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Behavioral inhibition as modified by melanocyte-stimulating hormone (MSH) and light-dark conditions
Physiology and Behavior. Vol. 6, lap. 45--48. Pergamon Press, 1971. Printed in Great Britain
Behavioral Inhibition as Modified by Melanocyte- Stimulating Hormone (MSH) and Light-Dark Conditions C U R T A. S A N D M A N
Department of Psychiatry and Biobehavioral Sciences, Louisiana State University School of Medicine in New Orleans, New Orleans, Louisiana, U.S.A. A B B A J. K A S T I N A N D A N D R E W
V. S C H A L L Y
Endocrinology Section of the Medical Service, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, Louisiana, U.S.A. (Received 22 June 1970) A. J. KAS~N ANt>A. V. SCHALLYoBehavioralinhibition as modified by melanocyte-stimulating hormone (MSH) and light-dark conditions. Prn'SIOL.BEHAV. 6 (l) 45-48, 1971.--MSH was administered to rats tested with a SANDMAN, C.,
passive avoidance response (PAR) at the peak (one hour after dark) and lowest (in the middle of the light period) level of activity. MSH treatment significantly inhibited the PAR in the dark period. For all animals, inhibition of response was intricately related to phase of activity. This implies an effect on memory and/or the expression of fear, Melanocyte-stimulating hormone
Light-dark
Behavioral inhibition
MELANOCYTE-STIMULATINGhormone (MSH), secreted by the pituitary gland, is known to produce pigmentary changes in amphibians. Its function in mammals is not known [15] but learning behavior, such as the maintenance of an appetitive response [22] and a conditioned avoidance response [7-9] have resulted from administration of MSH to rats. Among the hypotheses suggested to account for the extrapigmentary functions of MSH in learning tasks are: (a) a positive effect on memory [9, 22]; (b) the inability to inhibit a response [9, 22]; and (c) the possibility that general motivational factors may cause the maintenance of a response [22]. The present study was designed to measure directly the last two possibilities by testing the ability of an animal to inhibit a response in a passive avoidance situation which minimized the influence of motivational components. Another major purpose of this investigation was to examine the interaction of diurnal variation with MSH by testing the same passive avoidance response (PAR) behavior. Numerous studies indicate that rats are nocturnal animals engaging in the greatest amount of activity under conditions of darkness [18]. It is well established that the pituitary release of some hormones follows a circadian rhythm [11, 18] and it is possible that factors contributing to pigmentation and the release of MSH reveal endogenous periodicity in amphibians [17, 19-21]. This has not yet been demonstrated in mammals although pituitary content of MSH is sensitive to light-dark periods
[16]. However, surprisingly few studies have investigated learning performance as related to phase of activity especially as modified by hormonal administration.
MATERIALS AND METHODS
Fifty male albino rats, thirty days old, were randomly assigned to receive intraperitoneal injections of ~-MSH (10t~g), 13-MSH (20t~g), lysine vasopressin (0.1 I~g), or a saline control solution. The dose of MSH was similar to that used in other studies [9, 10, 22]. Both the purified and synthetic ~-MSH preparations contained the MSH activity of 1 × l07 units/mg and the fI-MSH contained 5 × l06 units/mg. The vasopressin control group received an amount of vasopressin equivalent to that contained in the preparation of porcine 13-MSH (1.0 presser units/rag), an amount larger than that in the bovine ~,-MSH (0.02 presser units/mg). All of the rats were adapted to a 12 hr light-12 hr dark cycle for 15 days. One half of the animals were tested during the peak of activity (the first hour of darkness) and half during the lowest level of activity (the middle of the light period). A partial replication involving twenty additional animals was performed. The standard procedure for a P A R was employed and adapted for exploratory behavior as described by Weiss,
1Supported in part by NIH grants No. NS 07664 (AJK) and No. AM 07467 (AVS). SThe assistance of Dr. J. Coury and T. Sellers is appreciated. Reprint requests should be addressed to A. J. Kastin. 3Published in abstract form in Clin. Res. 18: 371, 1970. 45
46
SANDMAN, KASTIN AND S( H A I I h
McEwen, Silva and Kalbut [25]. In a double blind procedure each rat was injected 15 min prior to entry into the two chamber test apparatus. Each rat was placed in Chamber 1 for 15 sec, a guillotine door was raised and the latency, or time required for the animal to enter Chamber 2, was recorded. Timing began when the door was raised. After entry into Chamber 2, which was defined as having all four feet in contact with the grid floor, the rat was removed from the apparatus. On the second day as the rat entered Chamber 2 his latency time was recorded and a mild shock was delivered to his feet for 1.5 sec with a Foringer shocker. The rat was allowed to escape into Chamber 1, then removed. On the first two days a baseline latency score was established. The rats were rested on Days 3 and 4 but the injections were continued. On Days 5 and 6 the procedure was the same as for Day 1. Animals not responding after 200 sec were removed from the apparatus and a 200 sec latency score was recorded.
receiving the same treatment and lighting conditions were not significantly different between Days 1 and 2 and between Days 5 and 6. Thus, the main effect of days in Table I was due to the difference between Days I and 2 with Days 5 and 6. MSH groups, as seen in Fig. 1, did not differ under light and dark conditions. Also, under light conditions, there was no significant difference between MSH and control animals. Dark conditions however, caused control animals to respond significantly faster than control animals in light and significantly faster than all of the animals receiving MSH. Most of the variance then, was due to the inhibition of response of animals treated with MSH following shock under darkened conditions (ABC p < 0.01). Since individual comparisons between treatment groups revealed nonsignificant differences within MSH groups and within control groups, the data were summarized in combined form (Table 2). 150
RESULTS
The results were examined with a least squares solution for a 2 x 5 × 4 repeated measures analysis of variance design and indiv;.dual comparisons [26]. It is apparent from Table 1 that the experimental manipulations produced significant effects on the animal's performance of a PAR. Light and dark variations yielded a significant main effect (A p < 0.05) since in the dark the animals had a lower latency of response on Days 5 and 6. A strong main effect of treatment was accounted for by the inhibition of exploratory behavior of MSH treated animals (B p < 0.01), especially in the dark (AB p < 0.01). The data indicate that shock on Day 2 inhibited all of the animals on Days 5 and 6 (C p < 0.01). Individual comparisons of results obtained from animals
125
G
c~ Z 0 u hi
I00
MSH
t
(.D Z
"I,,-'
75
P
_.!
1
Z
z t
50
rY
CONTROL
TABLE 1 LEASTSQUARESANALYSISOF VARIANCEOF RESULTSOBTAINEDON A PASSIVE AVOIDANCERESPONSE FROM RATS RECEIVING MSH AND CONTROL SOLUTIONS UNDER CONDITIONS OF LIGHT AND DARK (2 X 5 X 4 REPEATEDMEASURESDESIGN)
Source Between animals A (Light and dark) B (Treatment) AB (Light and dark x treatment) Animals within groups Within animals C (Days) AC (Light and dark x days) BC (Treatment x days) ABC (Light and dark x treatment × days) C x Animals within groups
Degrees of Freedom 69 1 4
Mean Square
F Ratio
p
13507.4 97118.5
4.32 31.04
0.05 0.01 0.01
4 60 210 3
29431.9 3128.6
9.41
78386.8
59.06
0.01
3 12
4738.0 20924.5
3.56 15.77
0.05 0.01
12
10795.5
195
1327.2
25
I
I
LIGHT
DARK
FIG. 1. Mean latency of response on a passive avoidance response (4- standard error) from combined groups of rats receiving MSH and control solutions on Day 5. TABLE 2 MEAN LATENCY OF RESPONSE ON A PASSIVE AVOIDANCE RESPONSE FROM COMBINED GROUPS OF RATS RECEIVING M S H AND CONTROL SOLUTIONS UNDER CONDITIONSOF LIGHT AND DARK
Day 1
8.13
0.01
MSH (n - 15) Control (n = 20)
15.8 9.8
MSH (n = 20) Control (n = 15)
9.9 10.1
2 Light 11.8 6.0
5
6
128.3 116,8*
t01.7 96.4*
102.7t 38.2
90,5t 33.4
Dark 9.7 11.6
*p < 0.01 when compared with same condition in dark. tP < 0.01 when compared with control group.
MSH AND BEHAVIOR
47 DISCUSSION
In an earlier study the role of memory, inhibition and motivation were suggested to account for the maintenance of an appetitive response in animals injected with MSH [22]. The present study clearly demonstrates that animals injected with MSH are capable of inhibiting a response when tested with a task specifically designed to determine inhibition. Administration of ACTH, which shares an active segment with MSH [10], causes a similar behavior on a P A R [25] as was found with MSH. Since the preparation of MSH used in this study contained no extrinsic ACTH, it is possible that the effects of A C T H on behavior [1, 5, 8, 25] are due to the intrinsic MSH activity of ACTH. A motivational hypothesis does not well explain the results observed in previous studies involving appetitive and active avoidance behavior [22]. The design of this study minimized the motivational factors and militated against the sporadic hyperactivity observed previously [22]. Thus, of the three possible explanations for the results observed with MSH on learning behavior, it appears that difficulty in inhibiting a response and motivation can be ruled out, leaving the role of memory to be directly tested. In addition to memory, another possibility to explain the behavioral results with MSH is emotionality. Animals injected with MSH may become more "emotional" in the stressful testing situation and revert to species specific defense reactions [6]. Accordingly, the animals may either flee or freeze depending upon which behavior is successful in reducing the stress. It is conceivable that in tasks such as active avoidance and appetitive behavior, in which activity is successful in reducing stress, a response may be perseverated. Similarly, the inhibition of activity is most efficient for the reduction of stress in the P A R situation and this response may be exaggerated in a highly emotional animal. The mechanisms by which animals become more emotional or by which memory may be implicated are not known. However, recent electroencephalographic data [23] suggest the limbic system, specifically the hippocampus, discharges following MSH injections. Additional studies of the effects of MSH on the central nervous system are currently in progress to further investigate hypothalamic and other limbic structure
involvement as well as the permeability of the blood-brain barrier to MSH. The second aspect of this study involves the role of periodicity with respect to learning tasks. The results indicate that in darkness, during the peak of activity, normal (control) animals are less capable of withholding a response than during the lowest level of activity in light. Noxious stimulation is more effective during the period of darkness [12, 14, 24, 27]. One explanation for these paradoxical findings for normal animals is that the tendency for the animal to inhibit a response is overwhelmed by high levels of activity. However, it has also been suggested that rats tend to explore stimuli which evoke fear [13]. This suggests that the expression of fear differs under light and dark conditions for normal animals but is undifferentiated for animals treated with MSH. The results obtained for normal animals in this study are opposite to those found for mice in what was essentially a P A R [24] but similar for rats in an open field study [4]. In addition, maze performance in mice [24] and earthworms [2] was influenced by phase of activity. This study demonstrates that under dark conditions administration of MSH to rats interferes with a normal response of a PAR. The exact mechanism of this action of MSH is not known. Since the effects of drugs and toxic agents are maximal during peak activity in rats [3, 14], it may be that the behavioral differences are a reflection of increased potency of MSH under conditions of light and dark. Interestingly, MSH is secreted by the pituitary under dark conditions and injections of MSH may be effective in inhibiting the pituitary release of MSH [16]. Therefore exogenous M S H may inhibit the release of endogenous MSH from the pituitary during the period of peak release. Consideration of circadian activity may serve to resolve controversies with respect to studies of learning and hormonal influence on behavior. The current debate on the influence of A C T H on fear-motivated behavior [5, 25] might be resolved by an explication of the conditions under which the animals were tested, especially with reference to phase of activity. Halberg's [11 ] claim that knowledge of the effects of circadian activity provides a refined index if evaluated and a confusing source of error if ignored is well illustrated in the present study with MSH. MSH is found to influence behavior involving memory or emotionality which intricately interacts with diurnal variation.
REFERENCES 1. Ader, R. Adrenocortical function and the measurement of emotionality. Ann. N . Y . Acad. Sci. 159: 791-805, 1969. 2. Arbit, J. Diurnal cycles and learning in earthworms. Science 126: 654-655, 1957. 3. Aschoof, J. Comparative physiology: diurnal rhythms. Ann. Rev. Physiol. 25: 581-600, 1963. 4. Battig, K. Drug effects on exploration of a combined maze and open field system by rats. Ann. N . Y . Acad. Sci. 159: 880-897, 1969. 5. Bohus, B., Sc. Nyakas and E. Endroczi. Effects of adrenocorticotropic hormones on avoidance behavior of intact and adrenalectomized rats. lnt. J. Neuropharm. 7: 307-314, 1968. 6. Bolles, R. C. Species-specific defense reactions and avoidance learning. PsychoL Rev. 77: 32-48, 1970. 7. DeWied, D. The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. lnt. J. Neuropharm. 4: 157-167, 1965.
8. DeWied, D. Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats. Proc. Soc. exp. Biol. Med. 122: 28-32, 1969. 9. DeWied, D. and B. Bohus. Long term and short term effects on retention of a conditioned avoidance response in rats by treatment with long acting pitressin and aMSH. Nature 213: 1484-1486, 1966. 10. Grevan, H. M. and D. DeWied. The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behavior in rats. Fur. J. Pharmac. 2: 14-16, 1967. 11. Halberg, F. The 24-hour scale: A time dimension of adaptive functional organization. Perspect. Biol. Med. 3: 491-527, 1960. 12. Halberg, F., E. Jacobsen, G. Wadsworth and J. J. Bittnea. Audiogenic abnormality spectra, twenty-four hour periodocity and lighting. Science 128: 657-658, 1958.
48 13. Halliday, M. S. Exploration and fear in the rat. Proc. Zool. Soc. Lond. 18: 45-59, 1966. 14. Haus, E. Periodicity in response and susceptibility to environmental stimuli. Ann. N.Y. Acad. Sci. 117: 292-315, 1964. 15. Kastin, A. J., S. Kullander, N. E. Borglin, B. Dahlberg, K. Dyster-Aas, C. E. T. Krakau, D. H. Ingvar, M. C. Miller, C. Y. Bowers and A. V. Schally. Extrapigmentary effects of melanocyte-stimulating hormone in amenorrhoeic women Lancet 1: 1007-1010, 1968. 16. Kastin, A. J., A. V. Schally, S. Viosca, L. Barrett and T. W. Redding. MSH activity in the pituitaries of rats exposed to constant illumination. Neuroendocrinology. 2: 257-262, 1967. 17. Kleinholz, L. H. Studies in the pigmentary system of crustacea. Biol. Bull. 72: 176-189, 1937. 18. Marler, P. R. and W. J. Hamilton. Mechanisms of Animal Behavior. New York: Wiley, 1967, pp. 25-72. 19. Rahn, H. and F. Rosendale. Diurnal rhythms of melanophore hormone secretion in the Anolis pituitary. Proc. Soc. exp. Biol. Med. 48: 100-102, 1941. 20. Reed, B. L. The control of circadian pigment changes in the pencil fish. Life Sci. 7: 961-973, 1968.
SANDMAN, KASTIN ANI) S('I:IAI~I '~ 21. Reinberg, A. and J. Ghata. Bh~logi~al Rh),thm~. New York: Walker, 1964, pp. 25 42. 22. Sandman, C., A. J. Kastin and A. V. Schally. Mehmocytestimulating hormone and learned appetitive behavior. Ea'perientia 25: 1001-1002, 1969. 23. Sandman, C. A., P. M. Denman, 1_. H. Miller, J. R. Knott. A. V. Schally and A. J. Kastin. Electroencephatographic indicants of melanocyte-stimulating horn]one activity, in preparation. 24. Stephens, G., J. L. McGaugh and H. P. Alpern. l'criodicity and memory in mice. Psychonom. Sci. 8: 201-202, 1967. 25. Weiss, J. M., B. S. McEwen, M. T. A. Silva and M. F. Kalbut. Pituitary-adrenal influences on fear responding. Sciettce 163: 197-199, 1969. 26. Winer, B. J. Statistical Principles in Experitnental Design. New York: McGraw-Hill, 1962. 27. Wolley, D. E. and P. S. Timeras. Estrous and circadian periodicity and electroshock convulsion in rats. Am. J. Pltysiol. 203: 379-382, 1962.
Behavioral Inhibition as Modified by Melanocyte- Stimulating Hormone (MSH) and Light-Dark Conditions C U R T A. S A N D M A N
Department of Psychiatry and Biobehavioral Sciences, Louisiana State University School of Medicine in New Orleans, New Orleans, Louisiana, U.S.A. A B B A J. K A S T I N A N D A N D R E W
V. S C H A L L Y
Endocrinology Section of the Medical Service, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, Louisiana, U.S.A. (Received 22 June 1970) A. J. KAS~N ANt>A. V. SCHALLYoBehavioralinhibition as modified by melanocyte-stimulating hormone (MSH) and light-dark conditions. Prn'SIOL.BEHAV. 6 (l) 45-48, 1971.--MSH was administered to rats tested with a SANDMAN, C.,
passive avoidance response (PAR) at the peak (one hour after dark) and lowest (in the middle of the light period) level of activity. MSH treatment significantly inhibited the PAR in the dark period. For all animals, inhibition of response was intricately related to phase of activity. This implies an effect on memory and/or the expression of fear, Melanocyte-stimulating hormone
Light-dark
Behavioral inhibition
MELANOCYTE-STIMULATINGhormone (MSH), secreted by the pituitary gland, is known to produce pigmentary changes in amphibians. Its function in mammals is not known [15] but learning behavior, such as the maintenance of an appetitive response [22] and a conditioned avoidance response [7-9] have resulted from administration of MSH to rats. Among the hypotheses suggested to account for the extrapigmentary functions of MSH in learning tasks are: (a) a positive effect on memory [9, 22]; (b) the inability to inhibit a response [9, 22]; and (c) the possibility that general motivational factors may cause the maintenance of a response [22]. The present study was designed to measure directly the last two possibilities by testing the ability of an animal to inhibit a response in a passive avoidance situation which minimized the influence of motivational components. Another major purpose of this investigation was to examine the interaction of diurnal variation with MSH by testing the same passive avoidance response (PAR) behavior. Numerous studies indicate that rats are nocturnal animals engaging in the greatest amount of activity under conditions of darkness [18]. It is well established that the pituitary release of some hormones follows a circadian rhythm [11, 18] and it is possible that factors contributing to pigmentation and the release of MSH reveal endogenous periodicity in amphibians [17, 19-21]. This has not yet been demonstrated in mammals although pituitary content of MSH is sensitive to light-dark periods
[16]. However, surprisingly few studies have investigated learning performance as related to phase of activity especially as modified by hormonal administration.
MATERIALS AND METHODS
Fifty male albino rats, thirty days old, were randomly assigned to receive intraperitoneal injections of ~-MSH (10t~g), 13-MSH (20t~g), lysine vasopressin (0.1 I~g), or a saline control solution. The dose of MSH was similar to that used in other studies [9, 10, 22]. Both the purified and synthetic ~-MSH preparations contained the MSH activity of 1 × l07 units/mg and the fI-MSH contained 5 × l06 units/mg. The vasopressin control group received an amount of vasopressin equivalent to that contained in the preparation of porcine 13-MSH (1.0 presser units/rag), an amount larger than that in the bovine ~,-MSH (0.02 presser units/mg). All of the rats were adapted to a 12 hr light-12 hr dark cycle for 15 days. One half of the animals were tested during the peak of activity (the first hour of darkness) and half during the lowest level of activity (the middle of the light period). A partial replication involving twenty additional animals was performed. The standard procedure for a P A R was employed and adapted for exploratory behavior as described by Weiss,
1Supported in part by NIH grants No. NS 07664 (AJK) and No. AM 07467 (AVS). SThe assistance of Dr. J. Coury and T. Sellers is appreciated. Reprint requests should be addressed to A. J. Kastin. 3Published in abstract form in Clin. Res. 18: 371, 1970. 45
46
SANDMAN, KASTIN AND S( H A I I h
McEwen, Silva and Kalbut [25]. In a double blind procedure each rat was injected 15 min prior to entry into the two chamber test apparatus. Each rat was placed in Chamber 1 for 15 sec, a guillotine door was raised and the latency, or time required for the animal to enter Chamber 2, was recorded. Timing began when the door was raised. After entry into Chamber 2, which was defined as having all four feet in contact with the grid floor, the rat was removed from the apparatus. On the second day as the rat entered Chamber 2 his latency time was recorded and a mild shock was delivered to his feet for 1.5 sec with a Foringer shocker. The rat was allowed to escape into Chamber 1, then removed. On the first two days a baseline latency score was established. The rats were rested on Days 3 and 4 but the injections were continued. On Days 5 and 6 the procedure was the same as for Day 1. Animals not responding after 200 sec were removed from the apparatus and a 200 sec latency score was recorded.
receiving the same treatment and lighting conditions were not significantly different between Days 1 and 2 and between Days 5 and 6. Thus, the main effect of days in Table I was due to the difference between Days I and 2 with Days 5 and 6. MSH groups, as seen in Fig. 1, did not differ under light and dark conditions. Also, under light conditions, there was no significant difference between MSH and control animals. Dark conditions however, caused control animals to respond significantly faster than control animals in light and significantly faster than all of the animals receiving MSH. Most of the variance then, was due to the inhibition of response of animals treated with MSH following shock under darkened conditions (ABC p < 0.01). Since individual comparisons between treatment groups revealed nonsignificant differences within MSH groups and within control groups, the data were summarized in combined form (Table 2). 150
RESULTS
The results were examined with a least squares solution for a 2 x 5 × 4 repeated measures analysis of variance design and indiv;.dual comparisons [26]. It is apparent from Table 1 that the experimental manipulations produced significant effects on the animal's performance of a PAR. Light and dark variations yielded a significant main effect (A p < 0.05) since in the dark the animals had a lower latency of response on Days 5 and 6. A strong main effect of treatment was accounted for by the inhibition of exploratory behavior of MSH treated animals (B p < 0.01), especially in the dark (AB p < 0.01). The data indicate that shock on Day 2 inhibited all of the animals on Days 5 and 6 (C p < 0.01). Individual comparisons of results obtained from animals
125
G
c~ Z 0 u hi
I00
MSH
t
(.D Z
"I,,-'
75
P
_.!
1
Z
z t
50
rY
CONTROL
TABLE 1 LEASTSQUARESANALYSISOF VARIANCEOF RESULTSOBTAINEDON A PASSIVE AVOIDANCERESPONSE FROM RATS RECEIVING MSH AND CONTROL SOLUTIONS UNDER CONDITIONS OF LIGHT AND DARK (2 X 5 X 4 REPEATEDMEASURESDESIGN)
Source Between animals A (Light and dark) B (Treatment) AB (Light and dark x treatment) Animals within groups Within animals C (Days) AC (Light and dark x days) BC (Treatment x days) ABC (Light and dark x treatment × days) C x Animals within groups
Degrees of Freedom 69 1 4
Mean Square
F Ratio
p
13507.4 97118.5
4.32 31.04
0.05 0.01 0.01
4 60 210 3
29431.9 3128.6
9.41
78386.8
59.06
0.01
3 12
4738.0 20924.5
3.56 15.77
0.05 0.01
12
10795.5
195
1327.2
25
I
I
LIGHT
DARK
FIG. 1. Mean latency of response on a passive avoidance response (4- standard error) from combined groups of rats receiving MSH and control solutions on Day 5. TABLE 2 MEAN LATENCY OF RESPONSE ON A PASSIVE AVOIDANCE RESPONSE FROM COMBINED GROUPS OF RATS RECEIVING M S H AND CONTROL SOLUTIONS UNDER CONDITIONSOF LIGHT AND DARK
Day 1
8.13
0.01
MSH (n - 15) Control (n = 20)
15.8 9.8
MSH (n = 20) Control (n = 15)
9.9 10.1
2 Light 11.8 6.0
5
6
128.3 116,8*
t01.7 96.4*
102.7t 38.2
90,5t 33.4
Dark 9.7 11.6
*p < 0.01 when compared with same condition in dark. tP < 0.01 when compared with control group.
MSH AND BEHAVIOR
47 DISCUSSION
In an earlier study the role of memory, inhibition and motivation were suggested to account for the maintenance of an appetitive response in animals injected with MSH [22]. The present study clearly demonstrates that animals injected with MSH are capable of inhibiting a response when tested with a task specifically designed to determine inhibition. Administration of ACTH, which shares an active segment with MSH [10], causes a similar behavior on a P A R [25] as was found with MSH. Since the preparation of MSH used in this study contained no extrinsic ACTH, it is possible that the effects of A C T H on behavior [1, 5, 8, 25] are due to the intrinsic MSH activity of ACTH. A motivational hypothesis does not well explain the results observed in previous studies involving appetitive and active avoidance behavior [22]. The design of this study minimized the motivational factors and militated against the sporadic hyperactivity observed previously [22]. Thus, of the three possible explanations for the results observed with MSH on learning behavior, it appears that difficulty in inhibiting a response and motivation can be ruled out, leaving the role of memory to be directly tested. In addition to memory, another possibility to explain the behavioral results with MSH is emotionality. Animals injected with MSH may become more "emotional" in the stressful testing situation and revert to species specific defense reactions [6]. Accordingly, the animals may either flee or freeze depending upon which behavior is successful in reducing the stress. It is conceivable that in tasks such as active avoidance and appetitive behavior, in which activity is successful in reducing stress, a response may be perseverated. Similarly, the inhibition of activity is most efficient for the reduction of stress in the P A R situation and this response may be exaggerated in a highly emotional animal. The mechanisms by which animals become more emotional or by which memory may be implicated are not known. However, recent electroencephalographic data [23] suggest the limbic system, specifically the hippocampus, discharges following MSH injections. Additional studies of the effects of MSH on the central nervous system are currently in progress to further investigate hypothalamic and other limbic structure
involvement as well as the permeability of the blood-brain barrier to MSH. The second aspect of this study involves the role of periodicity with respect to learning tasks. The results indicate that in darkness, during the peak of activity, normal (control) animals are less capable of withholding a response than during the lowest level of activity in light. Noxious stimulation is more effective during the period of darkness [12, 14, 24, 27]. One explanation for these paradoxical findings for normal animals is that the tendency for the animal to inhibit a response is overwhelmed by high levels of activity. However, it has also been suggested that rats tend to explore stimuli which evoke fear [13]. This suggests that the expression of fear differs under light and dark conditions for normal animals but is undifferentiated for animals treated with MSH. The results obtained for normal animals in this study are opposite to those found for mice in what was essentially a P A R [24] but similar for rats in an open field study [4]. In addition, maze performance in mice [24] and earthworms [2] was influenced by phase of activity. This study demonstrates that under dark conditions administration of MSH to rats interferes with a normal response of a PAR. The exact mechanism of this action of MSH is not known. Since the effects of drugs and toxic agents are maximal during peak activity in rats [3, 14], it may be that the behavioral differences are a reflection of increased potency of MSH under conditions of light and dark. Interestingly, MSH is secreted by the pituitary under dark conditions and injections of MSH may be effective in inhibiting the pituitary release of MSH [16]. Therefore exogenous M S H may inhibit the release of endogenous MSH from the pituitary during the period of peak release. Consideration of circadian activity may serve to resolve controversies with respect to studies of learning and hormonal influence on behavior. The current debate on the influence of A C T H on fear-motivated behavior [5, 25] might be resolved by an explication of the conditions under which the animals were tested, especially with reference to phase of activity. Halberg's [11 ] claim that knowledge of the effects of circadian activity provides a refined index if evaluated and a confusing source of error if ignored is well illustrated in the present study with MSH. MSH is found to influence behavior involving memory or emotionality which intricately interacts with diurnal variation.
REFERENCES 1. Ader, R. Adrenocortical function and the measurement of emotionality. Ann. N . Y . Acad. Sci. 159: 791-805, 1969. 2. Arbit, J. Diurnal cycles and learning in earthworms. Science 126: 654-655, 1957. 3. Aschoof, J. Comparative physiology: diurnal rhythms. Ann. Rev. Physiol. 25: 581-600, 1963. 4. Battig, K. Drug effects on exploration of a combined maze and open field system by rats. Ann. N . Y . Acad. Sci. 159: 880-897, 1969. 5. Bohus, B., Sc. Nyakas and E. Endroczi. Effects of adrenocorticotropic hormones on avoidance behavior of intact and adrenalectomized rats. lnt. J. Neuropharm. 7: 307-314, 1968. 6. Bolles, R. C. Species-specific defense reactions and avoidance learning. PsychoL Rev. 77: 32-48, 1970. 7. DeWied, D. The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. lnt. J. Neuropharm. 4: 157-167, 1965.
8. DeWied, D. Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats. Proc. Soc. exp. Biol. Med. 122: 28-32, 1969. 9. DeWied, D. and B. Bohus. Long term and short term effects on retention of a conditioned avoidance response in rats by treatment with long acting pitressin and aMSH. Nature 213: 1484-1486, 1966. 10. Grevan, H. M. and D. DeWied. The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behavior in rats. Fur. J. Pharmac. 2: 14-16, 1967. 11. Halberg, F. The 24-hour scale: A time dimension of adaptive functional organization. Perspect. Biol. Med. 3: 491-527, 1960. 12. Halberg, F., E. Jacobsen, G. Wadsworth and J. J. Bittnea. Audiogenic abnormality spectra, twenty-four hour periodocity and lighting. Science 128: 657-658, 1958.
48 13. Halliday, M. S. Exploration and fear in the rat. Proc. Zool. Soc. Lond. 18: 45-59, 1966. 14. Haus, E. Periodicity in response and susceptibility to environmental stimuli. Ann. N.Y. Acad. Sci. 117: 292-315, 1964. 15. Kastin, A. J., S. Kullander, N. E. Borglin, B. Dahlberg, K. Dyster-Aas, C. E. T. Krakau, D. H. Ingvar, M. C. Miller, C. Y. Bowers and A. V. Schally. Extrapigmentary effects of melanocyte-stimulating hormone in amenorrhoeic women Lancet 1: 1007-1010, 1968. 16. Kastin, A. J., A. V. Schally, S. Viosca, L. Barrett and T. W. Redding. MSH activity in the pituitaries of rats exposed to constant illumination. Neuroendocrinology. 2: 257-262, 1967. 17. Kleinholz, L. H. Studies in the pigmentary system of crustacea. Biol. Bull. 72: 176-189, 1937. 18. Marler, P. R. and W. J. Hamilton. Mechanisms of Animal Behavior. New York: Wiley, 1967, pp. 25-72. 19. Rahn, H. and F. Rosendale. Diurnal rhythms of melanophore hormone secretion in the Anolis pituitary. Proc. Soc. exp. Biol. Med. 48: 100-102, 1941. 20. Reed, B. L. The control of circadian pigment changes in the pencil fish. Life Sci. 7: 961-973, 1968.
SANDMAN, KASTIN ANI) S('I:IAI~I '~ 21. Reinberg, A. and J. Ghata. Bh~logi~al Rh),thm~. New York: Walker, 1964, pp. 25 42. 22. Sandman, C., A. J. Kastin and A. V. Schally. Mehmocytestimulating hormone and learned appetitive behavior. Ea'perientia 25: 1001-1002, 1969. 23. Sandman, C. A., P. M. Denman, 1_. H. Miller, J. R. Knott. A. V. Schally and A. J. Kastin. Electroencephatographic indicants of melanocyte-stimulating horn]one activity, in preparation. 24. Stephens, G., J. L. McGaugh and H. P. Alpern. l'criodicity and memory in mice. Psychonom. Sci. 8: 201-202, 1967. 25. Weiss, J. M., B. S. McEwen, M. T. A. Silva and M. F. Kalbut. Pituitary-adrenal influences on fear responding. Sciettce 163: 197-199, 1969. 26. Winer, B. J. Statistical Principles in Experitnental Design. New York: McGraw-Hill, 1962. 27. Wolley, D. E. and P. S. Timeras. Estrous and circadian periodicity and electroshock convulsion in rats. Am. J. Pltysiol. 203: 379-382, 1962.