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Antiopioid activity of the cardioexcitatory peptide in central modulation of cardiovascular functions
European Journal of Pharmacology, 130 (1986) 315-318
315
Elsevier EJO 470SC
Short communication
Antiopioid activity of the cardioexcitatory peptide in central modulation of cardiovascular functions S o n g - H a i Chai, Jian T a n g a n d J i - S h e n g H a n * Department of Physiology', Beijing Medical University, 38, Xue Yuan Road, Beijing 100083, China Received 12 August 1986, accepted 2 September 1986
Intracerebroventricular (i.c.v.) injection of [D-Ala2,LeuS]enkephalin produced depressor and bradycardiac responses which were prevented by naloxone or FMRF-NH 2 pretreatment. Intrathecal injection of FMRF-NH 2 to rats in hemorrhagic shock resulted in a slight but significant increase in blood pressure, and prevented the further decrease in heart rate which was observed in the control group. It is concluded that FMRF-NH 2 may act as an antiopioid substance in central modulation of cardiovascular functions. Cardioexcitatory peptide; Antiopioid activity; Naloxone; Blood pressure; Heart rate
1. Introduction
The tetrapeptide Phe-Met-Arg-Phe-NH2 ( F M R F - N H 2 ) was originally isolated from the ganglia of the clam Macrocallista nimbosa by Price and Greenberg (1977). It was shown to have positive inotropic, positive chronotropic and antiarrythmic actions on the heart of many mollusc species, hence was given the name cardioexcitatory peptide (Painter and Greenberg, 1982). Tang et al. (1984) have demonstrated that both F M R F - N H 2 and an immunoreactive F M R F NHz-like peptide extracted from bovine brain could antagonize opioid-induced analgesia in the rat. They have also shown that intracerebroventricular (i.c.v.) or intrathecal injection of antibodies directed to F M R F - N H 2 induced a longlasting analgesia and postponed the development of tolerance to morphine analgesia. Kavaliers et al. (1985) have found that i.c.v, administration of F M R F - N H 2 in mice inhibited both morphineand food deprivation-induced feeding. The latter has been shown to be at least partially mediated * -To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
by opioid mechanisms. These findings suggest an antiopioid activity for F M R F - N H 2 in the central modulation of nociception and feeding behavior. The present study was undertaken to evaluate the effects of F M R F - N H 2 on the central cardiovascular actions of exogenously administered opioid peptides as well as on the hemorrhagic hypotension and bradycardia, in which endogenous opioids have been implicated (Faden and Holady, 1979; Han et al., 1982).
2. Materials and methods
2.1. Animals Albino rats of both sexes, weighing 180-300 g, were used. Sodium pentobarbital (30 m g / k g i.p.) or urethane (1.25 g / k g i.p.) was used for anesthesia in experiments with [D-Ala2,LeuS]enkephalin (DALE) and hemorrhagic shock, respectively. 2.2. Preparations The anesthetized rats were implanted stereotaxically with a guide cannula of 0.8 m m outer
316
diameter, directed to the left ventricle. The cannula was fixed on the skull with dental acrylic cement. Injections were made through an injection tube inserted into the cannula, extending 1.5 m m beyond the tip of the cannula to reach the ventricle. Drugs dissolved in artificial cerebrospinal fluid (CSF) were injected in volumes of 12.5/~1 over 1 min. The sites of injection were verified by dye injection and surgical exposure after the experiments. Intrathecal injection was accomplished by catheterization of the spinal subarachnoid space according to Yaksh and Rudy (1976). A polyethylene catheter (PE 10, 12 cm in length) was inserted through a slit made on the cisternal membrane into the spinal subarachnoid space and passed caudally 6.5 cm to reach the thoraco-lumbar level. Drugs were dissolved in 10 /L1 of artificial CSF and injected over 1 min. The catheter was then flushed with 5 #1 of CSF to ensure complete drug delivery. Blood pressure was recorded through a pressure transducer (Nihon Kohden, MPU-0.5) connected with a polyethylene catheter filled with physiological saline containing 0.2% heparin, located in the left common carotid artery. Heart rate was calculated from the electrocardiogram recorded with an XDH-3 (Shanghai) electrocardiograph. For induction of hypovolemic shock, the carotid catheter was connected through a three-way stopcock to a reservoir placed at a certain height above the animal. Blood pressure (BP) was reduced to 45-50 m m Hg and maintained at the same level for 1 h. The reservoir was then disconnected and drug effects tested on the animal under a state of shock.
3. Results
3.1. Antagonism of cardiovascular actions of i.c.v. DALE by naloxone or FMRF-NH, Four groups of six-seven rats each were injected i.c.v, with (a) artificial CSF, 12.5 /~l, (b) naloxone, 10/~g (27.5 nmol), (c) F M R F - N H 2, 3.6 /~g (6.0 nmol), and (d) F M R F - N H 2, 7.2 /~g, respectively, followed 6 min later by DALE, 50 /~g (87.9 nmol), administered via the same route. A group of six rats receiving two consecutive injections of 12.5/~1 Of CSF was used as control. In the CSF-pretreated group, i.c.v, injection of 50 /~g D A L E produced a drastic decrease in BP, reaching its lowest level ( - 50 _+ 5%) in 20 min. A 50% recovery took place in 1 h. When i.c.v, injection of D A L E was preceded by naloxone (10/~g), the depressor response to D A L E was completely abolished (fig. 1). Figure 1 also shows that DALE-induced hypotension could be partially blocked by F M R F - N H 2 in a dose-dependent manner. Similar changes were found in heart rate (HR), where DALE-induced bradycardia was completely blocked by naloxone, 10/~g, and par-
% changein HR
% changein B'P 0
.......................
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-40 -50
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20
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2.3. Peptides and drugs F M R F - N H 2 and D A L E were supplied by Peninsula Laboratories (San Carlos, USA), naloxone HC1 (NX) by Endo Laboratories.
2.4. Data analysis The data are presented as means + S.E.M. The significance of differences between two groups was assessed by Student's t-test.
Fig. 1. Influence of F M R F - N H 2 on the hypotensive and b r a d y c a r d i a c effects of D A L E injected i.c.v, in rats. Baseline BP were 144.8+2.6, 131.8+4.9, 135.3+6.0, 141.4+5.1 and 131.2_-t-6.2 m m H g for the CSF-, naloxone-, F M R F - N H 2 (3.6 ~ g a n d 7.2 tLg)-pretreated group and the CSF control group, respectively. The c o r r e s p o n d i n g H R were 4 2 8 + 1 6 , 417_+26, 455 + 18, 443 + 13 and 400_+ 29 b e a t s / m i n , respectively. C o m p a r e d with C S F + D A L E group: * P < 0.05, * * P < 0.01, * * * P < 0.001. O C S F 12.5 / x l + D A L E 50 /~g (6); • F M R F - N H 2 3.6 p ~ g + D A L E 50 /~g (6); • F M R F - N H 2 7.2 / ~ g + D A L E 50 t~g (7); • N X 10 p.g+ D A L E 5 0 / ~ g (6); O . . . . . . O C S F 12.5 ~ I + C S F 12.5 t~l (6).
317
tially blocked by the two doses of F M R F - N H 2. N o significant cardiovascular responses were found when 3.6 ~g of F M R F - N H 2 was injected i.c.v, to the rat (n = 7, data not shown).
injected intrathecally with 10/~g of F M R F - N H 2, there were no significant differences in BP and H R from the CSF-injected group.
3.2. Reversal of hypotension and bradycardia in hypouolemic shock by intrathecal injection of FMRF-NH e
4. Discussion
Twenty eight urethane-anesthetized rats were bled through the carotid catheter to lower BP to a level of 45-50 m m Hg, which was maintained for 1 h. They were then randomly assigned to two groups of 14 each, given either 10/~1 of CSF or 10 /~g of F M R F - N H 2 by intrathecal bolus. The two groups of rats showed no statistically significant differences in body weight (189 + 6 g in CSF group vs. 180 + 4 g in drug group), baseline BP (102.9 + 3.0 vs. 95.5 + 3.8 m m Hg), baseline H R (398 + 10 vs. 387 + 12 beats/min), and total volume of blood removed (2.45 + 0.24 vs. 2.75 + 0.21 ml). In the control group injected with CSF, the BP remained at a low level and H R decreased progressively throughout the 1 h observation period whereas in the F M R F - N H 2treated group there was a steady and significant increase in BP (fig. 2), and the further decrease in H R was prevented (data not shown). When a group of six normotensive rats was
B-Pmm Hg II0 I00 ~ _ _ ~ 90
H
tl1 70
FMRF-NIJeI0 pg (141
o-o CSF ,O.u,(,4)
~ t%
injection
50
•
30
,
-70
•
,
i
- 6 0 I/ 0
i
i
I0 20 Time rnin
i
i
i
i
30
40
50
60
The results of the present study indicate that the cardiovascular effects of i.c.v, injection of the opioid peptide DALE, a decrease in both BP and H R could be antagonized by naloxone or by F M R F - N H 2 administered via the same route. Moreover, F M R F - N H 2 was shown to be effective in reversing the hypotension and alleviating the bradycardia in hypovolemic shock, where endogenous opioids are suggested to play a role (Faden and Holady, 1979; Han et al., 1982). These results, together with the evidence obtained in other physiological systems, such as those involved in nociception and feeding, strongly suggest that F M R F N H 2 is an antagonist to opioids. Immunohistochemical studies (Chronwall et al., 1984; Williams and Dockray, 1983) revealed FMRF-NH2-1ike immunoreactivity in rat brain, including the hypothalamus and lower brain-stem and in the intermediolateral column of the thoracic cord, forming a structural basis for the function of F M R F - N H 2 as an endogenous antiopioid substance in the central modulation of cardiovascular function. It is well known that the central cardiovascular effects of opioid peptides vary according to the site of administration. While i.c.v, administration of D A L E produced depressor effects (fig. 1), injection of enkephalins into the nucleus tractus solitarii produced a pressor effect (Petty and De Jong, 1983). In this context it is interesting to note that Y G G F M R F produced an increase in BP after fourth ventricular administration and a decrease in BP after intrathecal injection. Both effects were antagonized by F M R F - N H 2 administered via the same route (Chai et al., to be published).
Acknowledgements Fig. 2. Pressor effect of F M R F - N H 2 injected intrathecally to rats with hemorrhagic shock. Compared with control group: *P < 0.05, **P < 0.01.
We thank Peninsula Laboratories and Endo Laboratories for their generous gifts of peptides and naloxone.
318
References Chronwall, B.M., J.A. Olschowka and T.L. O'Donohue, 1984, Histochemical localization of FMRFamide-like immunoreactivity in the rat brain, Peptides 5, 569. Faden, A.I. and J.W. Holady, 1979, Opiate antagonists: A role in the treatment of hypovolemic shock, Science 205, 317. Han, J.S., J. Tang, H.X. Zhang, Q.S. Chan and S.J. Li, 1982, Pressor effect of intrathecal injection of naloxone in rabbits with hemorrhagic shock, Chin. Med. J. 62, 610. Kavaliers, M., M, Hirst and A. Mathers, 1985, Inhibitory influence of FMRFamide on morphine- and deprivationinduced feeding, Neuroendocrinology 40, 533. Painter, S.D. and M.J. Greenberg, 1982, A survey of the response of bivalve hearts to the molluscan neuropeptide FMRFamide and to 5-hydroxytryptamine, Biol. Bull 162, 311.
Petty, M.A. and W. De Jong, 1983, Enkephalins induce a centrally mediated rise in blood pressure in rats, Brain Res. 260, 322. Price, D.A. and M.J. Greenberg, 1977, The structure of a molluscan cardioexcitatory neuropeptide, Science 197, 670. Tang, J., H.-Y. Yang and E. Costa, 1984, Inhibition of spontaneous and opiate-modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-amide-like immunoreactivity, Proc. Nat. Acad. Sci. U.S.A. 81, 5002. Williams, R.G. and G.J. Dockray, 1983, Immunohistochemical studies of FMRFamide-like immunoreactivity in rat brain, Brain Res. 276, 213. Yaksh, T.L. and T.A. Rudy, 1976, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17, 1031.
315
Elsevier EJO 470SC
Short communication
Antiopioid activity of the cardioexcitatory peptide in central modulation of cardiovascular functions S o n g - H a i Chai, Jian T a n g a n d J i - S h e n g H a n * Department of Physiology', Beijing Medical University, 38, Xue Yuan Road, Beijing 100083, China Received 12 August 1986, accepted 2 September 1986
Intracerebroventricular (i.c.v.) injection of [D-Ala2,LeuS]enkephalin produced depressor and bradycardiac responses which were prevented by naloxone or FMRF-NH 2 pretreatment. Intrathecal injection of FMRF-NH 2 to rats in hemorrhagic shock resulted in a slight but significant increase in blood pressure, and prevented the further decrease in heart rate which was observed in the control group. It is concluded that FMRF-NH 2 may act as an antiopioid substance in central modulation of cardiovascular functions. Cardioexcitatory peptide; Antiopioid activity; Naloxone; Blood pressure; Heart rate
1. Introduction
The tetrapeptide Phe-Met-Arg-Phe-NH2 ( F M R F - N H 2 ) was originally isolated from the ganglia of the clam Macrocallista nimbosa by Price and Greenberg (1977). It was shown to have positive inotropic, positive chronotropic and antiarrythmic actions on the heart of many mollusc species, hence was given the name cardioexcitatory peptide (Painter and Greenberg, 1982). Tang et al. (1984) have demonstrated that both F M R F - N H 2 and an immunoreactive F M R F NHz-like peptide extracted from bovine brain could antagonize opioid-induced analgesia in the rat. They have also shown that intracerebroventricular (i.c.v.) or intrathecal injection of antibodies directed to F M R F - N H 2 induced a longlasting analgesia and postponed the development of tolerance to morphine analgesia. Kavaliers et al. (1985) have found that i.c.v, administration of F M R F - N H 2 in mice inhibited both morphineand food deprivation-induced feeding. The latter has been shown to be at least partially mediated * -To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
by opioid mechanisms. These findings suggest an antiopioid activity for F M R F - N H 2 in the central modulation of nociception and feeding behavior. The present study was undertaken to evaluate the effects of F M R F - N H 2 on the central cardiovascular actions of exogenously administered opioid peptides as well as on the hemorrhagic hypotension and bradycardia, in which endogenous opioids have been implicated (Faden and Holady, 1979; Han et al., 1982).
2. Materials and methods
2.1. Animals Albino rats of both sexes, weighing 180-300 g, were used. Sodium pentobarbital (30 m g / k g i.p.) or urethane (1.25 g / k g i.p.) was used for anesthesia in experiments with [D-Ala2,LeuS]enkephalin (DALE) and hemorrhagic shock, respectively. 2.2. Preparations The anesthetized rats were implanted stereotaxically with a guide cannula of 0.8 m m outer
316
diameter, directed to the left ventricle. The cannula was fixed on the skull with dental acrylic cement. Injections were made through an injection tube inserted into the cannula, extending 1.5 m m beyond the tip of the cannula to reach the ventricle. Drugs dissolved in artificial cerebrospinal fluid (CSF) were injected in volumes of 12.5/~1 over 1 min. The sites of injection were verified by dye injection and surgical exposure after the experiments. Intrathecal injection was accomplished by catheterization of the spinal subarachnoid space according to Yaksh and Rudy (1976). A polyethylene catheter (PE 10, 12 cm in length) was inserted through a slit made on the cisternal membrane into the spinal subarachnoid space and passed caudally 6.5 cm to reach the thoraco-lumbar level. Drugs were dissolved in 10 /L1 of artificial CSF and injected over 1 min. The catheter was then flushed with 5 #1 of CSF to ensure complete drug delivery. Blood pressure was recorded through a pressure transducer (Nihon Kohden, MPU-0.5) connected with a polyethylene catheter filled with physiological saline containing 0.2% heparin, located in the left common carotid artery. Heart rate was calculated from the electrocardiogram recorded with an XDH-3 (Shanghai) electrocardiograph. For induction of hypovolemic shock, the carotid catheter was connected through a three-way stopcock to a reservoir placed at a certain height above the animal. Blood pressure (BP) was reduced to 45-50 m m Hg and maintained at the same level for 1 h. The reservoir was then disconnected and drug effects tested on the animal under a state of shock.
3. Results
3.1. Antagonism of cardiovascular actions of i.c.v. DALE by naloxone or FMRF-NH, Four groups of six-seven rats each were injected i.c.v, with (a) artificial CSF, 12.5 /~l, (b) naloxone, 10/~g (27.5 nmol), (c) F M R F - N H 2, 3.6 /~g (6.0 nmol), and (d) F M R F - N H 2, 7.2 /~g, respectively, followed 6 min later by DALE, 50 /~g (87.9 nmol), administered via the same route. A group of six rats receiving two consecutive injections of 12.5/~1 Of CSF was used as control. In the CSF-pretreated group, i.c.v, injection of 50 /~g D A L E produced a drastic decrease in BP, reaching its lowest level ( - 50 _+ 5%) in 20 min. A 50% recovery took place in 1 h. When i.c.v, injection of D A L E was preceded by naloxone (10/~g), the depressor response to D A L E was completely abolished (fig. 1). Figure 1 also shows that DALE-induced hypotension could be partially blocked by F M R F - N H 2 in a dose-dependent manner. Similar changes were found in heart rate (HR), where DALE-induced bradycardia was completely blocked by naloxone, 10/~g, and par-
% changein HR
% changein B'P 0
.......................
'" ""
-,o
0
......
"
..
-2o
........
-30
.,.
\; ,.
-
ICv
,t.-U!
~
"
i
' ~
' 4'O '60'rain
-40 -50
O
20
40
60rain
6
2.3. Peptides and drugs F M R F - N H 2 and D A L E were supplied by Peninsula Laboratories (San Carlos, USA), naloxone HC1 (NX) by Endo Laboratories.
2.4. Data analysis The data are presented as means + S.E.M. The significance of differences between two groups was assessed by Student's t-test.
Fig. 1. Influence of F M R F - N H 2 on the hypotensive and b r a d y c a r d i a c effects of D A L E injected i.c.v, in rats. Baseline BP were 144.8+2.6, 131.8+4.9, 135.3+6.0, 141.4+5.1 and 131.2_-t-6.2 m m H g for the CSF-, naloxone-, F M R F - N H 2 (3.6 ~ g a n d 7.2 tLg)-pretreated group and the CSF control group, respectively. The c o r r e s p o n d i n g H R were 4 2 8 + 1 6 , 417_+26, 455 + 18, 443 + 13 and 400_+ 29 b e a t s / m i n , respectively. C o m p a r e d with C S F + D A L E group: * P < 0.05, * * P < 0.01, * * * P < 0.001. O C S F 12.5 / x l + D A L E 50 /~g (6); • F M R F - N H 2 3.6 p ~ g + D A L E 50 /~g (6); • F M R F - N H 2 7.2 / ~ g + D A L E 50 t~g (7); • N X 10 p.g+ D A L E 5 0 / ~ g (6); O . . . . . . O C S F 12.5 ~ I + C S F 12.5 t~l (6).
317
tially blocked by the two doses of F M R F - N H 2. N o significant cardiovascular responses were found when 3.6 ~g of F M R F - N H 2 was injected i.c.v, to the rat (n = 7, data not shown).
injected intrathecally with 10/~g of F M R F - N H 2, there were no significant differences in BP and H R from the CSF-injected group.
3.2. Reversal of hypotension and bradycardia in hypouolemic shock by intrathecal injection of FMRF-NH e
4. Discussion
Twenty eight urethane-anesthetized rats were bled through the carotid catheter to lower BP to a level of 45-50 m m Hg, which was maintained for 1 h. They were then randomly assigned to two groups of 14 each, given either 10/~1 of CSF or 10 /~g of F M R F - N H 2 by intrathecal bolus. The two groups of rats showed no statistically significant differences in body weight (189 + 6 g in CSF group vs. 180 + 4 g in drug group), baseline BP (102.9 + 3.0 vs. 95.5 + 3.8 m m Hg), baseline H R (398 + 10 vs. 387 + 12 beats/min), and total volume of blood removed (2.45 + 0.24 vs. 2.75 + 0.21 ml). In the control group injected with CSF, the BP remained at a low level and H R decreased progressively throughout the 1 h observation period whereas in the F M R F - N H 2treated group there was a steady and significant increase in BP (fig. 2), and the further decrease in H R was prevented (data not shown). When a group of six normotensive rats was
B-Pmm Hg II0 I00 ~ _ _ ~ 90
H
tl1 70
FMRF-NIJeI0 pg (141
o-o CSF ,O.u,(,4)
~ t%
injection
50
•
30
,
-70
•
,
i
- 6 0 I/ 0
i
i
I0 20 Time rnin
i
i
i
i
30
40
50
60
The results of the present study indicate that the cardiovascular effects of i.c.v, injection of the opioid peptide DALE, a decrease in both BP and H R could be antagonized by naloxone or by F M R F - N H 2 administered via the same route. Moreover, F M R F - N H 2 was shown to be effective in reversing the hypotension and alleviating the bradycardia in hypovolemic shock, where endogenous opioids are suggested to play a role (Faden and Holady, 1979; Han et al., 1982). These results, together with the evidence obtained in other physiological systems, such as those involved in nociception and feeding, strongly suggest that F M R F N H 2 is an antagonist to opioids. Immunohistochemical studies (Chronwall et al., 1984; Williams and Dockray, 1983) revealed FMRF-NH2-1ike immunoreactivity in rat brain, including the hypothalamus and lower brain-stem and in the intermediolateral column of the thoracic cord, forming a structural basis for the function of F M R F - N H 2 as an endogenous antiopioid substance in the central modulation of cardiovascular function. It is well known that the central cardiovascular effects of opioid peptides vary according to the site of administration. While i.c.v, administration of D A L E produced depressor effects (fig. 1), injection of enkephalins into the nucleus tractus solitarii produced a pressor effect (Petty and De Jong, 1983). In this context it is interesting to note that Y G G F M R F produced an increase in BP after fourth ventricular administration and a decrease in BP after intrathecal injection. Both effects were antagonized by F M R F - N H 2 administered via the same route (Chai et al., to be published).
Acknowledgements Fig. 2. Pressor effect of F M R F - N H 2 injected intrathecally to rats with hemorrhagic shock. Compared with control group: *P < 0.05, **P < 0.01.
We thank Peninsula Laboratories and Endo Laboratories for their generous gifts of peptides and naloxone.
318
References Chronwall, B.M., J.A. Olschowka and T.L. O'Donohue, 1984, Histochemical localization of FMRFamide-like immunoreactivity in the rat brain, Peptides 5, 569. Faden, A.I. and J.W. Holady, 1979, Opiate antagonists: A role in the treatment of hypovolemic shock, Science 205, 317. Han, J.S., J. Tang, H.X. Zhang, Q.S. Chan and S.J. Li, 1982, Pressor effect of intrathecal injection of naloxone in rabbits with hemorrhagic shock, Chin. Med. J. 62, 610. Kavaliers, M., M, Hirst and A. Mathers, 1985, Inhibitory influence of FMRFamide on morphine- and deprivationinduced feeding, Neuroendocrinology 40, 533. Painter, S.D. and M.J. Greenberg, 1982, A survey of the response of bivalve hearts to the molluscan neuropeptide FMRFamide and to 5-hydroxytryptamine, Biol. Bull 162, 311.
Petty, M.A. and W. De Jong, 1983, Enkephalins induce a centrally mediated rise in blood pressure in rats, Brain Res. 260, 322. Price, D.A. and M.J. Greenberg, 1977, The structure of a molluscan cardioexcitatory neuropeptide, Science 197, 670. Tang, J., H.-Y. Yang and E. Costa, 1984, Inhibition of spontaneous and opiate-modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-amide-like immunoreactivity, Proc. Nat. Acad. Sci. U.S.A. 81, 5002. Williams, R.G. and G.J. Dockray, 1983, Immunohistochemical studies of FMRFamide-like immunoreactivity in rat brain, Brain Res. 276, 213. Yaksh, T.L. and T.A. Rudy, 1976, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17, 1031.