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Effects of GABA blockade on lateral hypothalamic self-stimulation
628
Bram Re,search, 107 (1976) 628 -632 ,(~ Elsewer Scientific Publishing Company, Amsterdam - Printed In The Netherlands
Effects of GABA blockade on lateral hypothalamic self-stimulation
ERNEST W. KENT AND PAUL FEDINETS Department of Psychology, Universtty of llhnois at Chicago, Chicago, 111.60680 (U.S.A.)
(Accepted February 4th, 1976)
Gamma-aminobutyric acid (GABA) has been identified in many brain structures, including several which are closely identified with the self-stimulation phenomenon. In particular, it has been reported in the lateral hypothalamus 3 and in the mgro-striatal system, where it has been suggested as a neurotransmitter in the output of the caudate to both the globus pallidus and the substantia nigra 1,4. Further, it has been suggested that the role of striato-nigral GABAergic fibers is to provide a feedback regulation of the dopaminergic neurons of the nigro-striatal system 5, which in turn have received attention as a possible substrate of self-stimulation. It seemed timely, therefore, to investigate the role of GABA in self-stimulation. As a first step in this investigation, we have studied the effects of two GABA blocking agents (picrotoxin and bicuculline) administered in various dosages by intra-peritoneal injection. Since it is clear that GABA blockade administered in this fashion might have many effects in addition to a direct effect on a self-stimulation 'reward' system, and that these effects might confound our results by interfering with the performance of the lever pressing response in any of a variety of ways, we also investigated the effects of these same drugs and dosages on an identical lever pressing task which offered a 1-sec escape from footshock as the reinforcer. The results of both of these studies are reported and discussed in this article. Subjects. Ten adult male Sprague-Dawley albino rats were employed as subjects. The animals were individually housed in an air conditioned windowless vivarium, and maintained on ad libitum Purina Lab Chow and water. Continuous illumination was provided to randomize diurnal activity cycles. Six subjects were prepared with hypothalamic stimulating electrodes as described below, and served as the self-stimulation group. The remaining subjects were employed in the shock-escape test. The six subjects of the self-stimulation group were implanted with four electrodes each in the lateral hypothalamus, two electrodes to a side. Electrodes consisted of varnish insulated stainless steel wire (62/am diameter), exposed at the cut tip. A stainless steel screw implanted in the frontal sinus served as the indifferent electrode. Electrodes were implanted with standard stereotaxic techniques under Nembutal anesthesia at coordinates: A 5.4, H - - 2.6, L 1.7 (De Groot system). One week was allowed for recovery.
629
Apparatus. Two identical operant conditioning boxes (LeHigh Valley) were employed. One was fitted with a flexible cable for delivering stimulation, and the other was fitted with a shock source (I mA constant current) delivered to the grid floor through a rotary shock scrambler in the form of 0.1 msec pulses. Administration of the shock was interrupted for an adjustable period of time by depression of the lever manipulandum, which was effective only after the onset of shock. In the self-stimulation box, each lever press was rewarded by a 250 msec train of 0.3-msec monophasic pulses delivered at 100 Hz through a capacitive coupling. The pulses were generated by a constant current device, and current was adjusted to produce the desired level of responding in each animal. The range of current intensities employed was from 60 to 200 #A. Lever pressing activityin both boxes was continuously monitored by a PDP-12 computer which cumulated responses in 30-sec time bins, converted the numbers to equivalent rates/min, and recorded, plotted and analyzed the results. Drugs. The drugs employed were picrotoxin (Sigma), and bicuculline (Regis). Picrotoxin was dissolved in the desired concentrations in sterile physiological saline, bicuculline was dissolved in 0.01 N HC1, titrated to pH 4.5 with NaOH, and diluted to the desired concentrations with physiological saline. Both vehicles were also employed alone as control substances. Drugs and control substances were administered by intra-peritoneal injection in 1 ml of vehicle. Dosages employed were: picrotoxin, 0.5, 1 or 2 mg/kg and bicuculline, 1, 2 or 4 mg/kg. All drugs were freshly prepared before injection. Training and testing. Training in the self-stimulation apparatus was carried out with conventional shaping techniques after determining the intensity at which the typical 'alerting' response to rewarding brain stimulation was obtained. After stable responding was developed, the animal's rate-intensity curve was determined, and a current intensity was selected that produced reliable responding at a rate midway between threshold and asymptotic rate. In the shock-escape apparatus, the animals were initially trained with a long (30 sec) escape from shock for each lever press. The length of this reward period was gradually shortened, until the animal was working reliably for a reward consisting of one second's interruption of the shock. The animals responded almost immediately, producing a lever pressing rate of nearly 60 responses/ min. In either apparatus, test trials were conducted by introducing the animal into the apparatus and allowing a 10-min 'warm up' period during which responding was not recorded. The animal's responding was then recorded for a baseline period of 20 min, following which the animal was briefly removed from the apparatus, injected and replaced. Responding was then recorded for a total period of 2 h. During all testing sessions, the animal was alone in a closed room. Behavior was continually monitored from the computer console by closed circuit television for signs of seizure or other aberrant activity. Tests of different drugs and dosages were administered in counterbalanced order. Self-stimulation test results. All animals demonstrated a dose-dependent depression of responding within a few minates of drug administration. This was followed by
63O A:
SELF- 6TIMULATION PICROTDXIN
BICUCULLINE
VEHICLE
SALINE IOO-
IOOR A
0-
0
T
I N
i 2 MG/ KG
I MG/KS
E "~ I00 a
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O-
0 4MG/KG
2MG/KG
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'1
2 HOURS
HOURS
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EBCAPE
P|CROTOXIN
R A H
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o
BICUCULLINE
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"1 HOURR
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Fig. 1. A: effects of veh]cle and two doses of p]crotoxm and bicuculline on responding for self-stimulation of lateral hypothalamus. Individual runs on the same animal are shown. B" individual runs of an animal responding on an identical lever pressing task for l-sec relief from footshock. Effects of the highest doses shown m part A are presented. a more gradual return to baseline rates, or, in some cases, to supra-baseline levels. This pattern of effect was shown in response to both drugs, although differences in time course and dose-effect curves were apparent. In the case o f picrotoxin, depression o f responding began approximately 5-10 min after injection, and developed to maximal effect within 20 min. The duration of the effect was variable; response rates began to rise within 30-40 min, in most cases, but full recovery was slow and frequently was not complete within 2 h. In the case of bicuculline, onset of the depression was much more rapid, usually occurring within 1 rain after injection, and reaching maximal effect within 3-5 min. On the other hand, the duration o f the depressive effect was shorter with this drug (15-20 min), and recovery was more rapid (full recovery was usually seen within 30 min). Moreover, recovery was frequently to slightly supra-baseline levels, and often showed a pronounced overshoot for a few minutes following resumption o f responding in those cases in which responding had completely ceased. Picrotoxin was about twice as effective as bicuculline in terms of dose-response measures. With picrotoxin, most animals showed no effect at doses of 0.5 mg/kg, a barely discernible to moderate depression at 1 mg/kg, and severe depression to complete elimination of responding at 2 mg/kg. Bicuculline, on the other hand, typically showed no effect at 1 mg/kg, mild to moderate effects at 2 mg/kg, and strong depres-
631 sion to elimination at 4 mg/kg. Some variation was seen in the effectiveness of different dosages of both drugs among subjects, but the effects were reliably replicable within repeated tests on the same subject, indicating slight individual differences in susceptibility to the drug. Representative examples of individual tests with both drugs on the same animal are shown for vehicle, low and high dosage conditions in Fig. 1A. Observation of the animals' behavior showed no evidence of seizure activity or other abnormal behavior. During periods of depressed responding, the animals explored the chamber, groomed or rested quietly. Even when responding was essentially eliminated, the animals occasionally returned to the lever, pressed once or twice and then moved away. Stimulation did not have any noticeable aversive consequences during the period of response suppression. The behavior of the animals during this period was indistinguishable from that of animals following a reduction of stimulation intensity to sub-threshold levels. Lever press escape test results. In this situation, very stable responding was maintained by all animals, and neither drug disrupted lever pressing, even at the highest doses employed. Only one difference in the animals' behavior was noted: undrugged animals invariably stood attentively by the lever with only infrequent excursions into the other parts of the chamber. For a period following drug administration, however, the animals were more prone to wander about, or in some cases lie down near the lever. Responding during these effects was also prompt and executed without apparent difficulty. The slightly greater response latencies incurred by greater distance from the lever at shock onset account for the small perturbations of response rate seen following drug administration. Representative examples of these results are shown for high doses of both drugs on individual tests of the same animal in Fig. 1B. These results demonstrate that two different GABA blocking agents have very similar inhibitory effects on responding for lateral hypothalamic self-stimulation. Moreover, the lack of effect of these drugs on the shock-escape reinforced lever pressing task demonstrates that this loss of lever pressing behavior cannot be ascribed to impairment of any sensory or motor abilities essential to performance of the lever pressing response. The behavior of the self-stimulating animals suggests that the stimulation had lost its 'rewarding' character; i.e., they returned to the lever at intervals for one or two 'test' presses but did not continue responding. The same pattern of behavior is seen in animals working for variable reinforcement during periods of low stimulus intensity, and during extinction. During the period of response elimination, stimulation administered by the experimenter retained its alerting and activating effects, but was ineffective in shaping or 'priming' lever pressing behavior. In this regard, the present effects are similar to those seen in loss of responding for lateral hypothalamic stimulation following parasagittal knife cuts at the medial border of the internal capsuleL The painful stimulus of footshock, on the other hand, clearly retained its punishing properties, and produced prompt and well executed responding. It is possible, however, that the tendency towards exploration or relaxed postures produced by the drugs may indicate an interference with fear, and the effects on avoidance responding require investigation.
632 The relatively brief (20-30 min) duration of the effect ~s puzzling in the case of the relatively long-acting drug blcuculline. Several features of the bicuculline response (return to slightly above baseline, greater resistance to fatigue) suggest the possiblhty that the inhibitory effect may be terminated by a subsequent facilitatory effect. With both picrotoxin and bicuculline it was noted that, even in the case of a low close which produced little or no depression of responding (see Fig. lA, l mg/kg picrotoxln and 2 mg/kg blcuculline), the fatigue effects on responding in the second hour which were frequently seen with vehicle injections were absent or reduced. Also, m the case of picrotoxin, it has been demonstrated (Kent and Fedinets, in preparation) that doses of only 0.05 #g refused into a discrete area (zona reticulata of the substantla mgra) can produce a behaviorally indistmgmshable depression of responding for lateral hypothalamic stimulation which persists for a much longer period.
1 KANAZAWA, I , AND TOYOKURA, Y., Quantitative hlstochemlstry of 7-ammobutyrJc aod (GABA) m the human substantia mgra and globus palhdus In Proc 6th Symp. Int. Soc. Res. Stereoencephalatomy, Tokyo, 1973, Part I, Confin Neurol. (Basel), 36 (1974) 273-281. 2 KENT, E., AND GROSgMAN, S., Ehmination of learned behaviors after transection of fibers crossing the lateral border of the hypothalamus, Physiol. Behav., l0 (1973) 953-963. 3 KIMURA,H., AND KUR1YAMA,K., Distribution o f gamma-ammobutyrlc acid (GABA) m the rat hypothalamus: functional correlates of GABA with activities of appetite controlling mechamsms, J. Neurochem., 24 (1975) 903-907. 4 YOSmDA,M., Functional aspects of, and role of transmitter m, the basal ganglia, Confin. Neurol. (Basel), 36 (1974) 282-291 5 YOSHIDA,M., AND PRECHT,W., Monosynaptic inhibition of neurons of the substantm mgra by caudato-nigral fibers, Brain Research, 32 0971) 225-228.
Bram Re,search, 107 (1976) 628 -632 ,(~ Elsewer Scientific Publishing Company, Amsterdam - Printed In The Netherlands
Effects of GABA blockade on lateral hypothalamic self-stimulation
ERNEST W. KENT AND PAUL FEDINETS Department of Psychology, Universtty of llhnois at Chicago, Chicago, 111.60680 (U.S.A.)
(Accepted February 4th, 1976)
Gamma-aminobutyric acid (GABA) has been identified in many brain structures, including several which are closely identified with the self-stimulation phenomenon. In particular, it has been reported in the lateral hypothalamus 3 and in the mgro-striatal system, where it has been suggested as a neurotransmitter in the output of the caudate to both the globus pallidus and the substantia nigra 1,4. Further, it has been suggested that the role of striato-nigral GABAergic fibers is to provide a feedback regulation of the dopaminergic neurons of the nigro-striatal system 5, which in turn have received attention as a possible substrate of self-stimulation. It seemed timely, therefore, to investigate the role of GABA in self-stimulation. As a first step in this investigation, we have studied the effects of two GABA blocking agents (picrotoxin and bicuculline) administered in various dosages by intra-peritoneal injection. Since it is clear that GABA blockade administered in this fashion might have many effects in addition to a direct effect on a self-stimulation 'reward' system, and that these effects might confound our results by interfering with the performance of the lever pressing response in any of a variety of ways, we also investigated the effects of these same drugs and dosages on an identical lever pressing task which offered a 1-sec escape from footshock as the reinforcer. The results of both of these studies are reported and discussed in this article. Subjects. Ten adult male Sprague-Dawley albino rats were employed as subjects. The animals were individually housed in an air conditioned windowless vivarium, and maintained on ad libitum Purina Lab Chow and water. Continuous illumination was provided to randomize diurnal activity cycles. Six subjects were prepared with hypothalamic stimulating electrodes as described below, and served as the self-stimulation group. The remaining subjects were employed in the shock-escape test. The six subjects of the self-stimulation group were implanted with four electrodes each in the lateral hypothalamus, two electrodes to a side. Electrodes consisted of varnish insulated stainless steel wire (62/am diameter), exposed at the cut tip. A stainless steel screw implanted in the frontal sinus served as the indifferent electrode. Electrodes were implanted with standard stereotaxic techniques under Nembutal anesthesia at coordinates: A 5.4, H - - 2.6, L 1.7 (De Groot system). One week was allowed for recovery.
629
Apparatus. Two identical operant conditioning boxes (LeHigh Valley) were employed. One was fitted with a flexible cable for delivering stimulation, and the other was fitted with a shock source (I mA constant current) delivered to the grid floor through a rotary shock scrambler in the form of 0.1 msec pulses. Administration of the shock was interrupted for an adjustable period of time by depression of the lever manipulandum, which was effective only after the onset of shock. In the self-stimulation box, each lever press was rewarded by a 250 msec train of 0.3-msec monophasic pulses delivered at 100 Hz through a capacitive coupling. The pulses were generated by a constant current device, and current was adjusted to produce the desired level of responding in each animal. The range of current intensities employed was from 60 to 200 #A. Lever pressing activityin both boxes was continuously monitored by a PDP-12 computer which cumulated responses in 30-sec time bins, converted the numbers to equivalent rates/min, and recorded, plotted and analyzed the results. Drugs. The drugs employed were picrotoxin (Sigma), and bicuculline (Regis). Picrotoxin was dissolved in the desired concentrations in sterile physiological saline, bicuculline was dissolved in 0.01 N HC1, titrated to pH 4.5 with NaOH, and diluted to the desired concentrations with physiological saline. Both vehicles were also employed alone as control substances. Drugs and control substances were administered by intra-peritoneal injection in 1 ml of vehicle. Dosages employed were: picrotoxin, 0.5, 1 or 2 mg/kg and bicuculline, 1, 2 or 4 mg/kg. All drugs were freshly prepared before injection. Training and testing. Training in the self-stimulation apparatus was carried out with conventional shaping techniques after determining the intensity at which the typical 'alerting' response to rewarding brain stimulation was obtained. After stable responding was developed, the animal's rate-intensity curve was determined, and a current intensity was selected that produced reliable responding at a rate midway between threshold and asymptotic rate. In the shock-escape apparatus, the animals were initially trained with a long (30 sec) escape from shock for each lever press. The length of this reward period was gradually shortened, until the animal was working reliably for a reward consisting of one second's interruption of the shock. The animals responded almost immediately, producing a lever pressing rate of nearly 60 responses/ min. In either apparatus, test trials were conducted by introducing the animal into the apparatus and allowing a 10-min 'warm up' period during which responding was not recorded. The animal's responding was then recorded for a baseline period of 20 min, following which the animal was briefly removed from the apparatus, injected and replaced. Responding was then recorded for a total period of 2 h. During all testing sessions, the animal was alone in a closed room. Behavior was continually monitored from the computer console by closed circuit television for signs of seizure or other aberrant activity. Tests of different drugs and dosages were administered in counterbalanced order. Self-stimulation test results. All animals demonstrated a dose-dependent depression of responding within a few minates of drug administration. This was followed by
63O A:
SELF- 6TIMULATION PICROTDXIN
BICUCULLINE
VEHICLE
SALINE IOO-
IOOR A
0-
0
T
I N
i 2 MG/ KG
I MG/KS
E "~ I00 a
Ioo--
O-
0 4MG/KG
2MG/KG
I00-
tOC~
0
O~INJ.
'1
2 HOURS
HOURS
B: l i H O C K
EBCAPE
P|CROTOXIN
R A H
ioo
g
o
BICUCULLINE
io i o
~lgJ.
"1 HOURR
'2
0
~ltJ,
'1 HOURI=
Fig. 1. A: effects of veh]cle and two doses of p]crotoxm and bicuculline on responding for self-stimulation of lateral hypothalamus. Individual runs on the same animal are shown. B" individual runs of an animal responding on an identical lever pressing task for l-sec relief from footshock. Effects of the highest doses shown m part A are presented. a more gradual return to baseline rates, or, in some cases, to supra-baseline levels. This pattern of effect was shown in response to both drugs, although differences in time course and dose-effect curves were apparent. In the case o f picrotoxin, depression o f responding began approximately 5-10 min after injection, and developed to maximal effect within 20 min. The duration of the effect was variable; response rates began to rise within 30-40 min, in most cases, but full recovery was slow and frequently was not complete within 2 h. In the case of bicuculline, onset of the depression was much more rapid, usually occurring within 1 rain after injection, and reaching maximal effect within 3-5 min. On the other hand, the duration o f the depressive effect was shorter with this drug (15-20 min), and recovery was more rapid (full recovery was usually seen within 30 min). Moreover, recovery was frequently to slightly supra-baseline levels, and often showed a pronounced overshoot for a few minutes following resumption o f responding in those cases in which responding had completely ceased. Picrotoxin was about twice as effective as bicuculline in terms of dose-response measures. With picrotoxin, most animals showed no effect at doses of 0.5 mg/kg, a barely discernible to moderate depression at 1 mg/kg, and severe depression to complete elimination of responding at 2 mg/kg. Bicuculline, on the other hand, typically showed no effect at 1 mg/kg, mild to moderate effects at 2 mg/kg, and strong depres-
631 sion to elimination at 4 mg/kg. Some variation was seen in the effectiveness of different dosages of both drugs among subjects, but the effects were reliably replicable within repeated tests on the same subject, indicating slight individual differences in susceptibility to the drug. Representative examples of individual tests with both drugs on the same animal are shown for vehicle, low and high dosage conditions in Fig. 1A. Observation of the animals' behavior showed no evidence of seizure activity or other abnormal behavior. During periods of depressed responding, the animals explored the chamber, groomed or rested quietly. Even when responding was essentially eliminated, the animals occasionally returned to the lever, pressed once or twice and then moved away. Stimulation did not have any noticeable aversive consequences during the period of response suppression. The behavior of the animals during this period was indistinguishable from that of animals following a reduction of stimulation intensity to sub-threshold levels. Lever press escape test results. In this situation, very stable responding was maintained by all animals, and neither drug disrupted lever pressing, even at the highest doses employed. Only one difference in the animals' behavior was noted: undrugged animals invariably stood attentively by the lever with only infrequent excursions into the other parts of the chamber. For a period following drug administration, however, the animals were more prone to wander about, or in some cases lie down near the lever. Responding during these effects was also prompt and executed without apparent difficulty. The slightly greater response latencies incurred by greater distance from the lever at shock onset account for the small perturbations of response rate seen following drug administration. Representative examples of these results are shown for high doses of both drugs on individual tests of the same animal in Fig. 1B. These results demonstrate that two different GABA blocking agents have very similar inhibitory effects on responding for lateral hypothalamic self-stimulation. Moreover, the lack of effect of these drugs on the shock-escape reinforced lever pressing task demonstrates that this loss of lever pressing behavior cannot be ascribed to impairment of any sensory or motor abilities essential to performance of the lever pressing response. The behavior of the self-stimulating animals suggests that the stimulation had lost its 'rewarding' character; i.e., they returned to the lever at intervals for one or two 'test' presses but did not continue responding. The same pattern of behavior is seen in animals working for variable reinforcement during periods of low stimulus intensity, and during extinction. During the period of response elimination, stimulation administered by the experimenter retained its alerting and activating effects, but was ineffective in shaping or 'priming' lever pressing behavior. In this regard, the present effects are similar to those seen in loss of responding for lateral hypothalamic stimulation following parasagittal knife cuts at the medial border of the internal capsuleL The painful stimulus of footshock, on the other hand, clearly retained its punishing properties, and produced prompt and well executed responding. It is possible, however, that the tendency towards exploration or relaxed postures produced by the drugs may indicate an interference with fear, and the effects on avoidance responding require investigation.
632 The relatively brief (20-30 min) duration of the effect ~s puzzling in the case of the relatively long-acting drug blcuculline. Several features of the bicuculline response (return to slightly above baseline, greater resistance to fatigue) suggest the possiblhty that the inhibitory effect may be terminated by a subsequent facilitatory effect. With both picrotoxin and bicuculline it was noted that, even in the case of a low close which produced little or no depression of responding (see Fig. lA, l mg/kg picrotoxln and 2 mg/kg blcuculline), the fatigue effects on responding in the second hour which were frequently seen with vehicle injections were absent or reduced. Also, m the case of picrotoxin, it has been demonstrated (Kent and Fedinets, in preparation) that doses of only 0.05 #g refused into a discrete area (zona reticulata of the substantla mgra) can produce a behaviorally indistmgmshable depression of responding for lateral hypothalamic stimulation which persists for a much longer period.
1 KANAZAWA, I , AND TOYOKURA, Y., Quantitative hlstochemlstry of 7-ammobutyrJc aod (GABA) m the human substantia mgra and globus palhdus In Proc 6th Symp. Int. Soc. Res. Stereoencephalatomy, Tokyo, 1973, Part I, Confin Neurol. (Basel), 36 (1974) 273-281. 2 KENT, E., AND GROSgMAN, S., Ehmination of learned behaviors after transection of fibers crossing the lateral border of the hypothalamus, Physiol. Behav., l0 (1973) 953-963. 3 KIMURA,H., AND KUR1YAMA,K., Distribution o f gamma-ammobutyrlc acid (GABA) m the rat hypothalamus: functional correlates of GABA with activities of appetite controlling mechamsms, J. Neurochem., 24 (1975) 903-907. 4 YOSmDA,M., Functional aspects of, and role of transmitter m, the basal ganglia, Confin. Neurol. (Basel), 36 (1974) 282-291 5 YOSHIDA,M., AND PRECHT,W., Monosynaptic inhibition of neurons of the substantm mgra by caudato-nigral fibers, Brain Research, 32 0971) 225-228.