in a facility employing NIH animal care standards with unrestricted access to standard feed and water and in a 12-hour lighting cycle. For ECS, rats were hand-held while smooth disc electrodes were applied to the ears with spring-held clips. Electrode paste was used to increase conductance, and the electrodes were cleaned with fine sandpaper before each ECS.
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
1150
Electroconvulsive Shock on TRH Content
Vol. 34, No. 12, 1984
To approximate constant current output, 1300 volts were passed for 1 second through an internal resistance of 40,000 ohms giving 30 m A output (a.c.) through a standard 5,000 ohm external resistance. When the external resistance was replaced by the animal, this current produced, sequentially, tonic extension of forelimbs, hindlimbs, then clonus, and transiently altered behavior for several minutes. Sham animals were treated identically except current was shunted through the external resistance. All rats were first adapted to handling. This avoids fear-induced aggression. Individual members of each caged pair of rats always received the same treatment. Sham ECS and ECS was given beginning at 1600 hrs. on alternate days for a total of 5 treatments. All rats continued to grow during the treatment, but ECS rats grew at 50% of the control rate. As we were not interested in acute post-ictal effects, the time chosen for sacrifice was 48 + 3 hrs. after the last treatment. Following decapitation, the brains were removed immediately following midcollicular transection and dissected on the surface of an ice-chilled Petri plate. The hypothalamus was dissected with a circular incision from the ventral surface. The hippocampi were reflected from each hemi-cortex and separated by blunt dissection. Striata were dissected with a special knife made by curving and sharpening the flat end of a weighing spatula. Cortical tissue anterior to a coronal cut at the mid-point of the ventricular aspect of the striatum was defined as anterior cortex. The remainder of the subcortical forebrain tissue was designated as thalamus + midbrain. Designated regions were quickly weighed, then frozen on solid CO 2 and stored at -70 ° until further processed. All tissues were extracted with acetic acid, and TRH was measured by radioimmunoassay (RIA) as previously described, using the original antibody at a final dilution of 1:100,400 (5,9). The assay, highly stereospecific for TRH, has a lower limit of sensitivity between 2-4 pg per tube. Assay analysis was performed using the NIH log-logit RIA program (i0) through Indiana University Computing Services. The interassay coefficient of variation was 13.2% (n=59). Values, uncorrected for recovery, are expressed as pg/mg wet weight, and statistical comparisons were performed using a 2-tailed Student t-test. Results and Discussion Two days after the last of 5 alternate-day ECS, tissue content of TRH was significantly elevated over the control level in two of the five brain regions examined (Table I). Tissue weights did not differ between the two treatment groups. The magnitude of the ECS effect was nearly 5-fold in hippocampus and 2-fold in striatum. As no effect was seen in the other regions examined, it TABLE I Effect of ECS on TRH Content in Five Regions of Rat Brain
Region Anterior Cortex Hippocampus Striatum Thalamus + Midbrain Hypothalamus
TRH (pg/mg wet weight) Sham ECS ECS 3.57 6.18 9.26 28.53 292.1
+ 0.50 + 1.01 + 1.37 ~ 2.14 + 26.3
(16) (15) (16) (16) (13)
4.39 *30.37 +19.01 35.50 299.7
+ 0.50 + 2.75 + 2.35 ~ 3.83 + 19.2
(15) (15) (15) (13) (17)
Results are means + SEM (number of rats) pooled from three separate experiments. Differences from sham ECS were: *p <0.0005 and +p <0.005.
Vol. 34, No. 12, 1984
Electroconvulsive Shock on TRH Content
1151
would appear that the effect of ECS on brain TRH is selective. Furthermore, the effect is prolonged for at least 2 days after completion of the treatment. Further work is planned to characterize the time course of the increase and possible decline in TRH content of these and other relevant brain regions. The regional selectivity of the ECS effect has been further characterized by preliminary data in which ECS-induced increases of TRH in hippocampus and striatum were extended to include amygdala-pyriform cortex and the remainder of cerebral cortex, but no effect was observed in n. accumbens. Importantly, subconvulsive shock, although stressful to the rats, had no effect on TRH content in any of these regions except for striatum where the increase was as great or greater than that following ECS (II). Prolonged and large increases in an endogenous brain peptide of the magnitude reported here are unusual. Assaying immunoreactive met-enkephalin in rat brain, Hong et al. found almost a 2-fold increase 24 hours after I0 daily ECS in hypothalamus, and smaller increases in the accumbens, septum, caudate, and amygdala. Subconvulsive shock had no effect in these regions (12). Although their ECS paradigm differs from ours, it seems likely that the regional effects of ECS on TRH differ from the effects on met-enkephalin. In contrast with the latter, we have not observed any ECS-induced increase in TRH in n. accumbens or in whole hypothalamus. However, because of the very high initial content of TRH in whole hypothalamus, it is possible that a change in TRH content was concealed in one or more hypothalamic nuclei. The mechanism responsible for accumulation of TRH in specific CNS loci following ECS is not presently understood. Possible neurochemical explanations include increased rate of TRH synthesis, or decreased rate of metabolism or release. The neurophysiological meaning of such increased TRH content is unknown. Spindel et al. have reported greater than a 2-fold increase in rat striatal TRH 5 days following hemisection of the nigrostriatal tract. This increased tissue content of TRH was hypothesized to be due to loss of GABAergic inhibition of TRH release (13). Additionally, the acute release of biogenic amines and possibly other transmitters during each seizure might initiate neuromodulatory effects that result, over time, in the observed accumulation of TRH. Functionally-induced long-term modulation of transmitter activity has been described in simple nervous systems (14). Alternatively, seizures may have a more direct effect on TRH itself. TRH has been shown to interact with and modulate catecholamine, cholinergic, and possibly indoleamine systems in brain (1,2,9). Since these transmitter systems are believed to be involved in affective disorders, it will be important to obtain further data concerning the interactions of TRH with these systems. In summary, the present results provide the first evidence that ECS, given in a temporal paradigm used clinically, can induce significant and prolonged elevations of TRH in specific brain loci. This initial finding suggests that alterations of TRH content might contribute to our understanding of the antidepressant effect of ECT, and further implicates TRH as a neurotransmitter and/or neuromodulator that is relevant to affective function. Acknowledsements Supported by MH 29126, Indiana University Biomedical Research Grant, and the Mental Health Research and Education Foundation, Indianapolis. We thank Donald Etchison and Marianna Zaphiriou for technical assistance, and James Norton, Ph.D. for statistical advice.
1152
Electroconvulsive Shock on TRH Content
Vol. 34, No. 12, 1984
References I. 2. 3. 4.
C.B. NEMEROFF, P.T. LOOSEN, G. BISSETTE, P.J. MANBERG, I.C. WILSON, M.A. LIPTON and A.J. PRANGE, Jr., Psychoneuroendocrinol. 3 279-310 (1979). G.G. YARBROUGH, Progr. in Neurobiol. 12 291-312 (1979). C. KIRKEGAARD, Psychoneuroendocrinol. 6 189-212 (1981). M.J. BROWNSTEIN, M. PALKOVITS, J.M. SAAVEDRA, R.M. BASSIRI and R.D. UTIGER, Science 185 167-169 (1974). M.J. KUBEK, M.A. LORINCZ and J.F. WlLBER, Brain Reso 126 196-200 (1977). M.J. KUBEK, J.F. WILBER and J.M. GEORGE, Endocrinol. 105 537-540 (1979). F.H. FRANKEL, Electroconvulsive Therapy: Report of the Task Force on Electroconvulsive Therapy of the American Psychiatric Association, The American Psychiatric Association, Washington, DC (1978). M.J. KUBEK, D. ETCHISON and A. SATTIN, Abstr. Soc. Neurosci. 7 379 (1981). J.F. WILBER, E. MONTOYA, N.P. PLONTNIKOFF, W.F. WHITE, R. GENDRICH, L. RENAUD and J.B. MARTIN, Rec. Prog. Horm. Res. 32 117-159 (1976). D. RODBARD, Clin. Chem. 20 1255-1270 (1974). M.Jo KUBEK, J.L. MEYERHOFF and A. SATTIN, Abstr. Soc. Neurosci. 8 982 (1982). J.S. HONG, J.C. GILLIN, Y.-Y. YANG and E. COSTA, Brain Res. 177 273-278 (1979). EoR. SPINDEL, D.J. PETTIBONE and R.J. WURTMAN, Brain Res. 216 323-331 (1981). E.R. KANDEL and J.H. SCHWARTZ, Science 218 433-443 (1982). J.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14.