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Changes in total serum immunoreactive neurophysins and corticosterone levels after restraint stress in rats
P,vchoneuroendocrinolog ~. Vol. 8, No. 4, pp. 447 450. 1983. Printed in Great Britain.
SHORT CHANGES
COMMUNICATION
IN T O T A L
NEUROPHYSINS
0306 - 4530/83 $3.00 + 0.00 Cc 1983Pergamon Press Ltd.
SERUM IMMUNOREACTIVE
AND CORTICOSTERONE
RESTRAINT
LEVELS AFTER
S T R E S S IN R A T S
ANDR[~ F. CRINE*I", FRANCINE LOUISt, JOSE SULON:[: and
JEAN-JACQUES
LEGROS~
*Research Assistant at the N a t i o n a l Fund for Scientific Research, Belgium, } N e u r o e n d o c r i n o l o g y Section, C H U , 4 / 1 2 University o f Liege, Sart T i l m a n , B-4000 Li6ge, Belgium and :[:Department o f Medical P a t h o l o g y , University o f Liege, 1 rue des Bonnes Villes, B-4000 Li6ge, Belgium
(Received 3 September 1982; in.final.form 10 January 1983) SUMMARY This study indicates that total i m m u n o r e a c t i v e circulating n e u r o p h y s i n s (IRN) increased in male rats s u b m i t t e d to acute restraint stress. Total i m m u n o r e a c t i v e serum c o r t i c o s t e r o n e levels also were elevated, c o n f i r m i n g the stressful c h a r a c t e r o f the restraint situation. It is speculatively suggested that IRN elevations due to stress are the reflection of a c o r t i c o t r o p i n releasing factor ( C R F ) - I R N product.
INTRODUCTION
IN RATS, the partial suppression of movements easily obtained by placing the animal in an appropriate container is largely used as a strong stressful situation to study, among other things, changes in endocrine functions responsive to environmental stress. Several researchers have reported changes in corticosterone, prolactin, LH, FSH, growth hormone and TSH levels. All these hormones show different secretion patterns, which depend on procedural variations such as duration, intensity and acute or repeated exposure to restraint, as well as on their own responsiveness to the stressful stimulation (Krulich et al., 1974; Tache et al., 1978; Collu et al., 1979; Seggie & Brown, 1982). In this respect, strangely enough, the hypothalamoneurohypophyseal axis does not appear to have been investigated yet. This may be due to the apparent absence of changes in central and peripheral levels of radioimmunoassayable vasopressin and oxytocin in stressful situations (Keil & Severs, 1977; Husain et al., 1979). Our study demonstrated, however, that the total serum immunoreactive neurophysins concentrations (1RN), which are regarded as an indirect measure of hypothalamoneurohypophyseal activity (Legros, 1979), are high in rats subjected to various durations of restraint stress. Total serum corticosterone immunoreactivity also has been determined. Gradual increases confirm the stressful character of the procedure used. METHODS
Subjects F i f t y - f o u r male W i s t a r rats (400 - 450 g) were used. They were singly housed in an a n i m a l r o o m . T e m p e r a t u r e (23 - 1 °C), h u m i d i t y (40 - 50 °) and light - d a r k cycle (L: 0700 to 1900 hr) were controlled. F o o d and w a t e r were p r o v i d e d ad lib. except d u r i n g the periods of restraint. 447
448
ANI)RI: F. CRIN[:, FRAN( INt: 1 OtiS, J{)s/ S[:I ON and JI:AN-JA{ QUtS [.t:{;ROS
.qpl~urattt~ The restrainer had a p r i s m a t i c s h a p e ( l l ~ 20cm). h',',as made of transparent plexiglas',~ith air-conducting holes. Removable inserts prevented escape of the animals. The restrainer was located in a quiet, darkened room adjacenl to the animal room. [Jrocedlll'e
Behavioral procedure. The rat v, as carried in its home cage from the animal room to the experimental room, then manually introduced into the restrainer and left alone for the given period of time. Nine rats for each period of restraint were acutely exposed during either 5, 10, 20, 30 or 40 rain. To minimize diurnal variations in hormone levels, each of the nine rats within each group was tested at different times between 0900 and 1200 hr. Blood samplin~ and radioitnmunoasstLvs. As soon as the ral was removed from the restrainer, it was transported into another room and placed for 4 5 - 90 sec in a jar saturated with ether. A quantity of 1.0 1.5 cm' of blood was drav, n by means of a micro hematocrit capillary tube via retroorbital puncture {Pettit, 1913}. The blood collected in the plastic tube '.~,as stored at 4°C and then centrifuged at 20(Xt rev/min for 10 rain. Serum ',,,as frozen at 20°C until corticosterone and IRN determinations Bere performed. Corticosterone v.a,, measured directly on dried dichloromethane extracts by radioimmunoassay. Tire specificity of the anti corticosterone serum has been described previously ISulon et ul., 1978). l h e IRN radioimmunoassay also has been described in detail elsewhere (Legros, 1976). Control ,.alues v, ere obtained from rats left totally undisturbed ira flleir home cage until blood sampling. The maximum time required for ether anaesthesia and drav, ing blood ',,,as less than 2 min. 5lttlisti{~s Statistically significant differences ',sere e,.aluated by a correlation analysis and a one-way analysis of variance {ANOVA). Because of the nonhomogeneity of ',ariances, the data were subjected to a logarithmic transformation before statistical analysis. Subsequent mean comparisons ~ere made by the Dunnett's tesl (tv.o sided).
RESL;I.TS A N O V A revealed that there was a significant effect of restraint both on corticosterone levels 1F(5,48) = 121.9, p < 0.0011 and IRN concentrations [F(5,48) = 2.68, p < 0.05] (Fig. !). The dynamics were, however, different. Corticosterone levels showed a gradual increase which was significantly higher than control values whatever the duration of restraint (2p < 0.01). IRN levels increased rapidly to reach a two-fold rise after 5 min of restraint (2p < 0.05). Additionally, only the elevations observed after 20 and 40 rain of restraint were significantly different from controls (2p < 0.05). Furthermore, there was a significant correlation between both hormone levels, best characterized by a power function (v = 0.325 x °-~-'5, r = 0.478, p < 0.001). DISCUSSION
Total immunoreactive blood neurophysins (IRN) concentrations as assayed by Legros's (1976) technique were elevated in rats submitted to an acute restraint stress. This report confirms previously reported data showing elevated circulating IRN in rats trained in a mild stressful situation based on their 'innate' preference for darkness (Crine et al., 1981, 1982). Ether anaesthesia by itself did not cause an)' increase in IRN levels. IRN concentrations previously determined in unanaesthetized rats are relatively similar to those o f our present controls (Legros & Dreifttss, 1975). Further, basal I R N values appear to increase with age (Crine, in preparation). As vasopressin and oxytocin levels do not seem to be consistently altered after various stressful Greidanus
circumstances
(Keil
&
et al., 1979; D o g t e r o m
Severs,
1977;
Husain
et a l . ,
& B u i j s , 1980; H a l d a r & B a d e ,
1979;
Van
Wimersma
1981), t w o s p e c u l a t i v e
NEUROPHYSINS AND RESTRAINT
4.49
t
7
800
z5
t
CC
t -300
-200
CORI'JCO8TEROflE (B)
*
NEUI~3~WN8 (LR.N.) •
O
DURATION OF FIESTR.MNT lien)
Fl¢~. I. The effect of restraint duration on total serum immunoreactive neurophysins and corticosterone concentrations. Each point represents the mean value from nine rats. Vertical bars represent the standard error of the mean. hypotheses concerning the 1RN elevation can be proposed. First, neurophysins might not only be the protein carriers of neurohypophyseal hormones; they also could play an independent role in the biochemical adaptation of the organism to the inescapability. Second, total immunoreactive neurophysins determined by Legros's technique could be the reflection not only of circulating neurophysins concentrations but also of unknown biologically active substances such as fragments of neurophysins. Furthermore, these two hypotheses can be moderated by a previous report suggesting the existence of a CRF-associated neurophysin (Watkins et al., 1974). Similarly, Krish (1980) demonstrated the existence of a vasopressin-like substance released from the supraoptic perikarya after 5 min of immobilization stress and suggested ~hat this substance could be a CRF cofactor or a factor with CRF-like activity, in addition, restraint stress is capable of changing catecholaminergic metabolism in paraventricular, supraoptic and ventromedial nuclei (Keim & Sigg, 1976; Kvetnansky et al., 1977), which are physiologically involved in ACTH and neurophysin synthesis (e.g. Dornhorst et al., 1981 ; Carlson et al., 1982). Accordingly, it can be speculatively proposed that elevated circulating IRN after stress are the reflection of one or several CRF-IRN substances. The nonlinear correlation
450
ANDRE F. CRINI, ]=RAN(INE LOUIS, JOSE SUI ON and JEAN-JA( QUt!S LtC;ROS
between serum IRN and corticosterone are responsive
to restraint
corticosterone
levels w o u l d
levels, w h i c h c o u l d m e a n that I R N c o n c e n t r a t i o n s
stress especially
immediately
b e still s e n s i t i v e t o f u r t h e r
after
being applied,
application
whereas
of stress, perhaps
reinforces this hypothesis. This research was supported by grant 3.4507.79 from the Belgian F.R.S.M. The authors would like to express appreciation to Marc Sojic for statistical analysis. REFERENCES CARLSON, D. E., DORNHORSt, A., SELF, S. M., ROmNSON, A. G. & GAnn, D. S. (1982) Vasopressin-dependent and -independent control of the release of adrenocorticotropin. Endocrinology 110, 6 8 0 - 682. Col t u, R., DurwlsseAu, P. & TACm:, Y. (1979) Role of putative neurotransmitters in prolactin, GH and LH response to acute immobilization stress in male rats. Neuroendoerinolog_v 28, 178 186. CRIN[-, A. F., PERIKEI, J. J., Louis, F. & LE(.iROS, J. J. (1981) Modulation du systeme hypothalamoneurohypophysaire ~t la suite de modifications environnementales chez le rat. C. r. Soc. Biol. Paris 175, 882-888. CRINE, A. F., LOUIS, F. & LE(iROS, J. J. (1982) Peripheral neurophysins changes under mild stress conditions. Biol. Psvchol. 14, 311 - 315. DOGTErOSl, J. & ButJs, R. M. (1980) Vasopressin and oxytocin distribution in rat brain: radioimmunoassay and immunocytochemical studies. In Neuropeptides and Neural Transmission, Marsan, C. A. & Traczyk, W. Z. (Eds.), pp. 307 314, Raven Press, New York. DORNHORSF, A., CARLSON, D. E., Sl~ll~, S. M., ROBINSON, A. G., ZIMMFRMAN, E. A. & GANN, D. A. (1981) Control of release of adrenocorticotropin and vasopressin by the supraoptic and paraventricular nuclei. Endocrinology 11)8, 1420- 1424. Hat DAR, J. & BADI=, V. (1981) Involvemem of opioid peptides in the inhibition of oxytocin release by heal stress in lactating mice (41227). Proe. Soc. exp. Biol. Med. 168, 10- 14. HUSAIN, M. K., MANGER, W. M., ROCK, T. W., WEISS, R. J. & FRANTZ, A. G. (1979) Vasopressin release due to manual restraint in the rat: role of body compression and comparison with other stressful stimuli. Endocrinology 10, 641 - 644. Kell, t.. & SEVERS, W. B. (1977) Reduction in plasma vasopressin levels of dehydrated rats following acute stress. Endocrinology 100, 30 38. KHM, K. L. & SIGc;, E. B. (1976) Physiological and biochemical concomitants of restraint stress in rats. PharmacoL Biochem. Behav. 4, 289-297. KRISH, B. (1980) Are there indications for a granule-free form of vasopressin? J. Histochem. (~vtochem. 28, 472 - 474. KruI J~H, J., HLFCO, E., It I NLR, P. & READ, C. B. (1974) The effects of acute stress on the secretion of LH, FSH, prolactin and GH in the normal male rat, with comments on their statistical evaluation. Nearoendocrinolog.v 16, 293 311. KVE-TNaNSK',, R., PAt.KOVITS, M., MJtro, A., TORt)A, T. & MIl,:ul AJ, L. (1977) Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats. Neuroendocrinolog.v 23, 257 - 267. Lt-c;ros, J. J. (1976) Les Nearoph.vsines. Reeherches Methodologique.{, Exp&imentales et Cliniques. Masson, Paris. l.t(;ros, J. J. (1979) The radioimmunoassay of neurophysin as a tool for posterior pituitary investigation. Biochem. Pharmac. 27, 1009 1018. I.t:(;ROS, J. J. & DrHFUSS, J. J. (1975) Water deprivation, in rats: elevated plasma neurophysin levels. E.vperientia 31,603 - 605. PETTIT, A. (1913) Proced6 simple pour pr6lever du sang chez les petits rongeurs. ('. r. Soc. Biol. Paris 74, 11. SeC;~;IE, J. & Brov,'n, G. M. (1982) Profiles of hormone stress response: recruitement or pathway specificity. In Brain Peptides and Hormones, Collu, R., Ducharme, J. R., Barbeau, A. & Tollis, G. (Eds.), pp. 277 - 285, Raven Press, New York. SUl (}N, J., DEMt Y-PONSART, L., BEAUI}UIN, P. & SODOYI:Z, J. C. (1978} Radioimmunosassay of corticosterone, cortisol and cortisone: their application to human cord and maternal plasma. J. Steroid Biochem, 9, 671 676. TA(.ttI:, Y., DURtJISSEAU, P., DU(_HARMt, J. R. & COI IU, R. (1978) Pattern of adenohypophyseal hormone changes in male rats following chronic stress. Neuroendocrinology 26, 208 - 219. VAN WIM~-RSMAGRHt)ANUS, T. B., CroJsH G., GOI:DEMANS, H. & DO,.;Tero",~, J. (1979) Vasopressin levels in peripheral blood and in cerebrospinal fluid during passive and active avoidance behavior in rats. Horm. Behav. 12, 103 - 111. W,',,t~dns, W. B., SCHV,AmD,",I, P. & Boca., R. (1974) hnmunohistochemical demonstration of a CRF-associated neurophysin in the external zone of the rat median eminence. Cell. I'i~s,e Res. 152, 411 421.
SHORT CHANGES
COMMUNICATION
IN T O T A L
NEUROPHYSINS
0306 - 4530/83 $3.00 + 0.00 Cc 1983Pergamon Press Ltd.
SERUM IMMUNOREACTIVE
AND CORTICOSTERONE
RESTRAINT
LEVELS AFTER
S T R E S S IN R A T S
ANDR[~ F. CRINE*I", FRANCINE LOUISt, JOSE SULON:[: and
JEAN-JACQUES
LEGROS~
*Research Assistant at the N a t i o n a l Fund for Scientific Research, Belgium, } N e u r o e n d o c r i n o l o g y Section, C H U , 4 / 1 2 University o f Liege, Sart T i l m a n , B-4000 Li6ge, Belgium and :[:Department o f Medical P a t h o l o g y , University o f Liege, 1 rue des Bonnes Villes, B-4000 Li6ge, Belgium
(Received 3 September 1982; in.final.form 10 January 1983) SUMMARY This study indicates that total i m m u n o r e a c t i v e circulating n e u r o p h y s i n s (IRN) increased in male rats s u b m i t t e d to acute restraint stress. Total i m m u n o r e a c t i v e serum c o r t i c o s t e r o n e levels also were elevated, c o n f i r m i n g the stressful c h a r a c t e r o f the restraint situation. It is speculatively suggested that IRN elevations due to stress are the reflection of a c o r t i c o t r o p i n releasing factor ( C R F ) - I R N product.
INTRODUCTION
IN RATS, the partial suppression of movements easily obtained by placing the animal in an appropriate container is largely used as a strong stressful situation to study, among other things, changes in endocrine functions responsive to environmental stress. Several researchers have reported changes in corticosterone, prolactin, LH, FSH, growth hormone and TSH levels. All these hormones show different secretion patterns, which depend on procedural variations such as duration, intensity and acute or repeated exposure to restraint, as well as on their own responsiveness to the stressful stimulation (Krulich et al., 1974; Tache et al., 1978; Collu et al., 1979; Seggie & Brown, 1982). In this respect, strangely enough, the hypothalamoneurohypophyseal axis does not appear to have been investigated yet. This may be due to the apparent absence of changes in central and peripheral levels of radioimmunoassayable vasopressin and oxytocin in stressful situations (Keil & Severs, 1977; Husain et al., 1979). Our study demonstrated, however, that the total serum immunoreactive neurophysins concentrations (1RN), which are regarded as an indirect measure of hypothalamoneurohypophyseal activity (Legros, 1979), are high in rats subjected to various durations of restraint stress. Total serum corticosterone immunoreactivity also has been determined. Gradual increases confirm the stressful character of the procedure used. METHODS
Subjects F i f t y - f o u r male W i s t a r rats (400 - 450 g) were used. They were singly housed in an a n i m a l r o o m . T e m p e r a t u r e (23 - 1 °C), h u m i d i t y (40 - 50 °) and light - d a r k cycle (L: 0700 to 1900 hr) were controlled. F o o d and w a t e r were p r o v i d e d ad lib. except d u r i n g the periods of restraint. 447
448
ANI)RI: F. CRIN[:, FRAN( INt: 1 OtiS, J{)s/ S[:I ON and JI:AN-JA{ QUtS [.t:{;ROS
.qpl~urattt~ The restrainer had a p r i s m a t i c s h a p e ( l l ~ 20cm). h',',as made of transparent plexiglas',~ith air-conducting holes. Removable inserts prevented escape of the animals. The restrainer was located in a quiet, darkened room adjacenl to the animal room. [Jrocedlll'e
Behavioral procedure. The rat v, as carried in its home cage from the animal room to the experimental room, then manually introduced into the restrainer and left alone for the given period of time. Nine rats for each period of restraint were acutely exposed during either 5, 10, 20, 30 or 40 rain. To minimize diurnal variations in hormone levels, each of the nine rats within each group was tested at different times between 0900 and 1200 hr. Blood samplin~ and radioitnmunoasstLvs. As soon as the ral was removed from the restrainer, it was transported into another room and placed for 4 5 - 90 sec in a jar saturated with ether. A quantity of 1.0 1.5 cm' of blood was drav, n by means of a micro hematocrit capillary tube via retroorbital puncture {Pettit, 1913}. The blood collected in the plastic tube '.~,as stored at 4°C and then centrifuged at 20(Xt rev/min for 10 rain. Serum ',,,as frozen at 20°C until corticosterone and IRN determinations Bere performed. Corticosterone v.a,, measured directly on dried dichloromethane extracts by radioimmunoassay. Tire specificity of the anti corticosterone serum has been described previously ISulon et ul., 1978). l h e IRN radioimmunoassay also has been described in detail elsewhere (Legros, 1976). Control ,.alues v, ere obtained from rats left totally undisturbed ira flleir home cage until blood sampling. The maximum time required for ether anaesthesia and drav, ing blood ',,,as less than 2 min. 5lttlisti{~s Statistically significant differences ',sere e,.aluated by a correlation analysis and a one-way analysis of variance {ANOVA). Because of the nonhomogeneity of ',ariances, the data were subjected to a logarithmic transformation before statistical analysis. Subsequent mean comparisons ~ere made by the Dunnett's tesl (tv.o sided).
RESL;I.TS A N O V A revealed that there was a significant effect of restraint both on corticosterone levels 1F(5,48) = 121.9, p < 0.0011 and IRN concentrations [F(5,48) = 2.68, p < 0.05] (Fig. !). The dynamics were, however, different. Corticosterone levels showed a gradual increase which was significantly higher than control values whatever the duration of restraint (2p < 0.01). IRN levels increased rapidly to reach a two-fold rise after 5 min of restraint (2p < 0.05). Additionally, only the elevations observed after 20 and 40 rain of restraint were significantly different from controls (2p < 0.05). Furthermore, there was a significant correlation between both hormone levels, best characterized by a power function (v = 0.325 x °-~-'5, r = 0.478, p < 0.001). DISCUSSION
Total immunoreactive blood neurophysins (IRN) concentrations as assayed by Legros's (1976) technique were elevated in rats submitted to an acute restraint stress. This report confirms previously reported data showing elevated circulating IRN in rats trained in a mild stressful situation based on their 'innate' preference for darkness (Crine et al., 1981, 1982). Ether anaesthesia by itself did not cause an)' increase in IRN levels. IRN concentrations previously determined in unanaesthetized rats are relatively similar to those o f our present controls (Legros & Dreifttss, 1975). Further, basal I R N values appear to increase with age (Crine, in preparation). As vasopressin and oxytocin levels do not seem to be consistently altered after various stressful Greidanus
circumstances
(Keil
&
et al., 1979; D o g t e r o m
Severs,
1977;
Husain
et a l . ,
& B u i j s , 1980; H a l d a r & B a d e ,
1979;
Van
Wimersma
1981), t w o s p e c u l a t i v e
NEUROPHYSINS AND RESTRAINT
4.49
t
7
800
z5
t
CC
t -300
-200
CORI'JCO8TEROflE (B)
*
NEUI~3~WN8 (LR.N.) •
O
DURATION OF FIESTR.MNT lien)
Fl¢~. I. The effect of restraint duration on total serum immunoreactive neurophysins and corticosterone concentrations. Each point represents the mean value from nine rats. Vertical bars represent the standard error of the mean. hypotheses concerning the 1RN elevation can be proposed. First, neurophysins might not only be the protein carriers of neurohypophyseal hormones; they also could play an independent role in the biochemical adaptation of the organism to the inescapability. Second, total immunoreactive neurophysins determined by Legros's technique could be the reflection not only of circulating neurophysins concentrations but also of unknown biologically active substances such as fragments of neurophysins. Furthermore, these two hypotheses can be moderated by a previous report suggesting the existence of a CRF-associated neurophysin (Watkins et al., 1974). Similarly, Krish (1980) demonstrated the existence of a vasopressin-like substance released from the supraoptic perikarya after 5 min of immobilization stress and suggested ~hat this substance could be a CRF cofactor or a factor with CRF-like activity, in addition, restraint stress is capable of changing catecholaminergic metabolism in paraventricular, supraoptic and ventromedial nuclei (Keim & Sigg, 1976; Kvetnansky et al., 1977), which are physiologically involved in ACTH and neurophysin synthesis (e.g. Dornhorst et al., 1981 ; Carlson et al., 1982). Accordingly, it can be speculatively proposed that elevated circulating IRN after stress are the reflection of one or several CRF-IRN substances. The nonlinear correlation
450
ANDRE F. CRINI, ]=RAN(INE LOUIS, JOSE SUI ON and JEAN-JA( QUt!S LtC;ROS
between serum IRN and corticosterone are responsive
to restraint
corticosterone
levels w o u l d
levels, w h i c h c o u l d m e a n that I R N c o n c e n t r a t i o n s
stress especially
immediately
b e still s e n s i t i v e t o f u r t h e r
after
being applied,
application
whereas
of stress, perhaps
reinforces this hypothesis. This research was supported by grant 3.4507.79 from the Belgian F.R.S.M. The authors would like to express appreciation to Marc Sojic for statistical analysis. REFERENCES CARLSON, D. E., DORNHORSt, A., SELF, S. M., ROmNSON, A. G. & GAnn, D. S. (1982) Vasopressin-dependent and -independent control of the release of adrenocorticotropin. Endocrinology 110, 6 8 0 - 682. Col t u, R., DurwlsseAu, P. & TACm:, Y. (1979) Role of putative neurotransmitters in prolactin, GH and LH response to acute immobilization stress in male rats. Neuroendoerinolog_v 28, 178 186. CRIN[-, A. F., PERIKEI, J. J., Louis, F. & LE(.iROS, J. J. (1981) Modulation du systeme hypothalamoneurohypophysaire ~t la suite de modifications environnementales chez le rat. C. r. Soc. Biol. Paris 175, 882-888. CRINE, A. F., LOUIS, F. & LE(iROS, J. J. (1982) Peripheral neurophysins changes under mild stress conditions. Biol. Psvchol. 14, 311 - 315. DOGTErOSl, J. & ButJs, R. M. (1980) Vasopressin and oxytocin distribution in rat brain: radioimmunoassay and immunocytochemical studies. In Neuropeptides and Neural Transmission, Marsan, C. A. & Traczyk, W. Z. (Eds.), pp. 307 314, Raven Press, New York. DORNHORSF, A., CARLSON, D. E., Sl~ll~, S. M., ROBINSON, A. G., ZIMMFRMAN, E. A. & GANN, D. A. (1981) Control of release of adrenocorticotropin and vasopressin by the supraoptic and paraventricular nuclei. Endocrinology 11)8, 1420- 1424. Hat DAR, J. & BADI=, V. (1981) Involvemem of opioid peptides in the inhibition of oxytocin release by heal stress in lactating mice (41227). Proe. Soc. exp. Biol. Med. 168, 10- 14. HUSAIN, M. K., MANGER, W. M., ROCK, T. W., WEISS, R. J. & FRANTZ, A. G. (1979) Vasopressin release due to manual restraint in the rat: role of body compression and comparison with other stressful stimuli. Endocrinology 10, 641 - 644. Kell, t.. & SEVERS, W. B. (1977) Reduction in plasma vasopressin levels of dehydrated rats following acute stress. Endocrinology 100, 30 38. KHM, K. L. & SIGc;, E. B. (1976) Physiological and biochemical concomitants of restraint stress in rats. PharmacoL Biochem. Behav. 4, 289-297. KRISH, B. (1980) Are there indications for a granule-free form of vasopressin? J. Histochem. (~vtochem. 28, 472 - 474. KruI J~H, J., HLFCO, E., It I NLR, P. & READ, C. B. (1974) The effects of acute stress on the secretion of LH, FSH, prolactin and GH in the normal male rat, with comments on their statistical evaluation. Nearoendocrinolog.v 16, 293 311. KVE-TNaNSK',, R., PAt.KOVITS, M., MJtro, A., TORt)A, T. & MIl,:ul AJ, L. (1977) Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats. Neuroendocrinolog.v 23, 257 - 267. Lt-c;ros, J. J. (1976) Les Nearoph.vsines. Reeherches Methodologique.{, Exp&imentales et Cliniques. Masson, Paris. l.t(;ros, J. J. (1979) The radioimmunoassay of neurophysin as a tool for posterior pituitary investigation. Biochem. Pharmac. 27, 1009 1018. I.t:(;ROS, J. J. & DrHFUSS, J. J. (1975) Water deprivation, in rats: elevated plasma neurophysin levels. E.vperientia 31,603 - 605. PETTIT, A. (1913) Proced6 simple pour pr6lever du sang chez les petits rongeurs. ('. r. Soc. Biol. Paris 74, 11. SeC;~;IE, J. & Brov,'n, G. M. (1982) Profiles of hormone stress response: recruitement or pathway specificity. In Brain Peptides and Hormones, Collu, R., Ducharme, J. R., Barbeau, A. & Tollis, G. (Eds.), pp. 277 - 285, Raven Press, New York. SUl (}N, J., DEMt Y-PONSART, L., BEAUI}UIN, P. & SODOYI:Z, J. C. (1978} Radioimmunosassay of corticosterone, cortisol and cortisone: their application to human cord and maternal plasma. J. Steroid Biochem, 9, 671 676. TA(.ttI:, Y., DURtJISSEAU, P., DU(_HARMt, J. R. & COI IU, R. (1978) Pattern of adenohypophyseal hormone changes in male rats following chronic stress. Neuroendocrinology 26, 208 - 219. VAN WIM~-RSMAGRHt)ANUS, T. B., CroJsH G., GOI:DEMANS, H. & DO,.;Tero",~, J. (1979) Vasopressin levels in peripheral blood and in cerebrospinal fluid during passive and active avoidance behavior in rats. Horm. Behav. 12, 103 - 111. W,',,t~dns, W. B., SCHV,AmD,",I, P. & Boca., R. (1974) hnmunohistochemical demonstration of a CRF-associated neurophysin in the external zone of the rat median eminence. Cell. I'i~s,e Res. 152, 411 421.