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Comparison of the temporal profiles of vasopressin and oxytocin in the cerebrospinal fluid of the cat, monkey and rat
Brain Research, 261 (1983) 341-345
341
Elsevier Biomedical Press
Comparison of the temporal profiles of vasopressin and oxytocin in the cerebrospinal fluid of the cat, monkey and rat STEVEN M. REPPERT, WILLIAM J. SCHWARTZ, HENRY G. ARTMAN and DELBERT A. FISHER
Children's and Neurology Services, Massachusetts GeneralHospital and Harvard Medical School, Boston, MA 02114and (H. G.A. and D.A.F.) Department of Pediatrics, UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance, CA 90509(U.S.A.) (Accepted November 2nd, 1982)
Key words:vasopressin - oxytocin - cerebrospinal fluid - circadian rhythms - suprachiasmatic nuclei
The temporal profiles of oxytocin were examined in the cerebrospinal fluid (CSF) of the cat and rat. Unlike the marked daily
rhythm of oxytocin concentrations recently described in the CSF of the rhesus monkey, no daily rhythm of the peptide was evident in the CSF of either the cat or rat. The apparent species specificity of the CSF oxytoein profiles among these mammals is contrasted with the consistent expression of a daily rhythm of a rginine-vasopressinin the CSF of each of the three species.
Although structurally related, the posterior pituitary nonapeptides arginine-vasopressin and oxytocin have quite distinct functions following their release into blood; a primary effect of blood-borne vasopressin is its action on the kidney to conserve body water, whereas circulating oxytocin acts to induce both uterine contractility and milk ejection in the female. Recent evidence suggests that both peptides have important central nervous system (CNS) effects which are separate from their better known peripheral actions4-~6. Our investigations into the dynamic temporal profiles of the nonapeptides in the cerebrospinal fluid (CSF) of several mammalian species support the concept of separate peripheral and CNS functions. In the cat 8'9, m o n k e y 7 and rat ~2, vasopressin concentrations consistently exhibit a daily rhythm in the CSF; no such rhythm is expressed in the blood of either the cat or rat. The daily patterns of oxytocin levels have so far been characterized in only one mammal, the rhesus monkey, in which, like vasopressin, the peptide exhibits a clear daily rhythm in CSF but not in blood '.7. In the present report, we extend these CSF studies by describing the daily profiles of oxytocin in the CSF of the cat and rat. These results, combined with 0006-8993/ 83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
previous observations, are used to provide a comparison of vasopressin and oxytocin profiles in the CSF of the cat, monkey and rat. Adult male domesticated cats (b.w. = 3.5-5.5 kg) and Long-Evans rats (b.w. = 280-320 g) were housed singly and had free access to food and water; the time of day of their routine care was randomized. All animals were kept under a diurnal lighting schedule consisting of 12 h light per day. Illumination intensity at the mid-cage level was 400-600 lux and 600-700 lux for the cat and rat studies, respectively; light was provided by cool white fluorescent tubes. Our methods for continuous withdrawal of CSF from cats and serial removal of CSF from rats have been described in detail elsewhere9.~2. In each case, the animals were tethered but were free to move about their cages. Oxytocin concentrations were measured from 100 #1 portions of unextracted CSF samples by a previously described double antibody radioimmunoassay (RIA) systeml7; this is the same system that was used to measure the daily CSF oxytocin rhythm reported for the rhesus monkey~.7. Oxytocin concentrations are expressed in microunits based on USP posterior pituitary extract reference standard. The lower limit of assay sen-
342 sitivity was 0 . 2 / ~ U / t u b e (2.0/~U/ml CSF). All samples were analyzed in the same assay run: the intraassay coefficient of variation was 7%. The daily CSF profiles of oxytocin were examined in 5 cats and 5 rats, throughout 1 3 day periods of study in diurnal lighting. Visual inspection of the individual daily profiles of CSF oxytocin in the cat reveal no obvious daily rhythm of the peptide (Fig. 1). Rather, oxytocin levels appear to fluctuate randomly over a range of 3-15 # U / m l o f CSF. Similarly, there is no
32
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consistent 24-h pattern to the individual CSF oxytocin profiles in the rat (Fig. 2). As in the cat, the levels of this peptide in rodent CSF vary irregularly over a wide range (3-26 /~U/ml) throughout the day. W h a t will follow is a comparison of the temporal profiles of vasopressin and oxytocin in the CSF of the cat, m o n k e y and rat. We believe this is a valid analysis since the experimental conditions used in these studies were quite similar:
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in CSF vasopressin varies among species and is greatest in the cat, followed in order by the rat and monkey. An interesting feature of these rhythms is that, despite the diverse array of behavioral patterns exhibited by these 3 species (the monkey is day-active; the rat is night-active; the cat shows no clear daily activity pattern), daily increases in CSF vasopressin occur at approximately the same time of day in each. This preservation of phase among species, together with the consistent expression of the CSF vasopressin rhythm in each species examined, suggests that the peptide rhythm may reflect a basic biological process in the mammalian brain. Because the suprachiasmatic nuclei (SCN), the site of a putative circadian pacemaker in the mammalian hypothalamus ~, appear to synthesize va-
Fig. 3. Temporal profiles ofvasopressin and oxytocin in the CSF of rhesus monkeys and cats studied over a 24-h period in diurnal lighting. CSF was collected as 2-h fractions. Each value is the mean _ S.E. of 5 animals. For the vasopressin values in the monkey, a one-way analysis of variance showed a significant effect between intervals 18.00 to 02.00 h and 06.00 to 12.00 h (p<0.05). The mean values for vasopressin and oxytocin in the monkey and vasopressin in the cat were derived from individual 24-h profiles of the peptides previously reported 7.8. The mean oxytocin values in the cat are derived from the data in Fig. I.
only adult male animals were used; all animals were housed under diurnal lighting consisting of 12 h light per day, with light intensity ranging from 400 to 700 lux among the different studies; for each species, cisternal CSF was removed either continuously (cat and monkey) or serially (rat) from unanesthetized animals; the CSF oxytocin levels in all species were measured with the same RIA system; and the two different RIA systems used to measure CSF vaspressin both yielded virtually identical results*. As previously reported, a clear daily oscillation of vasopressin concentration is manifested in the CSF of the cat, monkey and rat. In all cases, the daily rhythm is characterized by high daytime levels and low nighttime values. During the nighttime hours of diurnal lighting, baseline CSF vasopressin levels are similar among the 3 species. The magnitude of the daytime increase
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Clock Time (hrs.) Fig. 4. Population profiles of vasopressin and oxvtocin in the CSF of Long-Evans rats studied in diurnal lighting. The vaso.pressin and oxytocin values are shown as individual data points from the daily profiles of 12 and 7 rats, respectively.
* Antibody R357 was used to measure CSF vasopressin in the cat and monkey 7.8. Skowsky antibody R71 was used to measure vasopressin in the CSF of the cat and rat 9.12.
344 sopressin6,13-j5, the rhythmic expression of the peptide in CSF has led us to conjecture that the rhythm reflects an output signal from this circadian clock. Unlike those ofvasopressin, the daily patterns ofCSF oxytocin appear to vary according to the species examined. In male rhesus monkeys, CSF oxytocin levels consistently manifest a pronounced daily rhythm which is synchronous with the CSF vasopressin rhythm (Fig. 3). In contrast, no obvious daily oxytocin rhythm is apparent in the CSF of either the cat or rat (Fig. 3 and 4). Even though there are marked differences in the CSF oxytocin patterns among these 3 species, the daily range of concentrations of this peptide in the CSF are similar in each. Also, within the CSF of each species, oxytocin levels are generally higher than those ofvasopressin. The dissociation between the vasopressin and oxytocin patterns in CSF in some species suggests that the release of each into the fluid is regulated differentially. Such a dissociation supports the notion that, as in the periphery, vasopressin and oxytocin have distinct functions in
the brain 2.3. The finding that oxytocin exhibits a daily rhythm in the cisternal fluid of the monkey but not in the cat or rat may reflect fundamental physiological differences of peptide function between these species. Interestingly, recent evidence suggests that the primate brain possesses a second circadian pacemaker in addition to that present in the SCN 5.~°. Since oxytocin is absent from the mammalian S C N 13, and since preliminary evidence suggests that SCN ablation has little effect on the CSF oxytocin rhythm in the monkey (Reppert, unpublished observations), it is conceivable that the rhythmic expression of oxytocin in primate CSF reflects an output signal from this second circadian pacemaker.
1 Artman, H. G., Reppert, S. M., Perlow, M. J., Swaminathan, S., Oddie, T. H. and Fisher, D. A., Further characterization of the daily oxytocin rhythm in primate cerebrospinal fluid, J: Neurosci., 2 (1982) 598--603. 2 Bohus, B., Kovacs, G. L. and de Wied, D., Oxytocin, vasopressin and memory: opposite effects on consolidation and retrieval processes, Brain Research, 157 (1978) 414417. 3 Bohus, B., Urban, I., van Wimersma Greidanus, Tj. B. and De Wied, D. Opposite effects of oxytocin and vasopresssin on avoidance behaviour and hippocampal theta rhythm in the rat, Neuropharmacolog)', 17 (1978) 239247. 4 De Wied, D., Peptides and behaviour, Life Sci., 20(1977) 195 204. 5 Fuller, C. A., Lydic, R., Sulzman, F. M., Albers, H. E., Tepper, B. and Moore-Ede, M., Circadian rhythm of body temperature persists after suprachiasmatic lesions in the squirrel monkey, Amer. J. Physiol., 241 (1981) R385-R391. 6 Hawthorn, J., Ang, V. T. Y. and Jenkins, J. S., Localization of vasopressin in the rat brain, Brain Research, 197 (1980) 75-8t. 7 Perlow, M. J., Reppert, S. M., Artman, H. A., Fisher. D. A., Seif, S. M. and Robinson, A. G., Oxytocin, vasopressin and estrogen-stimulated neurophysin: daily patterns of concentrations in cerebrospinal fluid, Science, 216 (1982) 1416-1418. 8 Reppert, S. M., Artman, H. G., Swaminathan, S. and Fisher, D. A., Vaopressin exhibits a rhythmic daily pat-
tern in cerebrospinal fluid but not in blood. Science, 213 (1981) 1256 1257. 9 Reppert, S. M., Coleman, R. J., Heath, H. W. and Keutmann, H. T., Circadian properties of vasopressin and melatonin rhythms in cat cerebrospinal fluid, Amer. J. PhysioL, in press. 10 Reppert, S. M., Perlow, M. J., Ungerleider, L. G., Mishkin, M., Tamarkin, L., Orloff, D. G., Hoffman, H. J. and Klein, D. C., Effects of damage to the suprachiasmatic area of the anterior hypothalamus on the daily melatonin and cortisol rhythms in the rhesus monkey, J. Neurosci., 1 (1981) 1414- 1425. I 1 Rusak, B. and Zucker, 1., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 12 Schwartz, W. J., Coleman, R. J. and Reppert, S. M., A daily vasopressin rhythm in rat cerebrospinal fluid, Brain Research, in press. 13 Sofroniew, M. V. and Weindl, A., Identification of parvocellular vasopressin and neurophysin neurons in the suprachiasmatic nucleus of a variety of mammals including primates, J. comp. Neurol., 193 (1980) 659-675. 14 van Leeuwen, F. W., Swaab. D. F. and de Raay, C., lmmunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus, Cell Tiss. Res., 193 (1978) 1-10. 15 Vandesande, F., Dierickx, K. and DeMey J., Identification of the vasopressin-neurophysin producing neurons of the rat suprachiasmatic nuclei, Cell Tiss. Res., 156 (1975) 377--380. 16 van Ree, J. M., Bohus, B., Versteeg, H. G. and de Wied,
S.M.R. is a Research Fellow of the Charles A. King Trust. W.J.S. is supported by N1NCDS Teacher-Investigator Development Award 1 K07 NS 00672-01. These studies were supported in part by PHS Grant HD 14427, the William Milton Fund of Harvard University, and March of Dimes Basil O'Connor Starter Research Grant 5 335.
345 D., Neurohypophyseal principles and memory processes, Biochem. Pharmacol. 27 (1978) 1793-1800. 17 Weitzman, R. E., Glatz, T. H. and Fisher, D. A., The ef-
fect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs, Endocrinology, !03 (1978) 2 ! 54-2160.
341
Elsevier Biomedical Press
Comparison of the temporal profiles of vasopressin and oxytocin in the cerebrospinal fluid of the cat, monkey and rat STEVEN M. REPPERT, WILLIAM J. SCHWARTZ, HENRY G. ARTMAN and DELBERT A. FISHER
Children's and Neurology Services, Massachusetts GeneralHospital and Harvard Medical School, Boston, MA 02114and (H. G.A. and D.A.F.) Department of Pediatrics, UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance, CA 90509(U.S.A.) (Accepted November 2nd, 1982)
Key words:vasopressin - oxytocin - cerebrospinal fluid - circadian rhythms - suprachiasmatic nuclei
The temporal profiles of oxytocin were examined in the cerebrospinal fluid (CSF) of the cat and rat. Unlike the marked daily
rhythm of oxytocin concentrations recently described in the CSF of the rhesus monkey, no daily rhythm of the peptide was evident in the CSF of either the cat or rat. The apparent species specificity of the CSF oxytoein profiles among these mammals is contrasted with the consistent expression of a daily rhythm of a rginine-vasopressinin the CSF of each of the three species.
Although structurally related, the posterior pituitary nonapeptides arginine-vasopressin and oxytocin have quite distinct functions following their release into blood; a primary effect of blood-borne vasopressin is its action on the kidney to conserve body water, whereas circulating oxytocin acts to induce both uterine contractility and milk ejection in the female. Recent evidence suggests that both peptides have important central nervous system (CNS) effects which are separate from their better known peripheral actions4-~6. Our investigations into the dynamic temporal profiles of the nonapeptides in the cerebrospinal fluid (CSF) of several mammalian species support the concept of separate peripheral and CNS functions. In the cat 8'9, m o n k e y 7 and rat ~2, vasopressin concentrations consistently exhibit a daily rhythm in the CSF; no such rhythm is expressed in the blood of either the cat or rat. The daily patterns of oxytocin levels have so far been characterized in only one mammal, the rhesus monkey, in which, like vasopressin, the peptide exhibits a clear daily rhythm in CSF but not in blood '.7. In the present report, we extend these CSF studies by describing the daily profiles of oxytocin in the CSF of the cat and rat. These results, combined with 0006-8993/ 83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
previous observations, are used to provide a comparison of vasopressin and oxytocin profiles in the CSF of the cat, monkey and rat. Adult male domesticated cats (b.w. = 3.5-5.5 kg) and Long-Evans rats (b.w. = 280-320 g) were housed singly and had free access to food and water; the time of day of their routine care was randomized. All animals were kept under a diurnal lighting schedule consisting of 12 h light per day. Illumination intensity at the mid-cage level was 400-600 lux and 600-700 lux for the cat and rat studies, respectively; light was provided by cool white fluorescent tubes. Our methods for continuous withdrawal of CSF from cats and serial removal of CSF from rats have been described in detail elsewhere9.~2. In each case, the animals were tethered but were free to move about their cages. Oxytocin concentrations were measured from 100 #1 portions of unextracted CSF samples by a previously described double antibody radioimmunoassay (RIA) systeml7; this is the same system that was used to measure the daily CSF oxytocin rhythm reported for the rhesus monkey~.7. Oxytocin concentrations are expressed in microunits based on USP posterior pituitary extract reference standard. The lower limit of assay sen-
342 sitivity was 0 . 2 / ~ U / t u b e (2.0/~U/ml CSF). All samples were analyzed in the same assay run: the intraassay coefficient of variation was 7%. The daily CSF profiles of oxytocin were examined in 5 cats and 5 rats, throughout 1 3 day periods of study in diurnal lighting. Visual inspection of the individual daily profiles of CSF oxytocin in the cat reveal no obvious daily rhythm of the peptide (Fig. 1). Rather, oxytocin levels appear to fluctuate randomly over a range of 3-15 # U / m l o f CSF. Similarly, there is no
32
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consistent 24-h pattern to the individual CSF oxytocin profiles in the rat (Fig. 2). As in the cat, the levels of this peptide in rodent CSF vary irregularly over a wide range (3-26 /~U/ml) throughout the day. W h a t will follow is a comparison of the temporal profiles of vasopressin and oxytocin in the CSF of the cat, m o n k e y and rat. We believe this is a valid analysis since the experimental conditions used in these studies were quite similar:
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in CSF vasopressin varies among species and is greatest in the cat, followed in order by the rat and monkey. An interesting feature of these rhythms is that, despite the diverse array of behavioral patterns exhibited by these 3 species (the monkey is day-active; the rat is night-active; the cat shows no clear daily activity pattern), daily increases in CSF vasopressin occur at approximately the same time of day in each. This preservation of phase among species, together with the consistent expression of the CSF vasopressin rhythm in each species examined, suggests that the peptide rhythm may reflect a basic biological process in the mammalian brain. Because the suprachiasmatic nuclei (SCN), the site of a putative circadian pacemaker in the mammalian hypothalamus ~, appear to synthesize va-
Fig. 3. Temporal profiles ofvasopressin and oxytocin in the CSF of rhesus monkeys and cats studied over a 24-h period in diurnal lighting. CSF was collected as 2-h fractions. Each value is the mean _ S.E. of 5 animals. For the vasopressin values in the monkey, a one-way analysis of variance showed a significant effect between intervals 18.00 to 02.00 h and 06.00 to 12.00 h (p<0.05). The mean values for vasopressin and oxytocin in the monkey and vasopressin in the cat were derived from individual 24-h profiles of the peptides previously reported 7.8. The mean oxytocin values in the cat are derived from the data in Fig. I.
only adult male animals were used; all animals were housed under diurnal lighting consisting of 12 h light per day, with light intensity ranging from 400 to 700 lux among the different studies; for each species, cisternal CSF was removed either continuously (cat and monkey) or serially (rat) from unanesthetized animals; the CSF oxytocin levels in all species were measured with the same RIA system; and the two different RIA systems used to measure CSF vaspressin both yielded virtually identical results*. As previously reported, a clear daily oscillation of vasopressin concentration is manifested in the CSF of the cat, monkey and rat. In all cases, the daily rhythm is characterized by high daytime levels and low nighttime values. During the nighttime hours of diurnal lighting, baseline CSF vasopressin levels are similar among the 3 species. The magnitude of the daytime increase
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Clock Time (hrs.) Fig. 4. Population profiles of vasopressin and oxvtocin in the CSF of Long-Evans rats studied in diurnal lighting. The vaso.pressin and oxytocin values are shown as individual data points from the daily profiles of 12 and 7 rats, respectively.
* Antibody R357 was used to measure CSF vasopressin in the cat and monkey 7.8. Skowsky antibody R71 was used to measure vasopressin in the CSF of the cat and rat 9.12.
344 sopressin6,13-j5, the rhythmic expression of the peptide in CSF has led us to conjecture that the rhythm reflects an output signal from this circadian clock. Unlike those ofvasopressin, the daily patterns ofCSF oxytocin appear to vary according to the species examined. In male rhesus monkeys, CSF oxytocin levels consistently manifest a pronounced daily rhythm which is synchronous with the CSF vasopressin rhythm (Fig. 3). In contrast, no obvious daily oxytocin rhythm is apparent in the CSF of either the cat or rat (Fig. 3 and 4). Even though there are marked differences in the CSF oxytocin patterns among these 3 species, the daily range of concentrations of this peptide in the CSF are similar in each. Also, within the CSF of each species, oxytocin levels are generally higher than those ofvasopressin. The dissociation between the vasopressin and oxytocin patterns in CSF in some species suggests that the release of each into the fluid is regulated differentially. Such a dissociation supports the notion that, as in the periphery, vasopressin and oxytocin have distinct functions in
the brain 2.3. The finding that oxytocin exhibits a daily rhythm in the cisternal fluid of the monkey but not in the cat or rat may reflect fundamental physiological differences of peptide function between these species. Interestingly, recent evidence suggests that the primate brain possesses a second circadian pacemaker in addition to that present in the SCN 5.~°. Since oxytocin is absent from the mammalian S C N 13, and since preliminary evidence suggests that SCN ablation has little effect on the CSF oxytocin rhythm in the monkey (Reppert, unpublished observations), it is conceivable that the rhythmic expression of oxytocin in primate CSF reflects an output signal from this second circadian pacemaker.
1 Artman, H. G., Reppert, S. M., Perlow, M. J., Swaminathan, S., Oddie, T. H. and Fisher, D. A., Further characterization of the daily oxytocin rhythm in primate cerebrospinal fluid, J: Neurosci., 2 (1982) 598--603. 2 Bohus, B., Kovacs, G. L. and de Wied, D., Oxytocin, vasopressin and memory: opposite effects on consolidation and retrieval processes, Brain Research, 157 (1978) 414417. 3 Bohus, B., Urban, I., van Wimersma Greidanus, Tj. B. and De Wied, D. Opposite effects of oxytocin and vasopresssin on avoidance behaviour and hippocampal theta rhythm in the rat, Neuropharmacolog)', 17 (1978) 239247. 4 De Wied, D., Peptides and behaviour, Life Sci., 20(1977) 195 204. 5 Fuller, C. A., Lydic, R., Sulzman, F. M., Albers, H. E., Tepper, B. and Moore-Ede, M., Circadian rhythm of body temperature persists after suprachiasmatic lesions in the squirrel monkey, Amer. J. Physiol., 241 (1981) R385-R391. 6 Hawthorn, J., Ang, V. T. Y. and Jenkins, J. S., Localization of vasopressin in the rat brain, Brain Research, 197 (1980) 75-8t. 7 Perlow, M. J., Reppert, S. M., Artman, H. A., Fisher. D. A., Seif, S. M. and Robinson, A. G., Oxytocin, vasopressin and estrogen-stimulated neurophysin: daily patterns of concentrations in cerebrospinal fluid, Science, 216 (1982) 1416-1418. 8 Reppert, S. M., Artman, H. G., Swaminathan, S. and Fisher, D. A., Vaopressin exhibits a rhythmic daily pat-
tern in cerebrospinal fluid but not in blood. Science, 213 (1981) 1256 1257. 9 Reppert, S. M., Coleman, R. J., Heath, H. W. and Keutmann, H. T., Circadian properties of vasopressin and melatonin rhythms in cat cerebrospinal fluid, Amer. J. PhysioL, in press. 10 Reppert, S. M., Perlow, M. J., Ungerleider, L. G., Mishkin, M., Tamarkin, L., Orloff, D. G., Hoffman, H. J. and Klein, D. C., Effects of damage to the suprachiasmatic area of the anterior hypothalamus on the daily melatonin and cortisol rhythms in the rhesus monkey, J. Neurosci., 1 (1981) 1414- 1425. I 1 Rusak, B. and Zucker, 1., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 12 Schwartz, W. J., Coleman, R. J. and Reppert, S. M., A daily vasopressin rhythm in rat cerebrospinal fluid, Brain Research, in press. 13 Sofroniew, M. V. and Weindl, A., Identification of parvocellular vasopressin and neurophysin neurons in the suprachiasmatic nucleus of a variety of mammals including primates, J. comp. Neurol., 193 (1980) 659-675. 14 van Leeuwen, F. W., Swaab. D. F. and de Raay, C., lmmunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus, Cell Tiss. Res., 193 (1978) 1-10. 15 Vandesande, F., Dierickx, K. and DeMey J., Identification of the vasopressin-neurophysin producing neurons of the rat suprachiasmatic nuclei, Cell Tiss. Res., 156 (1975) 377--380. 16 van Ree, J. M., Bohus, B., Versteeg, H. G. and de Wied,
S.M.R. is a Research Fellow of the Charles A. King Trust. W.J.S. is supported by N1NCDS Teacher-Investigator Development Award 1 K07 NS 00672-01. These studies were supported in part by PHS Grant HD 14427, the William Milton Fund of Harvard University, and March of Dimes Basil O'Connor Starter Research Grant 5 335.
345 D., Neurohypophyseal principles and memory processes, Biochem. Pharmacol. 27 (1978) 1793-1800. 17 Weitzman, R. E., Glatz, T. H. and Fisher, D. A., The ef-
fect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs, Endocrinology, !03 (1978) 2 ! 54-2160.