0013-7227/79/1052-0537$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 2 Printed in U.S.A.
The Distribution and Concentration of ThyrotropinReleasimg Hormone in Discrete Human Hypothalamic Nuclei* MICHAEL KUBEK, JOHN F. WILBER, AND JACK M. GEORGE Center for Endocrinology, Metabolism, and Nutrition, Chicago, Illinois; the Louisiana State University Medical Center, New Orleans, Louisiana 70112; and the Ohio State University School of Medicine, Columbus, Ohio
ABSTRACT. TRH has been measured by RIA in 8 discrete human hypothalamic nuclei, the mammillary complex, the median eminence, and infundibular stalk. Tissues were derived from 13 subjects during autopsy after sudden death. Serial micropunch brain samples were secured from 300-jum frozen sections with stainless steel cannulae 1000 or 2000 jiim in diameter. Pooled samples were extracted with 0.1 N HC1, lyophilized, and resuspended in phosphate-saline buffer for TRH determinations. Stability of TRH in postmortem tissues was assessed in rats sacrificed at varying time intervals before tissue extraction. TRH was detected in all structures examined. Highest concentrations of TRH were found in the median eminence area (3.139 ng/mg protein) and arcuate nucleus (2.034 ng/mg protein). Intermediate TRH concentrations, representing 5-10% of total
TRH activity, were localized to the paraventricular and periventricular nuclei. Lowest TRH concentrations were found in the mamillary complex and the posterior, supraoptic, and anterior hypothalamic nuclei. TRH activity in two rat central nervous system structures, the hypothalamus and cerebral cortex, appeared to be completely stable up to 16 h postmortem at 4 C. It is concluded that TRH is localized in part in those human hypothalamic nuclei corresponding precisely to areas implicated in TSH regulation in experimental animals. The presence of TRH in additional human hypothalamic sites also, however, suggests that TRH may be subserving other functions as well in the role of neurotransmitter or neuromodulator. (Endocrinology 105: 537, 1979)
I
T IS well established that the hypothalamus occupies a central role in the regulation of TSH secretion (1). Electrical stimulation and lesion studies as well as pituitary implant experiments in animals have established that the thyrotropic area is a diffuse region extending through the anterior and medial hypothalamus to include the supraoptic, paraventricular, suprachiasmatic, ventromedial, and arcuate nuclei (2-9). RIA studies of TRH activity in human hypothalamic tissue blocks previously have revealed a rather generalized distribution of TRH (10). However, exact localizations of TRH within specific human hypothalamic loci have not yet been accomplished, and assessment of TRH concentration in specific human nuclei would provide a more precise basis for ascertaining potential neuroendocrine and/or other nonendocrine roles of TRH in man. The micropunch biopsy technique of Palkovits, which allows discrete nuclei to be dissected from frozen sections (11), has been used recently to study the distribution of TRH, other
peptides, and neurotransmitters in specific hypothalamic nuclei in the rat (12-19). In the present report, data concerning the distribtuion and concentration of T R H in discrete nuclei of the human hypothalamus using the micropunch Palkovits technique are presented. M a t e r i a l s and Methods The hypothalamus and upper pituitary stalk were dissected and frozen from brains derived from 13 individuals undergoing autopsy after sudden death. In all cases, brains were judged to be essentially normal by both gross and microscopic examinations. No neuropharmacologically active agents were being administered at the time of death. The mean time between death and tissue processing was 8.7 h. Tissues were obtained from 11 males, aged 20-74 yr, and two females, aged 20 and 43 yr. Serial, alternating, 300- and 100-jum frozen sections were cut from hypothalamic blocks in the frontal plane using a cryostat at -10 C. The 100-jum frozen sections were stained to identify hypothalamic nuclei and areas according to the atlases of DeArmond et al. (20) and Carpenter (21). Serial micropunch samples were extirpated and pooled from the 300-/xm frozen sections with stainless steel cannulae, 1000 or 2000 jum in diameter, with the aid of a stereomicroscope. The cannulae diameters were well within the diameters of the hypothalamic
Received July 24, 1978. Address requests for reprints to: Dr. John F. Wilber, Division of Endocrinology and Metabolism, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112. * This work was supported in part by NIH Grants AM-106699 and AM-14997 and the V.A., Columbus, OH.
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KUBEK, WILBER, AND GEORGE
538
Endo • 1979 Vol 105 • No 2
nuclear areas sampled, providing assurance that the micropunch biopsies contained neural elements of the nuclear areas being examined. Neural elements were verified directly in stained sections after microdissection. Pooled samples from each microdissected area were homogenized in 0.4 ml 0.1 N HC1 by sonication. An aliquot was taken for protein determination by the micromethod of Lowry et al. (22), and the remainder was lyophilized and resuspended in 0.4 ml 0.01 M PO4-buffered saline, pH 7.5, for TRH determinations by specific RIA (23). To examine the stability of TRH activity in postmortem central nervous system tissues, male Charles River rats (250275 g) were killed by cervical dislocation, and carcasses were maintained intact at 4 C for 0, 4, 8, or 16 h after sacrifice. An additional group of 10 carcasses was maintained also at 25 C for 2 h after sacrifice. At each of these time intervals, whole hypothalami and equivalent sections of frontal cortex were removed, frozen on dry ice, weighed, and extracted as described previously (24). TRH measurements were performed using duplicate samples of all extracts.
Results
arc
TRH immunoactivity was identified in all eight human hypothalamic nuclear groups examined and also in the mamillary complex, median eminence, and infundibulum (Table 1). TRH concentrations, as anticipated, were not homogeneous but varied throughout different nuclei. The greatest TRH concentrations were found in two nonnuclear areas, median eminence and infundibulum. Of the eight nuclear areas examined, the highest TRH concentrations were found in the arcuate nucleus, paraventricular nucleus, and periventricular nucleus. The lowest concentrations of TRH were localized to the anterior hypothalamic nucleus. Concentrations of TRH in the remaining nuclei yielded intermediate values ranging between 0.448-0.264 ng/mg protein. The anatomic distribution of TRH activity, expressed as a percentage of total TRH activity, is depicted in Fig. 1. It can be seen that the stalk-median eminence area contained most of the TRH activity, representing approximately 49% of the total. The next most abundant TABLE 1. Concentration and distribution of TRH in specific nuclei of the human hypothalamus TRH (ng/mg protein ± SEM)
Median eminence Arcuate nucleus Infundibulum Paraventricular nucleus Periventricular nucleus Dorsomedial nucleus Ventromedial nucleus Mamillary complex Posterior nucleus Supraoptic nucleus Anterior hypothalamic nucleus
\
3.139 ± 2.034 ± 1.954 ± 0.814 ± 0.599 ± 0.491 ± 0.448 ± 0.362 ± 0.326 ± 0.264 ± 0.088 ±
0.967 (8) 0.478 (11) 0.406 (7) 0.164 (12) 0.200 (6) 0.157 (4) 0.031 (4) 0.182 (10) 0.080 (7) 0.073 (13) 0.029 (7)
vmn me PONS
TRH DISTRIBUTION 1111120-30% E£SI5-1O% 1-5% 10-20% FIG. 1. Percentage of distribution of TRH in a parasaggital section of the human hypothalamus, drawn according to Carpenter (21). ac, Anterior commissure; mi, massa intermedia; son, supraoptic nucleus; prn, paraventricular nucleus; arc, arcuate nucleus, me, median eminence; stk, infundibular stalk; vmr, ventromedial nucleus; dmn, dorsomedial nucleus; piv, periventricular nucleus; pn, posterior nucleus; mac, mammillary complex; oc, optic chiasm; It, lamma terminalis.
source of TRH was the arcuate nucleus, representing almost 20%. The paraventricular and periventricular nuclei each contributed 5-10%. The remaining five nuclei (supraoptic, anterior, dorsomedial, posterior, and ventromedial) and the mamillary complex each contained only 1-5% of the total TRH activity. The stability of TRH in rat central nervous system tissue extracts obtained at progressive postmortem time intervals is summarized in Table 2. There was no significant reduction in TRH activity in either hypothalamic or cortical extracts vs. time. The slight fall in the hypothalamic content of TRH, from a mean of 4.14 to 3.79 ng/mg protein, was found not to be statistically significant by analysis of variance. Similarly, no loss of TRH activity was observed in cerebal cortical extracts. The apparent higher TRH values at 2-16 h were not statisti-
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TRH IN HUMAN HYPOTHALAMIC NUCLEI TABLE 2. Stability of endogenous central nervous system TRH Time (h)
Temperature (C)
0 2 4 8 16
25 25 4 4 4
TRH (ng/mg protein ± SEM) Hypothalamus 4.14 3.82 4.26 3.65 3.79
± ± ± ± ±
0.58 0.54 0.72 0.66 0.45
(9) (10) (9) (9) (9)
Cerebral cortex 0.014 0.015 0.018 0.018 0.017
± ± ± ± ±
0.0024 0.0019 0.0019 0.0022 0.0024
(9) (10) (9) (8) (9)
Rats were sacrificed by cervical dislocation at 0 h and central nervous system tissues were maintained in situ at 4 C until acetic acid extraction at the times indicated. An additional group of carcasses was held at 25 C for 2 h before tissue extraction. The number in parentheses indicates the total number of individual brain extracts assayed at each time point. No significant differences were observed statistically between means as assessed by one-way analysis of variance.
cally different from the control TRH value of 0.014 ng/ mg protein.
Discussion The present study provides new information regarding the quantitative distribution of TRH in discrete nuclear structures of the human hypothalamus. Previously, we have reported that whole human hypothalamic extracts contained a substance which was indistinguishable from synthetic TRH by criteria of immunoidentity, molecular volume, and identity of bioactivity (24). The data herein do not establish that TRH is actually located within neurons, axons, or nerve terminals of the human brain. The present studies indicate that TRH in man appears to be found in those specific hypothalamic regions associated with the hypothalamic control of TSH secretion in experimental animals: the paraventricular and periventricular nuclei, the anterior nucleus, the anterior portion of the ventromedial nucleus, and the arcuate nucleus (2-9). Quantitatively, we would infer that those areas adjacent to the third ventricle, the paraventricular, periventricular, and arcuate nuclear groups are particularly important in the human. In this regard, it is pertinent that hypothalamic deafferentation studies by Hefco and coworkers (25, 26) have emphasized the major role of arcuate nucleus in TSH secretory regulation, and the arcuate nucleus has been found to be the nuclear group most endowed with TRH in the present studies. Our data are compatible qualitatively with the earlier and elegant micropunch studies in the rat by Brownstein and associates (13-15). However, rat hypothalamic nuclei contain higher concentrations of TRH than were observed herein in the human. This difference is unlikely to be related to postmortem autolysis, since no significant reductions in TRH content were observed in unbroken tissue preparations of rat hypothalamus or cerebral cortex up to 16 h after death (Table 2). Moreover, no alterations in TRH content were produced in intact
539
tissues incubated at 25 C for 1 h to simulate the time period of exposure to room temperature of the human brain tissues before their refrigeration at 4 C. Obviously, certain limitations must be kept in mind in using rat tissues as an index for these human studies, particularly since human brains are larger and therefore become cool more slowly. The quantitative discrepancies between our data in man and those in the rat may relate in part to biological variables involving both species and individual differences. Unfortunately, no control of potentially important biological determinants was possible in the present investigation. The identification of TRH in hypothalamic nuclei and regions not involved classically in thyroid regulation might imply that the dorsomedial, posterior nuclei, and mamillary bodies participate uniquely in human thyroid regulation. More intriguing, however, is the possibility that some TRH, even within hypothalamic nuclei, could be subserving functions other than those involved in neuroendocrine regulation, inferred from earlier studies in both animals and man (27, 28). Moreover, axonal collateral projections from hypothalamic tuberoinfundibular neurons to other hypothalamic, thalamic, and extrahypothalamic areas have been described in animals which could provide anatomical pathways for TRH distribution to distant central nervous system loci where TRH may be functioning in the capacity of a neurotransmitter and/or neuromodulator substance (29).
Acknowledgment The technical assistance of Ms. Margaret Lorincz is gratefully acknowledged.
References 1. Wilber, J. F., Thyrotropin-releasing hormone: secretion and actions, Annu Rev Med 24: 353, 1973. 2. Averill, R. L. W., H. D. Purves, and N. E. Sirrett, Relation of the hypothalamus to anterior pituitary thyrotropin secretion, Endocrinology 69: 735, 1961. 3. Halasz, B., L. Pupp, and S. Uhlarik, Hypophysiotropic area in the hypothalamus, J Endocrinol 25: 147, 1962. 4. Greer, M., Evidence of hypothalamic control of pituitary release of thyrotropin, Proc Soc Exp Biol Med 77: 603, 1951. 5. D'Angelo, S. A., J. Snyder, and J. M. Grodin, Electrical stimulation of the hypothalamus: simultaneous effects on the pituitary-adrenal and thyroid systems of the rat, Endocrinology 75: 417, 1964. 6. Martin, J. D., and S. Reichlin, Neural regulation of the pituitarythyroid axis, In Kinny, A., and R. Anderson (eds.), Proceedings of the Sixth Midwest Conference on the Thyroid and Endocrinology, University of Missouri, Columbia, MO, 1970, p. 1. 7. Wilber, J. F., and J. C. Porter, Thyrotropin and growth hormone releasing activity in hypophysical portal blood, Endocrinology 87: 807, 1970. 8. Szentagothai, J., B. Flerko, B. Mess, and B. Halasz (eds.), Hypothalamic Control of the Anterior Pituitary, Akademiai Kiado, Budapest, 1968, p. 156. 9. Reichlin, S., J. B. Martin, M. Mitnick, R. L. Boshans, Y. Grimm, J. Bollinger, J. Gordon, and J. Malacara, The hypothalamus in pituitary-thyroid regulation, Recent Prog Horm Res 28: 229, 1972. 10. Okon, E., and Y. Koch, Localization of gonadotropin-releasing and
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11. 12.
13. 14. 15. 16.
17. 18. 19. 20.
KUBEK, WILBER, AND GEORGE
thyrotropin-releasing hormones in human brain by radioimmunoassay, Nature 263: 345, 1976. Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res 59: 449, 1973. Palkovits, M., Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies, In Stumpf, W. E., and L. D. Grant (eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, p. 72. Brownstein, M., M. Palkovits, J. Saavedra, R. Bassiri, and R. Utiger, Thyrotropin-releasing hormone in specific nuclei of rat brain, Science 85: 267, 1974. Brownstein, M., A. Arimura, H. Sato, A. V. Schally, and J. S. Kizer, The regional distribution of somatostatin in the rat brain, Endocrinology 96: 1456, 1975. Brownstein, M., Neurotransmitters and hypothalamic hormones in the central nervous system, Fed Proc 36: I960, 1977. Palkovitz, M., A. Arimura, M. J. Brownstein, A. V. Schally, and J. M. Saavedra, Luteinizing hormone-releasing hormone (LHRH) content of the hypothalamic nuclei in rat, Endocrinology 96: 554, 1974. Saavedra, J. M., M. Palkovits, M. Brownstein, and J. Axelrod, Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Res 77: 157, 1974. George, J. M., and J. Forrest, Vasopressin and oxytocin content of microdissected hypothalamic areas in rats with hereditary diabetes insipidus, Neuroendocrinology 21: 275, 1976. George, J. M., Immunoreactive vasopressin and oxytocin: concentration in individual human hypothalamic nuclei, Science 200: 342, 1978. DeArmond, S. J., M. M. Fusco, and M. M. Dewey, Structure of the
Endo 1979 Vol 105 , No 2
Human Brain, Oxford University Press, New York, 1974, p. 98. 21. Carpenter, M. B., Human Neuroanatomy, Williams and Wilkins, Baltimore, 1976, p. 478. 22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 23. Montoya, E., M. J. Seibel, and J. F. Wilber, Thyrotropin-releasing hormone secretory physiology: studies by radioimmunoassay and affinity chromatography, Endocrinology 96: 1413, 1975. 24. Kubek, M., M. A. Lorincz, and J. F. Wilber, The identification of thyrotopin-releasing hormone (TRH) in hypothalamic and extrahypothalamic loci of the human CNS, Brain Res 126: 196, 1977. 25. Mess, B., Intrahypothalamic localization and onset of production of thyrotropin-releasing factor (TRF) in the albino rat, Hormones 1: 322, 1970. 26. Hefco, E., L. Krulich, and J. E. Aschenbrenner, Effect of hypothalamic deafferentation on the secretion of thyrotropin in resting conditions in the rat, Endocrinology 97: 1226, 1975. 27. Hefco, E., L. Krulich, and J. E. Aschenbrenner, Effect of hypothalamic deafferentation on the secretion of thyrotropin during thyroid blockade and exposure to cold in the rat, Endocrinology 97: 1234, 1975.
28. Wilber, J. F., E. Montoya, N. Plotnikoff, W. F. White, R. Gendrich, L. Renaud, and J. Martin, Gonadotropin-releasing hormone and thyrotropin-releasing hormone: distribution and effects in the central nervous system, Recent Prog Horm Res 32: 117, 1976. 29. Renaud, L. P., Martin, J. B., and Brazeau, P., Depressant action of TRH, LH-RH and somatostatin on activity of central neurons, Nature (Lond) 255: 233, 1975.
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Vol. 105, No. 2 Printed in U.S.A.
The Distribution and Concentration of ThyrotropinReleasimg Hormone in Discrete Human Hypothalamic Nuclei* MICHAEL KUBEK, JOHN F. WILBER, AND JACK M. GEORGE Center for Endocrinology, Metabolism, and Nutrition, Chicago, Illinois; the Louisiana State University Medical Center, New Orleans, Louisiana 70112; and the Ohio State University School of Medicine, Columbus, Ohio
ABSTRACT. TRH has been measured by RIA in 8 discrete human hypothalamic nuclei, the mammillary complex, the median eminence, and infundibular stalk. Tissues were derived from 13 subjects during autopsy after sudden death. Serial micropunch brain samples were secured from 300-jum frozen sections with stainless steel cannulae 1000 or 2000 jiim in diameter. Pooled samples were extracted with 0.1 N HC1, lyophilized, and resuspended in phosphate-saline buffer for TRH determinations. Stability of TRH in postmortem tissues was assessed in rats sacrificed at varying time intervals before tissue extraction. TRH was detected in all structures examined. Highest concentrations of TRH were found in the median eminence area (3.139 ng/mg protein) and arcuate nucleus (2.034 ng/mg protein). Intermediate TRH concentrations, representing 5-10% of total
TRH activity, were localized to the paraventricular and periventricular nuclei. Lowest TRH concentrations were found in the mamillary complex and the posterior, supraoptic, and anterior hypothalamic nuclei. TRH activity in two rat central nervous system structures, the hypothalamus and cerebral cortex, appeared to be completely stable up to 16 h postmortem at 4 C. It is concluded that TRH is localized in part in those human hypothalamic nuclei corresponding precisely to areas implicated in TSH regulation in experimental animals. The presence of TRH in additional human hypothalamic sites also, however, suggests that TRH may be subserving other functions as well in the role of neurotransmitter or neuromodulator. (Endocrinology 105: 537, 1979)
I
T IS well established that the hypothalamus occupies a central role in the regulation of TSH secretion (1). Electrical stimulation and lesion studies as well as pituitary implant experiments in animals have established that the thyrotropic area is a diffuse region extending through the anterior and medial hypothalamus to include the supraoptic, paraventricular, suprachiasmatic, ventromedial, and arcuate nuclei (2-9). RIA studies of TRH activity in human hypothalamic tissue blocks previously have revealed a rather generalized distribution of TRH (10). However, exact localizations of TRH within specific human hypothalamic loci have not yet been accomplished, and assessment of TRH concentration in specific human nuclei would provide a more precise basis for ascertaining potential neuroendocrine and/or other nonendocrine roles of TRH in man. The micropunch biopsy technique of Palkovits, which allows discrete nuclei to be dissected from frozen sections (11), has been used recently to study the distribution of TRH, other
peptides, and neurotransmitters in specific hypothalamic nuclei in the rat (12-19). In the present report, data concerning the distribtuion and concentration of T R H in discrete nuclei of the human hypothalamus using the micropunch Palkovits technique are presented. M a t e r i a l s and Methods The hypothalamus and upper pituitary stalk were dissected and frozen from brains derived from 13 individuals undergoing autopsy after sudden death. In all cases, brains were judged to be essentially normal by both gross and microscopic examinations. No neuropharmacologically active agents were being administered at the time of death. The mean time between death and tissue processing was 8.7 h. Tissues were obtained from 11 males, aged 20-74 yr, and two females, aged 20 and 43 yr. Serial, alternating, 300- and 100-jum frozen sections were cut from hypothalamic blocks in the frontal plane using a cryostat at -10 C. The 100-jum frozen sections were stained to identify hypothalamic nuclei and areas according to the atlases of DeArmond et al. (20) and Carpenter (21). Serial micropunch samples were extirpated and pooled from the 300-/xm frozen sections with stainless steel cannulae, 1000 or 2000 jum in diameter, with the aid of a stereomicroscope. The cannulae diameters were well within the diameters of the hypothalamic
Received July 24, 1978. Address requests for reprints to: Dr. John F. Wilber, Division of Endocrinology and Metabolism, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112. * This work was supported in part by NIH Grants AM-106699 and AM-14997 and the V.A., Columbus, OH.
537
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KUBEK, WILBER, AND GEORGE
538
Endo • 1979 Vol 105 • No 2
nuclear areas sampled, providing assurance that the micropunch biopsies contained neural elements of the nuclear areas being examined. Neural elements were verified directly in stained sections after microdissection. Pooled samples from each microdissected area were homogenized in 0.4 ml 0.1 N HC1 by sonication. An aliquot was taken for protein determination by the micromethod of Lowry et al. (22), and the remainder was lyophilized and resuspended in 0.4 ml 0.01 M PO4-buffered saline, pH 7.5, for TRH determinations by specific RIA (23). To examine the stability of TRH activity in postmortem central nervous system tissues, male Charles River rats (250275 g) were killed by cervical dislocation, and carcasses were maintained intact at 4 C for 0, 4, 8, or 16 h after sacrifice. An additional group of 10 carcasses was maintained also at 25 C for 2 h after sacrifice. At each of these time intervals, whole hypothalami and equivalent sections of frontal cortex were removed, frozen on dry ice, weighed, and extracted as described previously (24). TRH measurements were performed using duplicate samples of all extracts.
Results
arc
TRH immunoactivity was identified in all eight human hypothalamic nuclear groups examined and also in the mamillary complex, median eminence, and infundibulum (Table 1). TRH concentrations, as anticipated, were not homogeneous but varied throughout different nuclei. The greatest TRH concentrations were found in two nonnuclear areas, median eminence and infundibulum. Of the eight nuclear areas examined, the highest TRH concentrations were found in the arcuate nucleus, paraventricular nucleus, and periventricular nucleus. The lowest concentrations of TRH were localized to the anterior hypothalamic nucleus. Concentrations of TRH in the remaining nuclei yielded intermediate values ranging between 0.448-0.264 ng/mg protein. The anatomic distribution of TRH activity, expressed as a percentage of total TRH activity, is depicted in Fig. 1. It can be seen that the stalk-median eminence area contained most of the TRH activity, representing approximately 49% of the total. The next most abundant TABLE 1. Concentration and distribution of TRH in specific nuclei of the human hypothalamus TRH (ng/mg protein ± SEM)
Median eminence Arcuate nucleus Infundibulum Paraventricular nucleus Periventricular nucleus Dorsomedial nucleus Ventromedial nucleus Mamillary complex Posterior nucleus Supraoptic nucleus Anterior hypothalamic nucleus
\
3.139 ± 2.034 ± 1.954 ± 0.814 ± 0.599 ± 0.491 ± 0.448 ± 0.362 ± 0.326 ± 0.264 ± 0.088 ±
0.967 (8) 0.478 (11) 0.406 (7) 0.164 (12) 0.200 (6) 0.157 (4) 0.031 (4) 0.182 (10) 0.080 (7) 0.073 (13) 0.029 (7)
vmn me PONS
TRH DISTRIBUTION 1111120-30% E£SI5-1O% 1-5% 10-20% FIG. 1. Percentage of distribution of TRH in a parasaggital section of the human hypothalamus, drawn according to Carpenter (21). ac, Anterior commissure; mi, massa intermedia; son, supraoptic nucleus; prn, paraventricular nucleus; arc, arcuate nucleus, me, median eminence; stk, infundibular stalk; vmr, ventromedial nucleus; dmn, dorsomedial nucleus; piv, periventricular nucleus; pn, posterior nucleus; mac, mammillary complex; oc, optic chiasm; It, lamma terminalis.
source of TRH was the arcuate nucleus, representing almost 20%. The paraventricular and periventricular nuclei each contributed 5-10%. The remaining five nuclei (supraoptic, anterior, dorsomedial, posterior, and ventromedial) and the mamillary complex each contained only 1-5% of the total TRH activity. The stability of TRH in rat central nervous system tissue extracts obtained at progressive postmortem time intervals is summarized in Table 2. There was no significant reduction in TRH activity in either hypothalamic or cortical extracts vs. time. The slight fall in the hypothalamic content of TRH, from a mean of 4.14 to 3.79 ng/mg protein, was found not to be statistically significant by analysis of variance. Similarly, no loss of TRH activity was observed in cerebal cortical extracts. The apparent higher TRH values at 2-16 h were not statisti-
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TRH IN HUMAN HYPOTHALAMIC NUCLEI TABLE 2. Stability of endogenous central nervous system TRH Time (h)
Temperature (C)
0 2 4 8 16
25 25 4 4 4
TRH (ng/mg protein ± SEM) Hypothalamus 4.14 3.82 4.26 3.65 3.79
± ± ± ± ±
0.58 0.54 0.72 0.66 0.45
(9) (10) (9) (9) (9)
Cerebral cortex 0.014 0.015 0.018 0.018 0.017
± ± ± ± ±
0.0024 0.0019 0.0019 0.0022 0.0024
(9) (10) (9) (8) (9)
Rats were sacrificed by cervical dislocation at 0 h and central nervous system tissues were maintained in situ at 4 C until acetic acid extraction at the times indicated. An additional group of carcasses was held at 25 C for 2 h before tissue extraction. The number in parentheses indicates the total number of individual brain extracts assayed at each time point. No significant differences were observed statistically between means as assessed by one-way analysis of variance.
cally different from the control TRH value of 0.014 ng/ mg protein.
Discussion The present study provides new information regarding the quantitative distribution of TRH in discrete nuclear structures of the human hypothalamus. Previously, we have reported that whole human hypothalamic extracts contained a substance which was indistinguishable from synthetic TRH by criteria of immunoidentity, molecular volume, and identity of bioactivity (24). The data herein do not establish that TRH is actually located within neurons, axons, or nerve terminals of the human brain. The present studies indicate that TRH in man appears to be found in those specific hypothalamic regions associated with the hypothalamic control of TSH secretion in experimental animals: the paraventricular and periventricular nuclei, the anterior nucleus, the anterior portion of the ventromedial nucleus, and the arcuate nucleus (2-9). Quantitatively, we would infer that those areas adjacent to the third ventricle, the paraventricular, periventricular, and arcuate nuclear groups are particularly important in the human. In this regard, it is pertinent that hypothalamic deafferentation studies by Hefco and coworkers (25, 26) have emphasized the major role of arcuate nucleus in TSH secretory regulation, and the arcuate nucleus has been found to be the nuclear group most endowed with TRH in the present studies. Our data are compatible qualitatively with the earlier and elegant micropunch studies in the rat by Brownstein and associates (13-15). However, rat hypothalamic nuclei contain higher concentrations of TRH than were observed herein in the human. This difference is unlikely to be related to postmortem autolysis, since no significant reductions in TRH content were observed in unbroken tissue preparations of rat hypothalamus or cerebral cortex up to 16 h after death (Table 2). Moreover, no alterations in TRH content were produced in intact
539
tissues incubated at 25 C for 1 h to simulate the time period of exposure to room temperature of the human brain tissues before their refrigeration at 4 C. Obviously, certain limitations must be kept in mind in using rat tissues as an index for these human studies, particularly since human brains are larger and therefore become cool more slowly. The quantitative discrepancies between our data in man and those in the rat may relate in part to biological variables involving both species and individual differences. Unfortunately, no control of potentially important biological determinants was possible in the present investigation. The identification of TRH in hypothalamic nuclei and regions not involved classically in thyroid regulation might imply that the dorsomedial, posterior nuclei, and mamillary bodies participate uniquely in human thyroid regulation. More intriguing, however, is the possibility that some TRH, even within hypothalamic nuclei, could be subserving functions other than those involved in neuroendocrine regulation, inferred from earlier studies in both animals and man (27, 28). Moreover, axonal collateral projections from hypothalamic tuberoinfundibular neurons to other hypothalamic, thalamic, and extrahypothalamic areas have been described in animals which could provide anatomical pathways for TRH distribution to distant central nervous system loci where TRH may be functioning in the capacity of a neurotransmitter and/or neuromodulator substance (29).
Acknowledgment The technical assistance of Ms. Margaret Lorincz is gratefully acknowledged.
References 1. Wilber, J. F., Thyrotropin-releasing hormone: secretion and actions, Annu Rev Med 24: 353, 1973. 2. Averill, R. L. W., H. D. Purves, and N. E. Sirrett, Relation of the hypothalamus to anterior pituitary thyrotropin secretion, Endocrinology 69: 735, 1961. 3. Halasz, B., L. Pupp, and S. Uhlarik, Hypophysiotropic area in the hypothalamus, J Endocrinol 25: 147, 1962. 4. Greer, M., Evidence of hypothalamic control of pituitary release of thyrotropin, Proc Soc Exp Biol Med 77: 603, 1951. 5. D'Angelo, S. A., J. Snyder, and J. M. Grodin, Electrical stimulation of the hypothalamus: simultaneous effects on the pituitary-adrenal and thyroid systems of the rat, Endocrinology 75: 417, 1964. 6. Martin, J. D., and S. Reichlin, Neural regulation of the pituitarythyroid axis, In Kinny, A., and R. Anderson (eds.), Proceedings of the Sixth Midwest Conference on the Thyroid and Endocrinology, University of Missouri, Columbia, MO, 1970, p. 1. 7. Wilber, J. F., and J. C. Porter, Thyrotropin and growth hormone releasing activity in hypophysical portal blood, Endocrinology 87: 807, 1970. 8. Szentagothai, J., B. Flerko, B. Mess, and B. Halasz (eds.), Hypothalamic Control of the Anterior Pituitary, Akademiai Kiado, Budapest, 1968, p. 156. 9. Reichlin, S., J. B. Martin, M. Mitnick, R. L. Boshans, Y. Grimm, J. Bollinger, J. Gordon, and J. Malacara, The hypothalamus in pituitary-thyroid regulation, Recent Prog Horm Res 28: 229, 1972. 10. Okon, E., and Y. Koch, Localization of gonadotropin-releasing and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 14:45 For personal use only. No other uses without permission. . All rights reserved.
540
11. 12.
13. 14. 15. 16.
17. 18. 19. 20.
KUBEK, WILBER, AND GEORGE
thyrotropin-releasing hormones in human brain by radioimmunoassay, Nature 263: 345, 1976. Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res 59: 449, 1973. Palkovits, M., Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies, In Stumpf, W. E., and L. D. Grant (eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, p. 72. Brownstein, M., M. Palkovits, J. Saavedra, R. Bassiri, and R. Utiger, Thyrotropin-releasing hormone in specific nuclei of rat brain, Science 85: 267, 1974. Brownstein, M., A. Arimura, H. Sato, A. V. Schally, and J. S. Kizer, The regional distribution of somatostatin in the rat brain, Endocrinology 96: 1456, 1975. Brownstein, M., Neurotransmitters and hypothalamic hormones in the central nervous system, Fed Proc 36: I960, 1977. Palkovitz, M., A. Arimura, M. J. Brownstein, A. V. Schally, and J. M. Saavedra, Luteinizing hormone-releasing hormone (LHRH) content of the hypothalamic nuclei in rat, Endocrinology 96: 554, 1974. Saavedra, J. M., M. Palkovits, M. Brownstein, and J. Axelrod, Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Res 77: 157, 1974. George, J. M., and J. Forrest, Vasopressin and oxytocin content of microdissected hypothalamic areas in rats with hereditary diabetes insipidus, Neuroendocrinology 21: 275, 1976. George, J. M., Immunoreactive vasopressin and oxytocin: concentration in individual human hypothalamic nuclei, Science 200: 342, 1978. DeArmond, S. J., M. M. Fusco, and M. M. Dewey, Structure of the
Endo 1979 Vol 105 , No 2
Human Brain, Oxford University Press, New York, 1974, p. 98. 21. Carpenter, M. B., Human Neuroanatomy, Williams and Wilkins, Baltimore, 1976, p. 478. 22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 23. Montoya, E., M. J. Seibel, and J. F. Wilber, Thyrotropin-releasing hormone secretory physiology: studies by radioimmunoassay and affinity chromatography, Endocrinology 96: 1413, 1975. 24. Kubek, M., M. A. Lorincz, and J. F. Wilber, The identification of thyrotopin-releasing hormone (TRH) in hypothalamic and extrahypothalamic loci of the human CNS, Brain Res 126: 196, 1977. 25. Mess, B., Intrahypothalamic localization and onset of production of thyrotropin-releasing factor (TRF) in the albino rat, Hormones 1: 322, 1970. 26. Hefco, E., L. Krulich, and J. E. Aschenbrenner, Effect of hypothalamic deafferentation on the secretion of thyrotropin in resting conditions in the rat, Endocrinology 97: 1226, 1975. 27. Hefco, E., L. Krulich, and J. E. Aschenbrenner, Effect of hypothalamic deafferentation on the secretion of thyrotropin during thyroid blockade and exposure to cold in the rat, Endocrinology 97: 1234, 1975.
28. Wilber, J. F., E. Montoya, N. Plotnikoff, W. F. White, R. Gendrich, L. Renaud, and J. Martin, Gonadotropin-releasing hormone and thyrotropin-releasing hormone: distribution and effects in the central nervous system, Recent Prog Horm Res 32: 117, 1976. 29. Renaud, L. P., Martin, J. B., and Brazeau, P., Depressant action of TRH, LH-RH and somatostatin on activity of central neurons, Nature (Lond) 255: 233, 1975.
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