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Cholecystokinin octapeptide releases growth hormone from the pituitary in vitro
Life Sciences, Vol . 25, pp . 1201-1206 Printed in the U .S .A .
Pergamon Press
CHOLECYSTOKININ OCTAPEPTIDE RELEASES GROWTH HORMONE FROM THE PITUITARY IN VITRO John E . Morley', Shlomo Melmed', Jacqueline Briggs', Harold E . Carlson', Jerome M . Hershman', Travis E . Solomon 2, Cornelis Lamers 2 and David A . Damassa 2 'Endocrine Research Laboratory, Wadsworth VA Medical Center and Department of Medicine, University of California Los Angeles, CA 90073 2 Center for Ulcer Research and Education, University of California, Los Angeles, CA 90024 9 Brain Research Institute, University of California, Los Angeles, CA 90024 (Received in final form August 20, 1979) Summary Cholecyso kinin-octapeptide (CCK-8)(10-6 to 10 -8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH3 pituitary tumor cells . Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion in vitro . In the GH3 cell'line, CCK-8 (10 - 7M) reversed theT i fbitory effect of somatostatin (10-5M) on GH release . As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor .
n
Several peptides including somatostatin (1), thyrotropin-releasing hormone (2), substance P (3), bombesin (4), neurotensin (5), the enkephalins (6), vasoactive intestinal peptide (7) and cholecystokinin (8) have been found in the brain and the gastrointestinal tract . A number of these hormones influence both gastrointestinal function and pituitary hormone release (9-13) . For this reason we assessed the effects of five peptides originally isolated from the gastrointestinal tract on the secretion of hormones from incubated rat anterior pituitary quarters . We report here that cholecystokinin-octapeptide (CCK-8) produced a marked increase in growth hormone release in this system . Several analogues of CCK-8 also produced GH release . CCK-8 prevented the inhibitory effect of somatostatin on GH release from cultured GH3 rat pituitary tumor cells . Methods Acute anterior pituitary cultures : Anterior pituitaries were removed from male Sprague-Dawley rats weighing approximately 150 g . The pituitaries were quartered and the quarters randomized . Three quarters were placed in 10 ml stoppered Erlenmeyer flasks containing 1 ml modified Gey and Gey medium (14) which had been equilibrated to the culture conditions . The tissue portions were individually preincubated for 2 hours in a gyrotory water-bath shaker at 370C under 5% C02-95% 02 . The preincubation medium was then
0024-3205/79/141201-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Cholecystokinin Releases Growth Hormone
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replaced by 1 ml gassed medium containing either no additions (control) or one of the gastrointestinal hormones at varying concentrations . After 1 hour further incubation at 37oC the media were collected and stored frozen until assayed for growth hormone (GH), prolactin (PRL), thyrotropin (TSH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) . At the end of each experiment the pituitary tissue from each flask was weighed ; all results were calculated per mg wet weight of tissue and results are expressed as % of the mean control value . All the anterior pituitary hormones were measured by radioimmunoassay using materials and reference preparations supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethseda, Maryland . There was no cross-reaction of any of the gastrointestinal peptides used in any of the radioimmunoassays . Comparisons were made by using the Student's t-test . GH3 pituitary tumor cell cultures : Cells were supplied by ATCC and were cultured as monolayers in plastic multiwell 35 mm tissue culture plates (LUX), using Ham's F10 medium supplemented with 15% horse serum and 2 .5% fetal calf serum and containing penicillin (50 U/ml) and streptomycin (50 ug/ml) . Cells were maintained as described (15) . Experimental procedure : When cells were in late logarithmic phase of growth, medium was aspirated from each well and cells washed with serum-free Ham's F10 medium . Wells were then incubated with serum-free Ham's F10 medium containing appropriate dilutions of CCK-8 . Control wells contained medium alone . Incubations were in a humidified atmosphere, 95% air - 5% C02 at 37oC . At the end of each incubation, medium was aspirated for hormone assay . Monolayer of cells was harvested in saline suspension and counted in a Coulter counter . Medium and CCK solutions in Ham's F10 were equilibrated with 95% air - 5% C02 at 37oC before use . Results The results of the experiments using cultured pituitary quarters are given in Table I . In addition to the stimulatory effect of CCK-8 on GH release, tetragastrin (10-6M) also produced GH release . None of the other hor mones tested altered secretion of growth hormone . Bombesin, secretin, CCK-8 and pancreatic polypeptide all increased gonadotropin release at high concentrations . Tetragastrin and CCK-8 partially inhibited thyrotropin . Cholecystokinin-39 was also a potent releaser of GH (10-6 M, 253±60%, SEM and 10-7M, 160±20% of controls, p<0 .025) . High molecular weight forms are converted to smaller molecular weight forms in vitro (C . Lamers and J .H . Walsh : Personal Communication) and this may explain the effect of CCK-39 . Caerulein, a decapeptide from frog skin, with only 3 amino acids different from CCK-8, failed to release GH in 2 experiments . Gastrin-17 (10-6M) also enhanced growth hormone secretion (161±18% of controls, p<0 .05) . CCK-8 also produced GH release from monolayer cultures of GH3 pituitary tumor cells (Fig . 1) . In addition, the inhibitory effect of somatostatin (10 - 5M) was reversed by the addition of CCK-8 (10 - 7M) in the GH3 tumor system in vitro (Fig . 2) .
Cholecystokinin Releases Growth Hormone
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TABLE 1 The effect of CCK-8, tetragastrin, bombesin, secretin and pancreatic polypeptide on anterior pituitary hormone release . SUBSTANCE CCK-8 Tetragastrin Bombes1n Secretin Pancreatic Polypeptide
CONCENTRATION 1
N -
10 -6 10- 7 10- 6 10- 7 10- 7 10 -8 10-9 10- 6 10 - 7 10-8 10- 6
5 5 5 5 3 6 5 5 5 5 5
GH 205±19* 158±241 175±23 i 109±25 113±10 107±14 88±8 115±13 94±5 97±16 116±19
PRL TSH % of control) 82±9 120±14 96±14 76±14 119±21 104±18 68±14 120±15 92±10 110±7 107±17
79±5 i 42±lü:i 78±10 49±16 1 98±15 133±22 89±23 119±20 102±22 97±12 105t41
LH 174±14* 118±22 88±16 114±28 180±7* 118±33 78±14 262±48 i 118±15 161±30 157±14 t
FSH 134±2 143±2 88±13 147±19 112±11 119±8 160±14*
*p<0 .01, ip<0 .05, Mean±SEM Discussion Cholecystokinin immunoreactivity has been demonstrated to be present in the hypothalamus, with the predominant form co-chromatographing with CCK-8 on Sephadex G-50 (16,17) . Additionally, a substance with immunologic and chromatographic characteristics similar to gastrin has been demonstrated in the pituitary (18) . Using immunohistochemical techniques, Innis et al (19) found that the most dense collections of CCK-8 were in the medial hypothalamus and the periaqueductal gray . The reason for the high concentration of cholecystokinin in the brain is unknown . CCK-8 may be related to satiety (20,21) . Electrical stimulation of the ventromedial and arcuate nuclei in the pentobarbital anesthetized rat elicits GH secretion (22), and as satiety has been shown to be related to the ventromedial nucleus (23), a common peptide subserving these two functions would be an attractive hypothesis . Despite considerable physiologic and biochemical evidence for the existence of a growth hormone releasing factor, attempts to identify this substance have thus far been unsuccessful . A large number of peptides have been shown to release GH in vivo but only thyrotropin-releasing hormone and arginine vasopressin haveneen-Zemonstrated to have a direct effect on the pituitary in vitro (14,24) The recent demonstration that corticotropin releasing cttor activity in crude stalk median eminence is due to a multifactor system (25) suggests that a similar multi-factor hypothesis may explain the difficulty experienced in the search for a unique peptide with potent growth hormone releasing factor activity . The evidence presented here suggests that CCK-8 may play a role as an endogenous growth hormone releasing factor . The ability of CCK-8 (10-7M) to neutralize the inhibitory effect of somatostatin (10 - 5M) further indicates that CCK-8 may have a physiologic role in modulating GH secretion .
1204
Vol . 25, No . 14, 1979
Cholecystokinin Releases Growth Hormone
1 .50- p cO .01
c0 .05
NS
140. 130J GH CONTROL 120110100
10"M
W
M CCK -8
lag m
FIGURE 1 . Effect of CCK on GH release from GH3 cells . of 6 wells ± SEM . pc0.01
Each bar represents mean
pc 0.01
FIGURE 2 . Each bar represents Effect of somatostatin and CCK-8 on GH3 cells . mean of 4 wells (±SEM) . Cells were incubated using the experimental procedure as was described for Figure 1 .
Vol. 25, No . 14, 1979
Cholecystokinin Releases Growth Hormone
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Acknowledgements This study was supported in part by the VA Medical Research Service and USPHS Research Grand HD-7181 . C .B . Lamers was a recipient of a grant from the Netherlands Organisation for the Advancement of Pure Research (Z .W .0) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 .
A .G .E . Pears e and J .M . Polak, Histochem 41 :373-375 (1975) . A . Arimura, H . Sato, A . DuPont, N . NishnTand A .V . Schally, Science 189 : 1007-1009 (1975) . J .M . Polak, S .R . Bloom, S .N . Sullivan, P . Facer and A .G .E . Pearse, Lancet 1 :972-974 (1977) . S .I . Said and R .N . Rosenberg, Science 192 :907-908 (1976) . J .E . Morley, T .J . Garvin, A .E . Pekary and J .M . Hershman, Biochem Biophys Res Comm 79 :314-318 (1977) . G .L . Dockray, Nature 264 :568-570 (1976) . R . Carraway and S .E . eeman, J Biol Chem 251 :7045-7052 (1976) . M . Brown, R . Allen, J . Villarreal, J . RivTer and W . Vale, Life Sciences 23 :2721-2724 (1978) . J .E . Morley, J .H . Steinbach, E .J . Feldman and T .E . Solomon, Life Sciences 24 :1059-1066 (1979) . É. Vijayan, W .K . Samson and S .M . McCann, Life Sciences 23 :2225-2232, (1978) . A . Gomez-Pan, J .D . Reed, M . Albinus, B . Shaw, R . Hall, G .M . Besser, D .H . Coy, A .J . Kasten and A .V . Schally, Lancet 1 :888-891 (1975) . C . Rivier, J . Rivier and W . Vale, Endocrinology102 :519-522 (1978) . E . Vijayan, W .K . Samson, S .E . Said and S .M . McCann, Endocrinology 104 : 53-56 (1979) . H .E . Carlson, I .K . Mariz and W .H . Daughaday, Endocrinology 94 :1709-1713 (1974) . S . Melmed, G . Kurtzman, A . Reed and J .M . Hershman, Life Sciences _24 : 1947-1952 (1979) . E . Strauss and R .S . Yalow, Proc Natl Acad Sci USA 75 :186-189 (1978) . J .E . Morley, J .E . Briggs, T .E . Solomon, S . Melmed, C . Lamers, D .A . Damassa, H .E . Carlson and J .M . Hershman, Clin Res 27 :451A (1979) . J .F . Rehfeld, Nature 264 :568-569 (1976) . R .B . Innis, F .M .A . Correa, G .R . Uhl, B . Schneider and S .H . Snyder, Proc Natl Acad Sci USA 76 :521-525 (1979) . G .P . Smith, J . GibiT ss and R .C . Young, Fed Proc 33 :1146-1151 (1974) . E . Strauss and R . Yalow, Science 203 :68-69 (1975) . L .A . Frohman, L .L . Bernardis and .Kant, K Science 162 :580-582 (1968) . A .W . Hetherington and S .W . Ranson, Proc Soc Exp BioTMed 41 :465-472 L939) . J .B . Martin, P . Brazeau, G .S . Tannenbaum, J .O . Willoughby, J . Epelbaum, L .C . Terry and D . Durand, p . 329, in The Hypothalamus, S . Reichlin, R .J . Baldessarini and J .B . Martin (eds .), aven Press, New York (1978) . G . Gillies and P . Lowry, Nature 278 :463-464 (1979) .
Pergamon Press
CHOLECYSTOKININ OCTAPEPTIDE RELEASES GROWTH HORMONE FROM THE PITUITARY IN VITRO John E . Morley', Shlomo Melmed', Jacqueline Briggs', Harold E . Carlson', Jerome M . Hershman', Travis E . Solomon 2, Cornelis Lamers 2 and David A . Damassa 2 'Endocrine Research Laboratory, Wadsworth VA Medical Center and Department of Medicine, University of California Los Angeles, CA 90073 2 Center for Ulcer Research and Education, University of California, Los Angeles, CA 90024 9 Brain Research Institute, University of California, Los Angeles, CA 90024 (Received in final form August 20, 1979) Summary Cholecyso kinin-octapeptide (CCK-8)(10-6 to 10 -8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH3 pituitary tumor cells . Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion in vitro . In the GH3 cell'line, CCK-8 (10 - 7M) reversed theT i fbitory effect of somatostatin (10-5M) on GH release . As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor .
n
Several peptides including somatostatin (1), thyrotropin-releasing hormone (2), substance P (3), bombesin (4), neurotensin (5), the enkephalins (6), vasoactive intestinal peptide (7) and cholecystokinin (8) have been found in the brain and the gastrointestinal tract . A number of these hormones influence both gastrointestinal function and pituitary hormone release (9-13) . For this reason we assessed the effects of five peptides originally isolated from the gastrointestinal tract on the secretion of hormones from incubated rat anterior pituitary quarters . We report here that cholecystokinin-octapeptide (CCK-8) produced a marked increase in growth hormone release in this system . Several analogues of CCK-8 also produced GH release . CCK-8 prevented the inhibitory effect of somatostatin on GH release from cultured GH3 rat pituitary tumor cells . Methods Acute anterior pituitary cultures : Anterior pituitaries were removed from male Sprague-Dawley rats weighing approximately 150 g . The pituitaries were quartered and the quarters randomized . Three quarters were placed in 10 ml stoppered Erlenmeyer flasks containing 1 ml modified Gey and Gey medium (14) which had been equilibrated to the culture conditions . The tissue portions were individually preincubated for 2 hours in a gyrotory water-bath shaker at 370C under 5% C02-95% 02 . The preincubation medium was then
0024-3205/79/141201-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Cholecystokinin Releases Growth Hormone
Vol . 25, No . 14, 1979
replaced by 1 ml gassed medium containing either no additions (control) or one of the gastrointestinal hormones at varying concentrations . After 1 hour further incubation at 37oC the media were collected and stored frozen until assayed for growth hormone (GH), prolactin (PRL), thyrotropin (TSH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) . At the end of each experiment the pituitary tissue from each flask was weighed ; all results were calculated per mg wet weight of tissue and results are expressed as % of the mean control value . All the anterior pituitary hormones were measured by radioimmunoassay using materials and reference preparations supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethseda, Maryland . There was no cross-reaction of any of the gastrointestinal peptides used in any of the radioimmunoassays . Comparisons were made by using the Student's t-test . GH3 pituitary tumor cell cultures : Cells were supplied by ATCC and were cultured as monolayers in plastic multiwell 35 mm tissue culture plates (LUX), using Ham's F10 medium supplemented with 15% horse serum and 2 .5% fetal calf serum and containing penicillin (50 U/ml) and streptomycin (50 ug/ml) . Cells were maintained as described (15) . Experimental procedure : When cells were in late logarithmic phase of growth, medium was aspirated from each well and cells washed with serum-free Ham's F10 medium . Wells were then incubated with serum-free Ham's F10 medium containing appropriate dilutions of CCK-8 . Control wells contained medium alone . Incubations were in a humidified atmosphere, 95% air - 5% C02 at 37oC . At the end of each incubation, medium was aspirated for hormone assay . Monolayer of cells was harvested in saline suspension and counted in a Coulter counter . Medium and CCK solutions in Ham's F10 were equilibrated with 95% air - 5% C02 at 37oC before use . Results The results of the experiments using cultured pituitary quarters are given in Table I . In addition to the stimulatory effect of CCK-8 on GH release, tetragastrin (10-6M) also produced GH release . None of the other hor mones tested altered secretion of growth hormone . Bombesin, secretin, CCK-8 and pancreatic polypeptide all increased gonadotropin release at high concentrations . Tetragastrin and CCK-8 partially inhibited thyrotropin . Cholecystokinin-39 was also a potent releaser of GH (10-6 M, 253±60%, SEM and 10-7M, 160±20% of controls, p<0 .025) . High molecular weight forms are converted to smaller molecular weight forms in vitro (C . Lamers and J .H . Walsh : Personal Communication) and this may explain the effect of CCK-39 . Caerulein, a decapeptide from frog skin, with only 3 amino acids different from CCK-8, failed to release GH in 2 experiments . Gastrin-17 (10-6M) also enhanced growth hormone secretion (161±18% of controls, p<0 .05) . CCK-8 also produced GH release from monolayer cultures of GH3 pituitary tumor cells (Fig . 1) . In addition, the inhibitory effect of somatostatin (10 - 5M) was reversed by the addition of CCK-8 (10 - 7M) in the GH3 tumor system in vitro (Fig . 2) .
Cholecystokinin Releases Growth Hormone
Vol . 25, No . 14, 1979
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TABLE 1 The effect of CCK-8, tetragastrin, bombesin, secretin and pancreatic polypeptide on anterior pituitary hormone release . SUBSTANCE CCK-8 Tetragastrin Bombes1n Secretin Pancreatic Polypeptide
CONCENTRATION 1
N -
10 -6 10- 7 10- 6 10- 7 10- 7 10 -8 10-9 10- 6 10 - 7 10-8 10- 6
5 5 5 5 3 6 5 5 5 5 5
GH 205±19* 158±241 175±23 i 109±25 113±10 107±14 88±8 115±13 94±5 97±16 116±19
PRL TSH % of control) 82±9 120±14 96±14 76±14 119±21 104±18 68±14 120±15 92±10 110±7 107±17
79±5 i 42±lü:i 78±10 49±16 1 98±15 133±22 89±23 119±20 102±22 97±12 105t41
LH 174±14* 118±22 88±16 114±28 180±7* 118±33 78±14 262±48 i 118±15 161±30 157±14 t
FSH 134±2 143±2 88±13 147±19 112±11 119±8 160±14*
*p<0 .01, ip<0 .05, Mean±SEM Discussion Cholecystokinin immunoreactivity has been demonstrated to be present in the hypothalamus, with the predominant form co-chromatographing with CCK-8 on Sephadex G-50 (16,17) . Additionally, a substance with immunologic and chromatographic characteristics similar to gastrin has been demonstrated in the pituitary (18) . Using immunohistochemical techniques, Innis et al (19) found that the most dense collections of CCK-8 were in the medial hypothalamus and the periaqueductal gray . The reason for the high concentration of cholecystokinin in the brain is unknown . CCK-8 may be related to satiety (20,21) . Electrical stimulation of the ventromedial and arcuate nuclei in the pentobarbital anesthetized rat elicits GH secretion (22), and as satiety has been shown to be related to the ventromedial nucleus (23), a common peptide subserving these two functions would be an attractive hypothesis . Despite considerable physiologic and biochemical evidence for the existence of a growth hormone releasing factor, attempts to identify this substance have thus far been unsuccessful . A large number of peptides have been shown to release GH in vivo but only thyrotropin-releasing hormone and arginine vasopressin haveneen-Zemonstrated to have a direct effect on the pituitary in vitro (14,24) The recent demonstration that corticotropin releasing cttor activity in crude stalk median eminence is due to a multifactor system (25) suggests that a similar multi-factor hypothesis may explain the difficulty experienced in the search for a unique peptide with potent growth hormone releasing factor activity . The evidence presented here suggests that CCK-8 may play a role as an endogenous growth hormone releasing factor . The ability of CCK-8 (10-7M) to neutralize the inhibitory effect of somatostatin (10 - 5M) further indicates that CCK-8 may have a physiologic role in modulating GH secretion .
1204
Vol . 25, No . 14, 1979
Cholecystokinin Releases Growth Hormone
1 .50- p cO .01
c0 .05
NS
140. 130J GH CONTROL 120110100
10"M
W
M CCK -8
lag m
FIGURE 1 . Effect of CCK on GH release from GH3 cells . of 6 wells ± SEM . pc0.01
Each bar represents mean
pc 0.01
FIGURE 2 . Each bar represents Effect of somatostatin and CCK-8 on GH3 cells . mean of 4 wells (±SEM) . Cells were incubated using the experimental procedure as was described for Figure 1 .
Vol. 25, No . 14, 1979
Cholecystokinin Releases Growth Hormone
1205
Acknowledgements This study was supported in part by the VA Medical Research Service and USPHS Research Grand HD-7181 . C .B . Lamers was a recipient of a grant from the Netherlands Organisation for the Advancement of Pure Research (Z .W .0) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 .
A .G .E . Pears e and J .M . Polak, Histochem 41 :373-375 (1975) . A . Arimura, H . Sato, A . DuPont, N . NishnTand A .V . Schally, Science 189 : 1007-1009 (1975) . J .M . Polak, S .R . Bloom, S .N . Sullivan, P . Facer and A .G .E . Pearse, Lancet 1 :972-974 (1977) . S .I . Said and R .N . Rosenberg, Science 192 :907-908 (1976) . J .E . Morley, T .J . Garvin, A .E . Pekary and J .M . Hershman, Biochem Biophys Res Comm 79 :314-318 (1977) . G .L . Dockray, Nature 264 :568-570 (1976) . R . Carraway and S .E . eeman, J Biol Chem 251 :7045-7052 (1976) . M . Brown, R . Allen, J . Villarreal, J . RivTer and W . Vale, Life Sciences 23 :2721-2724 (1978) . J .E . Morley, J .H . Steinbach, E .J . Feldman and T .E . Solomon, Life Sciences 24 :1059-1066 (1979) . É. Vijayan, W .K . Samson and S .M . McCann, Life Sciences 23 :2225-2232, (1978) . A . Gomez-Pan, J .D . Reed, M . Albinus, B . Shaw, R . Hall, G .M . Besser, D .H . Coy, A .J . Kasten and A .V . Schally, Lancet 1 :888-891 (1975) . C . Rivier, J . Rivier and W . Vale, Endocrinology102 :519-522 (1978) . E . Vijayan, W .K . Samson, S .E . Said and S .M . McCann, Endocrinology 104 : 53-56 (1979) . H .E . Carlson, I .K . Mariz and W .H . Daughaday, Endocrinology 94 :1709-1713 (1974) . S . Melmed, G . Kurtzman, A . Reed and J .M . Hershman, Life Sciences _24 : 1947-1952 (1979) . E . Strauss and R .S . Yalow, Proc Natl Acad Sci USA 75 :186-189 (1978) . J .E . Morley, J .E . Briggs, T .E . Solomon, S . Melmed, C . Lamers, D .A . Damassa, H .E . Carlson and J .M . Hershman, Clin Res 27 :451A (1979) . J .F . Rehfeld, Nature 264 :568-569 (1976) . R .B . Innis, F .M .A . Correa, G .R . Uhl, B . Schneider and S .H . Snyder, Proc Natl Acad Sci USA 76 :521-525 (1979) . G .P . Smith, J . GibiT ss and R .C . Young, Fed Proc 33 :1146-1151 (1974) . E . Strauss and R . Yalow, Science 203 :68-69 (1975) . L .A . Frohman, L .L . Bernardis and .Kant, K Science 162 :580-582 (1968) . A .W . Hetherington and S .W . Ranson, Proc Soc Exp BioTMed 41 :465-472 L939) . J .B . Martin, P . Brazeau, G .S . Tannenbaum, J .O . Willoughby, J . Epelbaum, L .C . Terry and D . Durand, p . 329, in The Hypothalamus, S . Reichlin, R .J . Baldessarini and J .B . Martin (eds .), aven Press, New York (1978) . G . Gillies and P . Lowry, Nature 278 :463-464 (1979) .