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P-chlorophenylalanine effects on a conditioned emotional response in rats
Lüe Sciences Vol. 10, Part I, pp. 927-933, 1971. Printed in Great Britain
Pergamon Press
P{HLOROPHENYLALANINE EFFECTS ON A CONDITIONED EMOTIONAL RESPONSE IN RATS Roy J . Hartmann and Irving Geller Department of Experimental Pharmacology Southwest Foundation for Research and Education, San Antonio, Texo:
(Received 1 June 1971; in final form 6 July 1971) Sunmary . Hungry rats learned to press a lever for a liquid food reward on a 2~ninute variable-interval schedule of reinforcement . Lever pressing was suppressed in the presence of a tone stimulus by pairing the tone with brief electric shocks . p{hlorophenylalanine, the tryptophan hydroxylase inhibitor, produced an attenuation of the conditioned suppression . The effect was reversed in some animals by administration of 5-hydroxytryptophan . p{hlor+ophenylalanine (p{PA) depletes brain serotonin, presumably by interruption of the formation of 5-hydroxytryptophan (5-HTP), the serotonin precursor (1) .
Previous
studies have shown that p{PA abolishes a conditioned suppression of operont behavior which is established by punishing with electric shocks the lever responses of hungry rats (2) . Administration of 5-HTP reverses the effects of the p-CPA (3) . In this study, a conditioned suppression of the ° fear" or "anxiety" type (conditioned emotional response or CER) was eaablished in laboratory rats (4) . This was accomplished by repeated presentations of a tone stimulus which was terminated regularly with o brief elec tric shock . Administration of p{PA abolished the conditioned suppression, an effect that was reversed in some animals following injections of 5-HTP . Materials and Methods Ten male Sprague Dawley rats, approximately 90 days old, served as subjects .
They
were gradually reduced to 80% of their original starting body weight and maintained at this weight by limited feeding : after each experimental session . The procedure for establishing the CER has been described (~ .
927
The rats were given
92 8
Effect of p-Chlorophenylalanine on Behavior
Vol. 10, No . 16
training in small sound-resistant cubicles which contained a lever in the front wall, an automatic feeding device for the delivery of a liquid food reward, a small speaker for the presentation of auditory stimuli and a grid floor for the delivery of electric shocks .
Experimen-
tal sessions of 30 minutes duration were conducted on Monday through Friday of each week . The rats, 23 1~2 hours food deprived, learned to press a lever for a liquid food reward which was obtainable on a 2-minute variable-interval schedule of reinforcement (6) or once every two minutes on the average (2~ninute VI) .
When the lever-pressing rates became relatively
stable, a 3-minute tone stimulus was delivered into the experimental chamber 12 minutes after the start of the lever-pressing session .
Termination of the tone was accompanied by a
.25 sec shock of 40 volts (Scientific Prorotype Shock Generator S .P . 4008 J) delivered to the animal's feet through the grid floor of the apparatus . The suppression of lever pressing which developed gradually over a number of trials was expressed as a suppression ratio(S .R .) . Suppression ratios were calculated by dividing the number of responses during the stimulus period by the number of responses during the 3~ninute pre-stimulus period . A value of 1 .00 or more indicated no suppression, while a value of zero represented complete sup pression . Intermediate degrees of suppression were expressed by values from zero to 1 .00 . When a suppression ratio criterion of .10 or less was attained, the drug phase of the experiment was begun . Drug Administration The rats were given p{PA, 5-HTP, or p{PA plus 5-HTP in a mixed order so that the sequence of administrations differed for each subject . All doses were calculated as mg base/kg .
At least one month elapsed between each drug injection .
Control administra-
tions of 0 .5% carboxymethylcellulose (CMC) were also given . The dl form of p{PA was given orally as a 0 .59'o CMC suspension on the evening prior ro an experimental session . All rats received p{PA at 320 mg~kg except for Rat 3 who was given 400 mg/kg since the lower dose was ineffective in this animal .
The dl form
Vol . 10, No. 18
EYfect a~ p-Chloropheirylalanine on Behavior
929
of 5-HTP was administered intraperitoneally as a saline solution 2 to 6 hours prior to on experimental session in doses ranging from 15 to 25 mg/kg . The 15 to 25 mg/1cg doses were selected since in a previous study (3) this dose range was shown to reverse p-CPA effects . When 5-HTP was given in conjunction with p-CPA, the same time sequences and routes of administration were employed . Resu I is Data for a representative rat are shown in the cumulative records of Figure 1 .
The
pen offsets show the tone periods and the numbers represent suppression ratios . The control record with a suppression value of zero shows a complete absence of responding during the
RAT P-4
44 Hours After PCPA and 26 Hours After 5-HTP
CONTROL
68 Hours After PCPA and 50 Hours After 5-HTP
20 Hours After PCPA 320 mg/kg 22 Hours After PCPA 320 mg/kg and 2 .0 Hours After 5-HTP IS mg/ky
92 Hours After PCPA and 74 Hours After 5-HTP
FIG . 1 Cumulative response records showing effects of p-chlorophenylalanine and 5-hydroxytryptophan on a conditioned emotional response . The pen offsets indicate tone periods . The numbers show the suppression ratios for each trial .
93 0
Effect of p-Chlorophenylalanine on Behavior
tone period .
Vol. 10, No . 16
Twenty hours after p-CPA was given, the suppression was attenuated as is
indicated by the S .R . value of .40 . The subject was removed from the experimental chamber and injected with 15 mg/kg 5-HTP . The next trial, which occurred 2 hours post-S-HTP, showed a reinstatement of the suppression to a value of 0 .17 .
The 5-HTP reversal effect was
still evident 26 hours post-S-HTP when the S .R . value was 0 .27 . At 50 hours post-S-HTP and 68 hours post-p-CPA, the effects of the 5-HTP had dissipated completely andthe p{PA effect reached a maximum suppression ratio value of 0 .85 .
Ninety-two hours after p-CPA,
the suppression was reestablished almost completely, indicating that p-CPA effects had dissipated as well . In Table 1 are shown the suppression ratio values for all rats under the different treatment conditions .
Rat 3 received p{PA at 400 mg/kg, while the dose used for all
other rats was 320 mg/kg .
The 5-HTP dose was 25 mg/kg for the first six rats tested, 15
mg/kg for the next two, and 20 mg/lcg for the last two .
The times at which trials occurred
following drug administrations are shown in parentheses for p{PA and in brackets for 5-HTP . After p{PA, the conditioned suppression was attenuated for all rats, although at different time intervals post-injection .
Following 5-HTP, the conditioned suppression was reinstated
in nine animals in varying degrees .
For Rats 4, 7 and 8, p{PA effects were again evident
after 5-HTP effects had dissipated . Suppression ratio values approached control levels 116 hours post-p{PA for all rats with the exception of Rat 7, whose control level was attained 213 hours poster{PA . CMC or 5-HTP, when administered alone, had no effect on the conditioned suppression . Discussion The findings of this experiment, showing a p{PA-induced attenuation of a conditioned suppression of the CER type, are similar to those previously reported for a conditioned suppression maintained by punishment (3) .
Similarly, Wise et al . (7) found that p-CPA
Vol. 10, No, 18
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Effect of p-Chlorophenylalanine on Behavior
Vol. 10, No . 16
decreased the latency of a rat's licking response that had been suppressed previously with electric shock . A suggestion by Miller and Maickel (8) provides the basis for a possible interpretation to account for these data . They reported that the balance of free serotonin (5-HT) and free norepinephrine (NE) is related to abnormal behavior . They found that a depression of avoid ance behavior occurs when free 5-HTL free NE but not when free NE
free 5-HT . Other
investigators have shown. that emotional stress in animals which is induced by electric shock, produces a significant drop in brain NE but no effect on concentrations of serotonin . The conditioned suppression of lever pressing described in this experiment might refleet a stress-induced lowering of NE, resulting in free 5-HT ~ free NE . Restoration of the balance through p{PA administration, and presumably serotonin depletion, could account for the attenuation of the conditioned wppression . Further wpport for this speculation derives from the data of Rats 4, 7 and 8 since administration of 5-HTP reestablished the conditioned suppression in these animals. When the effects of the 5-HTP had worn off, the p-CPA effects were reinstated, thereby indicating that the conditioned wppression probably is related to levels of 5-HT . Acknowledgements This work was submitted by R .H . in partial fulfillment of requiranents for theM .S . degree and presented in part at the ASPET Fall Meetings, Palo Alto, California, 1970 . The authors are indebted to Dr . A . Weissman of Pfizer Laboratories for the generous supply of p{PA . This research was wpported by USPHS Grant MH 16430 and a grant from the Tom Slick Estate . Keferences 1.
B .K . KOE and A . WEISSMAN, J . Pharmacol . Exptl . Therap . 154, 499 (1966) .
2.
R, ROBICHAUD and K . SLEDGE, Life Sci . 8, 965 (1969) .
3.
I . GELLER and K . BLUM, Europ . J . Pharmacol . 9, 319 (1970) .
VoI. 10, No . 18
Effect of p-Chloropherrylalanine on Behavior
4.
H .F . HUNT and J .V . BRADY, J . Comp . Physiol . Psychol . 44, 88 (1951) .
5.
W .K . ESTES and B . F . SKINNER, J . Exptl . Psychol . 29, 390 (1941) .
6.
C . FERSTER and B . F . SKINNER, Schedules of Reinforcement, Appleton{entury-
9S3
Crofts, New York (1957) . 7.
C . D . WISE, B .D . BERGER and L . STEIN, Proceedings, 78th Annual Convention APA, 821 (1970) .
8.
F .P . MILLER and R . P, MAICKEL, Life Sci . 8, 487 (1969) .
Pergamon Press
P{HLOROPHENYLALANINE EFFECTS ON A CONDITIONED EMOTIONAL RESPONSE IN RATS Roy J . Hartmann and Irving Geller Department of Experimental Pharmacology Southwest Foundation for Research and Education, San Antonio, Texo:
(Received 1 June 1971; in final form 6 July 1971) Sunmary . Hungry rats learned to press a lever for a liquid food reward on a 2~ninute variable-interval schedule of reinforcement . Lever pressing was suppressed in the presence of a tone stimulus by pairing the tone with brief electric shocks . p{hlorophenylalanine, the tryptophan hydroxylase inhibitor, produced an attenuation of the conditioned suppression . The effect was reversed in some animals by administration of 5-hydroxytryptophan . p{hlor+ophenylalanine (p{PA) depletes brain serotonin, presumably by interruption of the formation of 5-hydroxytryptophan (5-HTP), the serotonin precursor (1) .
Previous
studies have shown that p{PA abolishes a conditioned suppression of operont behavior which is established by punishing with electric shocks the lever responses of hungry rats (2) . Administration of 5-HTP reverses the effects of the p-CPA (3) . In this study, a conditioned suppression of the ° fear" or "anxiety" type (conditioned emotional response or CER) was eaablished in laboratory rats (4) . This was accomplished by repeated presentations of a tone stimulus which was terminated regularly with o brief elec tric shock . Administration of p{PA abolished the conditioned suppression, an effect that was reversed in some animals following injections of 5-HTP . Materials and Methods Ten male Sprague Dawley rats, approximately 90 days old, served as subjects .
They
were gradually reduced to 80% of their original starting body weight and maintained at this weight by limited feeding : after each experimental session . The procedure for establishing the CER has been described (~ .
927
The rats were given
92 8
Effect of p-Chlorophenylalanine on Behavior
Vol. 10, No . 16
training in small sound-resistant cubicles which contained a lever in the front wall, an automatic feeding device for the delivery of a liquid food reward, a small speaker for the presentation of auditory stimuli and a grid floor for the delivery of electric shocks .
Experimen-
tal sessions of 30 minutes duration were conducted on Monday through Friday of each week . The rats, 23 1~2 hours food deprived, learned to press a lever for a liquid food reward which was obtainable on a 2-minute variable-interval schedule of reinforcement (6) or once every two minutes on the average (2~ninute VI) .
When the lever-pressing rates became relatively
stable, a 3-minute tone stimulus was delivered into the experimental chamber 12 minutes after the start of the lever-pressing session .
Termination of the tone was accompanied by a
.25 sec shock of 40 volts (Scientific Prorotype Shock Generator S .P . 4008 J) delivered to the animal's feet through the grid floor of the apparatus . The suppression of lever pressing which developed gradually over a number of trials was expressed as a suppression ratio(S .R .) . Suppression ratios were calculated by dividing the number of responses during the stimulus period by the number of responses during the 3~ninute pre-stimulus period . A value of 1 .00 or more indicated no suppression, while a value of zero represented complete sup pression . Intermediate degrees of suppression were expressed by values from zero to 1 .00 . When a suppression ratio criterion of .10 or less was attained, the drug phase of the experiment was begun . Drug Administration The rats were given p{PA, 5-HTP, or p{PA plus 5-HTP in a mixed order so that the sequence of administrations differed for each subject . All doses were calculated as mg base/kg .
At least one month elapsed between each drug injection .
Control administra-
tions of 0 .5% carboxymethylcellulose (CMC) were also given . The dl form of p{PA was given orally as a 0 .59'o CMC suspension on the evening prior ro an experimental session . All rats received p{PA at 320 mg~kg except for Rat 3 who was given 400 mg/kg since the lower dose was ineffective in this animal .
The dl form
Vol . 10, No. 18
EYfect a~ p-Chloropheirylalanine on Behavior
929
of 5-HTP was administered intraperitoneally as a saline solution 2 to 6 hours prior to on experimental session in doses ranging from 15 to 25 mg/kg . The 15 to 25 mg/1cg doses were selected since in a previous study (3) this dose range was shown to reverse p-CPA effects . When 5-HTP was given in conjunction with p-CPA, the same time sequences and routes of administration were employed . Resu I is Data for a representative rat are shown in the cumulative records of Figure 1 .
The
pen offsets show the tone periods and the numbers represent suppression ratios . The control record with a suppression value of zero shows a complete absence of responding during the
RAT P-4
44 Hours After PCPA and 26 Hours After 5-HTP
CONTROL
68 Hours After PCPA and 50 Hours After 5-HTP
20 Hours After PCPA 320 mg/kg 22 Hours After PCPA 320 mg/kg and 2 .0 Hours After 5-HTP IS mg/ky
92 Hours After PCPA and 74 Hours After 5-HTP
FIG . 1 Cumulative response records showing effects of p-chlorophenylalanine and 5-hydroxytryptophan on a conditioned emotional response . The pen offsets indicate tone periods . The numbers show the suppression ratios for each trial .
93 0
Effect of p-Chlorophenylalanine on Behavior
tone period .
Vol. 10, No . 16
Twenty hours after p-CPA was given, the suppression was attenuated as is
indicated by the S .R . value of .40 . The subject was removed from the experimental chamber and injected with 15 mg/kg 5-HTP . The next trial, which occurred 2 hours post-S-HTP, showed a reinstatement of the suppression to a value of 0 .17 .
The 5-HTP reversal effect was
still evident 26 hours post-S-HTP when the S .R . value was 0 .27 . At 50 hours post-S-HTP and 68 hours post-p-CPA, the effects of the 5-HTP had dissipated completely andthe p{PA effect reached a maximum suppression ratio value of 0 .85 .
Ninety-two hours after p-CPA,
the suppression was reestablished almost completely, indicating that p-CPA effects had dissipated as well . In Table 1 are shown the suppression ratio values for all rats under the different treatment conditions .
Rat 3 received p{PA at 400 mg/kg, while the dose used for all
other rats was 320 mg/kg .
The 5-HTP dose was 25 mg/kg for the first six rats tested, 15
mg/kg for the next two, and 20 mg/lcg for the last two .
The times at which trials occurred
following drug administrations are shown in parentheses for p{PA and in brackets for 5-HTP . After p{PA, the conditioned suppression was attenuated for all rats, although at different time intervals post-injection .
Following 5-HTP, the conditioned suppression was reinstated
in nine animals in varying degrees .
For Rats 4, 7 and 8, p{PA effects were again evident
after 5-HTP effects had dissipated . Suppression ratio values approached control levels 116 hours post-p{PA for all rats with the exception of Rat 7, whose control level was attained 213 hours poster{PA . CMC or 5-HTP, when administered alone, had no effect on the conditioned suppression . Discussion The findings of this experiment, showing a p{PA-induced attenuation of a conditioned suppression of the CER type, are similar to those previously reported for a conditioned suppression maintained by punishment (3) .
Similarly, Wise et al . (7) found that p-CPA
Vol. 10, No, 18
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Effect of p-Chlorophenylalanine on Behavior
Vol. 10, No . 16
decreased the latency of a rat's licking response that had been suppressed previously with electric shock . A suggestion by Miller and Maickel (8) provides the basis for a possible interpretation to account for these data . They reported that the balance of free serotonin (5-HT) and free norepinephrine (NE) is related to abnormal behavior . They found that a depression of avoid ance behavior occurs when free 5-HTL free NE but not when free NE
free 5-HT . Other
investigators have shown. that emotional stress in animals which is induced by electric shock, produces a significant drop in brain NE but no effect on concentrations of serotonin . The conditioned suppression of lever pressing described in this experiment might refleet a stress-induced lowering of NE, resulting in free 5-HT ~ free NE . Restoration of the balance through p{PA administration, and presumably serotonin depletion, could account for the attenuation of the conditioned wppression . Further wpport for this speculation derives from the data of Rats 4, 7 and 8 since administration of 5-HTP reestablished the conditioned suppression in these animals. When the effects of the 5-HTP had worn off, the p-CPA effects were reinstated, thereby indicating that the conditioned wppression probably is related to levels of 5-HT . Acknowledgements This work was submitted by R .H . in partial fulfillment of requiranents for theM .S . degree and presented in part at the ASPET Fall Meetings, Palo Alto, California, 1970 . The authors are indebted to Dr . A . Weissman of Pfizer Laboratories for the generous supply of p{PA . This research was wpported by USPHS Grant MH 16430 and a grant from the Tom Slick Estate . Keferences 1.
B .K . KOE and A . WEISSMAN, J . Pharmacol . Exptl . Therap . 154, 499 (1966) .
2.
R, ROBICHAUD and K . SLEDGE, Life Sci . 8, 965 (1969) .
3.
I . GELLER and K . BLUM, Europ . J . Pharmacol . 9, 319 (1970) .
VoI. 10, No . 18
Effect of p-Chloropherrylalanine on Behavior
4.
H .F . HUNT and J .V . BRADY, J . Comp . Physiol . Psychol . 44, 88 (1951) .
5.
W .K . ESTES and B . F . SKINNER, J . Exptl . Psychol . 29, 390 (1941) .
6.
C . FERSTER and B . F . SKINNER, Schedules of Reinforcement, Appleton{entury-
9S3
Crofts, New York (1957) . 7.
C . D . WISE, B .D . BERGER and L . STEIN, Proceedings, 78th Annual Convention APA, 821 (1970) .
8.
F .P . MILLER and R . P, MAICKEL, Life Sci . 8, 487 (1969) .