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Modulation of intracranial reward-punishment interaction by scopolamine
Physiology & Behavior, Vol. 23, pp. 223--228. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Modulation of Intracranial Reward-Punishment Interaction by Scopolamine D. L. H O F F M A N A N D J. A. T R O W I L L
The University of Denver, Department of Psychology, Denver, CO Received 5 July 1978 HOFFMAN, D. L. AND J. A. TROWlLL. Modulation of intracranial reward-punishment interaction by scopolamine. PHYSIOL. BEHAV. 23(2)223--228, 1979.--Rats were tested in a passive avoidance paradigm involving the alternation of punished and unpunished schedules of reinforcement. The rate of self-stimulating for 60 Hz sine wave current (15-130 p.a) to the lateral hypothalamus (LH) was lowered to 18% and 20% when response contingent aversive electrical stimulation to the medial hypothalamus (MH) (15--60 /za) and dorsal midbrain (DM) (25-90 p,a), respectively, was introduced. Scopolamine (1 mg/kg, IP) was found to significantly disinhibit the suppression of responding produced by the MH stimulation to 67%, but had no significant effect on suppression of responding produced by DM stimulation. Since approximately 50% of the placements in the MH were found to be positively reinforcing, a group of rats was run such that the rate of self-stimulation to the LH was increased to at least 130%when the positive MH stimulation was made contingent upon LH self-stimulation. Scopolamine had no effect on this response enhancement. The passive avoidance results demonstrating scopolamine's disinhibitory effect on suppression produced by MH stimulation support the hypothesis that cholinergic synapses within the MH form part of a periventricular system mediating the effects of punishment on "behavior. Self-stimulation
Passiveavoidance
Scopolamine
SEVERAL investigators have dealt with the competing effects of reward and punishment upon behavior, and the effects of drugs on this competition. Punishment, which occurs when a response produces an aversive stimulus, has the general effect of suppressing the response which produced the aversive stimulus. Miller [12] dealt with approach avoidance conflict between food rewards and punishing foot shock, such that two incompatible response tendencies occurred simultaneously, i.e., approaching the food and avoiding the shock. Food reward could not be obtained without receiving shocks, and thus approach behavior was suppressed. In several different paradigms, the conflict reducing effects of meprobamate and amobarbital were demonstrated. Geller and Seifter [7] also examined the effects of drugs upon a food shock conflict using a passive avoidance paradigm. In this paradigm, an unpunished schedule of reinforcement in which responding was rewarded with milk, was alternated with a punished schedule in which each response produced both a milk reward and a foot shock punisher. Responding was suppressed during the punished schedule. Drugs such as meprobamate and barbiturates were found to restore responding that had been suppressed by punishment, while amphetamine and promazine were found to further suppress responding during the punished interval. Margales and Stein [7], using a similar passive avoidance paradigm, examined the effects of direct MH chemical stimulation on shock induced suppression of responding for milk rewards. Cholinergic agonists such as carbachol, physostigmine, and musearine were found to enhance the suppressant effects of punishment, while cholinergic antagonists such as atropine and scopolamine had a disinhibitory effect
Aversivestimulation
on the suppressed behavior. They thus suggested that the MH forms part of a periventricular system involved in the mediation of behavioral suppression by punishment. Their findings stressed the importance of cholinergic synapses in the MH for the mediation of the behavioral suppression. Several investigators [4, 11, 15] have adopted the view that a cholinergic pathway suppressed behavior by antagonizing an adrenergic system for activation. According to this view, anticholinergics disinhibit behavior that has been suppressed by habituation, non-reward, and punishment by releasing an adrenergic system from inhibition. An increase in rate of LH self-stimulation has been demonstrated with anticholinergics in rats [13], as well as a dramatic decrease in self-stimulation rate with cholinergic agonists in rats [8] and dogs [20]. This rate decrease with cholinergic agonists has also been shown to be antagonized by scopolamine [15]. A decrement in LH self-stimulation with direct application of the cholinergic agonist carbachol to the DM has also been demonstrated in rats [18]. Thus evidence for a central cholinergic system involved in behavioral inhibition has been accumulating. The present study focuses upon the effects of the anticholinergic scopolamine on the punishment induced suppression of LH self-stimulation. Aversive stimulation to two different brain areas, the MH and the DM, was compared. A passive avoidance paradigm similar to that of Geller and Seifter [7] was used to measure any disinhibitory effects of scopolamine on the behavioral suppression. Since stimulation at many MH sites is positively reinforcing [1, 2, 3, 17], enhancement of LH self-stimulation, and scopolamine's effect upon it, were also examined.
Copyright © 1979 Brain Research Publications Inc.~0031-9384/79/080223-06501.10/0
224
HOFFMAN AND TROWILL METHOD
Animals and Surgery. Thirty-nine male albino Sprague-Dawley rats weighing approximately 330 g at the time of surgery were chronically implanted with monopolar electrodes constructed from No. 00 insect pins (Clay Adams). Electrodes were insulated with Insulex except for 0.5 mm at the tip. All rats were implanted with one electrode aimed at the LH, and a second electrode aimed at either the DM or MH. Coordinates for the LH were A. - 1 . 4 mm: L. 1.6 ram; H - 9 . 1 . For the DM, coordinates were A. - 4 . 4 mm: L. 0.5 ram: H. - 5 . 9 mm. Coordinates for the MH were varied between A. 1.0 to - 1.0: L. 0.6 ram: H. - 1 0 . 0 . All coordinates were in relation to bregma, midline, and skull top with the incisor bar 5.0 mm above the intraaural line. Ground electrodes consisted of 0--80× 1/8 in. machine screws (Small Parts Inc.) attached to the skull. Rats were implanted under sodium pentobarbital anaesthesia (50 mg/kg).
Histology At the completion of the study, each rat was sacrificed for determination of the location of the stimulating electrode tips. Rats were perfused with 10% Formalin solution under sodium pentobarbital anaesthesia. Brains were frozen sectioned at 48 p., mounted on glass slides, and stained with cresyl violet.
Apparatus A shuttle box (1.0×0.5x0.5 m) was used for screening and running of all animals. A central fulcrum created two compartments within the shuttle box. When the animal crossed from one side to the other, the shift in its weight activated microswitches which operated electromechanical relay equipment. The relay equipment was programmed to deliver intracranial stimulation either continuously or in 0.5 sec trains on a continuous reinforcement (CRF) or a variable ratio two (VR2) schedule in response to shuttle crossings. A commutator connecting the electrodes to the stimulator allowed for free movement in the shuttle box. Stimulation current was 60 Hz sine wave. The shuttle box was housed in a room separate from the relay equipment. Overhead lighting was provided by a 30 W red bulb.
Procedure Following seven days recovery from surgery, the rats were screened for aversiveness on the DM and MH electrodes. The screening procedure involved placing the rat in the shuttle box with the on-side set to deliver a continuous train of current to either the DM or MH. If the rat failed to make a response of crossing to the other side of the shuttle box within one minute the on and off sides were automatically switched, forcing the rat to escape the stimulation. Current level w a s i n c r e a s e d in 5 p.a steps until the animal would reliably (four out of five trials) escape the stimulation within 30 sec of its onset. The range of current intensities tested was 5-100/~a. The DM or MH placement was rated as aversive if during a 10 minute test the rat performed at least five consecutive escapes separated by an inter-trial interval of 1 rain, with escape latencies less than 30 sec. Thus the rats with aversive placements performed five consecutive e s c a p e s which were not interrupted by self-initiations of the stimula-
tion. If the criterion for aversiveness was not met, higher intensities were tested. Rats failing to meet the criterion due to continued reinitiation of the stimulation were rated as positive. All others were rated as neutral. Following the DM and MH screening, the rats were screened for positivity on the LH electrode. This involved allowing the rat to autoshape to 0.5 sec trains of LH stimulation of a CRF schedule such that each shuttle crossing produced a stimulus. Following shaping, current was adjusted until the rats reached a criterion of 80 responses/5 min. Rats were given at least three days, 1.5 hr/day to autoshape. Current intensities from five to 100 ~a were tested. Rats which reached criterion were rated positive while all others were rated neutral. On the basis of the two screening procedures, groups were formed for further testing. Rats with a positive LH electrode and either an aversive DM or aversive MH electrode were tested for suppression of LH self-stimulation by aversive stimulation. These rats were first trained to perform LH self-stimulation on a VR2 schedule such that one 0.5 sec train of brain stimulation was delivered for two shuttle crossings on the average. Training required a minimum of three daily 90 min sessions. When responding on the VR2 schedule stabilized, LH current intensity was adjusted to maintain response rate between 80 and 160 responses/5 min for a 45 min period. Testing for suppression was then begun. Daily suppression testing involved allowing the rats to self-stimulate on a VR2 schedule for a five minute warmup period for response stabilization, followed by a 5 min baseline rate measure (VR2). Then the first 5 rain suppression test followed in which 0.5 sec trains of current to the aversive placement were made contingent upon the responses that were not reinforced by the LH stimulation in the VR2 schedule. Thus the suppression tests were actually CRF schedules with one half of the responses reinforced with LH stimulation and the other half punished with either MH or DM stimulation. Following the first suppression test, a 5 min recovery period followed in which the rat could be primed (given free non-contingent LH stimulation) to reinstate responding. The recovery was followed by a second 5 min baseline (VR2) and a second 5 min suppression test. A third recovery, baseline, and suppression test (5 min each) was also run during the screening for operant suppression. For the suppression tests, current to the aversive DM or MH placement was increased in 5/za steps until an intensity was found which would reduce responding to less than 50% of the baseline rate for three consectuve suppression tests. Animals in which DM or MH stimulation failed to produce sufficient suppression were not tested further. Once current intensities for adequate suppression were established, five daily sessions were run to examine the effect of scopolamine (1 mg/kg, IP) upon the operant suppression by DM and MH stimulation. Daily sessions consisted of a 5 rain warmup (VR2), a 5 min baseline (VR2), a 5 rain suppression test, a 5 min recovery, a second baseline, a second suppression test, a second recovery, and a 5 min extinction test in which no brain stimulation was delivered. Thus daily sessions were 40 min. Two consecutive days of testing were conducted under equivolume saline injections (0.2 cc, IP). On the third day rats received scopolamine hydrochloride (1 mg/kg). On the fourth and fifth days saline injections were continued. All injections were given I hr prior to testing. Response rates were measured for baseline, suppression, and extinction intervals, and analyzed for drug ef-
INTRACRANIAL REWARD-PUNISHMENT INTERACTION fects with an ANOVA repeated measures design employing specific planned comparisons of drug and control sessions. In the second phase of the study, which was undertaken as a result of the high frequency of MH placements that were found to be positive, it was decided to determine the effect of the positive MH stimulation when made contingent upon operant responding for LH stimulation, as well as to examine the effect of scopolamine on this interaction. Thus rats with both a positive LH and a positive MH placement were tested in the same manner as the rats which were tested for operant suppression with aversive DM and MH placements. The second group was trained and screened in the same way as the first, except that instead of screening for suppression, they were screened for enhancement of LH selfstimulation with contingent delivery of positive MH stimulation. A reliable increase was defined as at least 130% of baseline on three enhancement tests/day for two consecutive days. The MH current intensity was increased in 5 p,a steps until the criterion was met, or until the current intensity reached I00/~a. Following the determination of the appropriate MH and LH current intensities, daily saline and scopolamine sessions were run exactly as with the first group to determine the effect of scopolamine on the enhancement of responding. Response rates during baseline, enhancement, recovery, and extinction were measured and analyzed for drug effects within an ANOVA repeated measures design utilizing gpecific planned comparisons.
225
RESULTS Out of 29 rats with MH placements, eight were classified as aversive, 16 as positive, and five as neutral. There were no apparent histological differences between the positive and aversive MH placements as there was considerable overlap between them. All placements were located medial to the fornix in the ventral hypothalamus (Fig. 1). Three of the rats with aversJve MH placements could not be used in the operant suppression paradigm (one lost electrode assembly while two had neutral LH placements). Thus five rats with aversive MH placements were tested for the effect of scopolamine on the operant suppression of LH selfstimulation. Out of 19 rats with DM placements, 16 were classified as aversive, one as positive, and two as neutral. Out of the 16 classified as aversive, 10 could not be used in the operant suppression paradigm (three showed no suppression, two had neutral LH placements, while five lost electrode assemblies). Thus six rats with aversive DM placements were tested for the effect of scopolamine on the operant suppression. The three rats with aversive DM placements which failed to produce operant suppression differed histologically from the six rats demonstrating suppression. Placements producing escape but no suppression of self-stimulation were found to lie in the superior colliculus, while those producing escape as well as suppression were more ventral in the dorsal lateral
FIG. 1. Electrode placements in the DM (above) and MH (below) classified as aversive 0, positive &, and aversive but failing to suppress LH selfstimulation • (from Pelligrino and Cushman [16]).
226
H O F F M A N AND TROWILL
TABLE 1 MEAN RATES OF RESPONDING PER FIVE M1N FOR MH AND DM ANIMALS ON BASELINE(B), SUPPRESSION(S), AND EXTINCTION (E) TESTS FOLLOWINGSALINE OR SCOPOLAMINE(SCOP) ADMINISTRATION. ± SEM IS INDICATED Saline
Scop 132 --- 16 152 _+ 17
B
MH DM
129 -+ 14 137 - 11
S
MH DM
32 -+ 12 35 -+ 9
87 - 13f 35 _+ 9
E
MH DM
24 _+ 2 19_+ 2
55 -+ 8T 21 _+ 6"
1.()()
I
I
I
t
©
/ 1
I
TI ME
I
!
1/
//
findicates p <0.01. central gray (Fig. 1). Placements in the central gray had much lower thresholds and elicited a more rapid escape than those in the superior coUiculus. Specific planned comparisons were conducted to contrast responding on the scopolamine day with responding on the four saline days. For baseline response rate there was no significant effect of scopolamine for either the MH, F(1.16)=0.15, p>0.05, or DM animals, F(1.20)=0.26, p>0.05 (Table 1). For responding during the suppression interval, MH rats showed a highly significant disinhibition as a function of scopolamine, F(1.16)=39.13, p <0.00! (Table 1, Fig. 2). In contrast, the DM rats showed no significant disinhibition of responding during suppression tests as a function of scopolamine, F(1.20)=0.01, p>0.05 (Table 1). For the four saline days, the MH and DM groups did not differ significantly from each other on baseline responding, F(1.9)=1.5, p>0.05. The MH group was suppressed to an average of 17.9% of baseline while the DM group was suppressed to 19.7% on the two saline test days preceeding scopolamine injections. The median current intensity delivered to the MH placements was 36/~a while for the DM placements the median intensity was 33/za. On the extinction tests, MH rats showed a significant resistance to extinction as a function of scopolamine, F(1.16)=24.9, p<0.001, while the DM rats showed no significant change as a function of scopolamine, F(1.20)=0.17, p>0.05 (Table 1). In the second phase of the study, eight rats which had both positive MH and LH placements were screened for response enhancement. Four rats met the criterion for consistent enhancement of operant rate with contingent MH stimulation. When tested under scopolamine, these four rats showed no significant changes in baseline rate, F(1.12)= 1.0, p >0.05, or rate during enhancement intervals, F(1.12)=0.4, p>0.05, as a function of scopolamine (Table 2). Resistance to extinction was significantly increased by scopolamine, F(1.12) = 11.1, p <0.01 (Table 2). In addition, rates during the recovery period following the enhancement were significantly greater under scopolamine as compared to saline days, F(1.12)=40.5, p<0.001 (Table 2). Of the four rats that did not show enhancement of responding with MH stimulation, two showed some suppression while two showed no effect of the contingent MH stimulation. Scopolamine disinhibited the suppressed responding but data were not analyzed for significance due to low number of subjects.
/
25
I
15(min )
|
•
30
FIG. 2. Representative cumulative response record from a rat with an aversive MH placement in the passive avoidance paradigm under conditions of saline (A) and scopolamine (B) i~ections demonstrating disinhibitionof suppressed responding with scopotamine~Region between pen offset marks indicates five rain suppression interval. Number above suppression interval indicates responses made during that interval.
TABLE 2 MEAN RATESOF RESPONDINGPER FIVEMIN FORANIMALSWITH POSITIVE MH PLACEMENTSON B A S E ~ (BASE), ENHANCEMENT (Enh), RECOVERY (Rec), AND EXTINCTION(Ext) TESTS FOLLOWINGSALINEOR SCOPOLAMINE(Scop) ADMINISTRATION: ± SEM IS INDICATED
Base Enh Rec Ext *indicates p<0.01. 1"indicatesp <0.001.
Saline
Scop
84 -+ 30 131 -+ 21 78 -+ 21 36 -+ l0
92 -+ 26 137 _+ 23 1tl +_ 26f 60 -+ 25*
INTRACRANIAL REWARD-PUNISHMENT INTERACTION
DISCUSSION The present findings demonstrate that electrical brain stimulation can serve as the rewarding and punishing stimuli in a passive avoidance paradigm. Response contingent electrical stimulation to two different brain areas, the MH and DM produced a suppression of LH self-stimulation to less than 20% of baseline rats. The finding that scopolamine at 1 mg/kg significantly disinhibited response suppression produced by MH but not DM stimulation lends support to the notion that cholinergic synapses within the MH play a role in response suppression [11]. These preliminary findings also suggest that response suppression due to punishing DM stimulation may have a different neurochemical substrate than suppression due to punishing MH stimulation, but larger doses of scopolamine as well as other neuropharmacological agents need to be tested before a more conclusive statement can be made. It is possible that the density of cholinergic cells in the DM is higher than those in the MH necessitating larger doses of scopolamine to produce disinhibition. Another possibility is that the DM stimulation activated a non-cholinergic efferent to the MH. Thus if cholinergic synapses within the MH are important for integrating positive and aversive stimuli and modulating behavioral inhibition [14] via the efferent fibers of the dorsal longitudinal fasciculus [5,21], then electrical stimulation of the DM could bypass any cholinergic integration in the MH. A serotonergic mediation of aversive stimuli in the midbrain has been suggested [9]. The serotonergic synthesis inhibitor pCPA significantly elevated aversive thresholds in a decremental bar press paradigm while the catecholamine synthesis inhibitor aMPT was without effect. One important difference found in the present study was that 80% of the DM placements were classified as aversive compared to 30% for the MH placements. The majority of the rats with MH placements failed to meet the criterion for aversiveness due to repeated initiation of the stimulation. Although many rats with MH placements consistently selfstimulated, their self-stimulation seemed ambivalent when compared to LH self-stimulation, as it was characterized by what seemed to be longer inter-stimulus intervals and shorter preferred durations of the stimulation when the animals were allowed to self select stimulation duration during the screening procedure. Rats with DM placements rarely selfstimulated and thus easily met the criterion for aversiveness. The rats with MH placements that did meet the criterion give support to other reports [6, 10, 11] which consider the MH as part of the neural substrate mediating responses to aversive stimuli. Thus qualitative differences may exist within what
227
has been described as a unified periventricular punishment system [11]. In the second portion of the study it was of interest to demonstrate a consistent enhancement of responding with the addition of contingent positive MH stimulation. This provided additional evidence for the positivity of this stimulation. When the positive MH stimulation was removed during the recovery intervals following the enhancement tests, a rapid rate decrease toward baseline normally occurred. Interestingly, under scopolamine the rats did not return as rapidly to baseline rates during the recovery and thus recovery rates under scopolamine were significantly greater than those under saline. This effect could also be interpreted as resulting from a reduced efficiency of a cholinergic system that inhibits behavior when reward is reduced or removed. Response rates during baseline and enhancement intervals were not significantly increased with scopolamine presumably because little or no inhibition was operative during these periods. It was also of interest to demonstrate a consistent suppression of responding with MH stimulation in two rats which were initially classified as positive at the MH placement. This indicated that these particular placements were more aversive than those resulting in enhancement of LH self-stimulation. Scopolamine's disinhibitory effect on responding during the suppression interval for these two rats is consistent with the present cholinergic hypothesis. With respect to the extinction tests, the significant resistance to extinction for the rats with aversive MH placements as well as those with positive MH placements is also in agreement with Carlton's model [4] of an inhibitory cholinergic system. But the lack of a significant resistance to extinction for the rats with the DM placements cannot be readily explained. It is possible that the aversive DM stimulation from the suppression interval had a carryover effect to the extinction test, thus counteracting any general disinhibitory effect of scopolamine. In conclusion, passive avoidance behavior has been demonstrated using intracranial reward and punishment. Scopolamine's disinhibitory effect on responding suppressed by MH stimulation has provided additional support for the notion that cholinergic synapses in the MH play a role in the suppression of operant behavior. In addition, the sensitivity of the passive avoidance paradigm to pharmacological manipulation has been demonstrated. The present study suggests the advantages of using pharmacological agents in exploring the neurochemical basis of aversive brain stimulation. The interrelationships of the several brain loci mediating aversive brain stimulation deserve further study.
REFERENCES 1. Atrens, D. M. A reinforcement analysis of rat hypothalamus. Am. J. Physiol. 224: 62-65, 1973. 2. Atrens, D. M. Modulation of reward and aversion processes in the rat diencephalon by amphetamine. Am. J. Physiol. 226: 874-880, 1974. 3. Ball, G. Self-stimulation in the ventromedial hypothalamus. Science 178: 72-73, 1972. 4. Carlton, P. L. Cholinergic mechanisms in the control of behavior by the brain. Psychol. Rev. 70: 19-39, 1963. 5. Cosby, E. C. and R. T. Woodburne. The mammalian midbrain and isthmus regions. J. "comp. Neurol. 94: 1-32, 1951.
6. Delgado, J. M. R., W. W. Roberts and N. E. Miller. Learning motivated by electricalstimulation of the brain. Am. J. Physiol. 179: 587-593, 1954. 7. Geller, I. and J. Seifter.The effects of meprobamate, barbiturates, d-amphetamine and promazine on experimentally induced conflict in the rat. Psychopharmacologia I: 482-492, 1960. 8. Jung, O. H. and E. S. Boyd. Effects of cholinergic drugs on self-stimulation response rates in rats. Am. J. Physiol. 210(3): 432--434, 1966. 9. Kiser, R. S. and R. M. Lebovitz. Monoaminergic mechanisms in aversive brain stimulation.Physiol. Behav. 15: 47-53, 1975.
228 10. Krasne, F. B. General disruption resulting from stimulation of the ventromedial hypothalamus. Science 138: 822-823, 1962. 11. Margules, D. L. and L. Stein. Cholinergic synapses of a periventricular punishment system in the medial hypothalamus. Am. J. Physiol. 217: 475-480, 1969. 12. Miller, N. E. Some recent studies of conflict behavior and drugs. Am. Psychol. 16: 12-24, 1960. 13. Olds, M. E. Comparative effects of amphetamine, scopolamine, chlordiazepoxide, and diphenylhydantoin on operant and extinction behavior with brain stimulation and food reward. Neuropharmacology 9- 519-532, 1970. 14. Olds, M. E. and J. Olds. Approach-avoidance analysis of rat diencephalon. J. comp. Neurol. 120: 259-295, 1%3. 15. Olds, M. E. and E. F. Domino. Differential effects of cholinergic agonists on self-stimulation and escape behavior. J. Pharmac. exp. Ther, 170: 157-167, 1969.
H O F F M A N A N D T R O W I LL 16. Pellegrino, L. J. and A. J, Cushman. A Stereotaxic Atlas ~/the Rat Brain. New York: Meredith Publishing Co., 1967. 17. Poschel, B. P. H. Comparison of reinforcing effects yielded by lateral versus medial hypothalamic stimulation. ,l. comp. physiol. Psychol. 61: 346-352, 1%6. 18. Routtenberg, A. and J. Olds. Stimulation of dorsal midbrain during septal and hypothalamic self-stimulation. J, comp. physiol. Psychol. 62: 25-255, 1966. 19. Shute, C. C. D. and P. R. Lewis. The ascending cholinergic reticular system: Neocortical, olfactory, and subcortieal projections. Brain 90: 497-520, 1%7. 20. Stark, P. and E. S. Boyd. Effects of cholinergic drugs on hypothalamic self-stimulation response rates of dogs. Am. J. Physiol. 205(4): 745-748, 1963. 21. Satin, J. The periventricular stratum of the hypothalamus. Int. Rev. Neurobiol. 9: 263-300, 1%6.
Modulation of Intracranial Reward-Punishment Interaction by Scopolamine D. L. H O F F M A N A N D J. A. T R O W I L L
The University of Denver, Department of Psychology, Denver, CO Received 5 July 1978 HOFFMAN, D. L. AND J. A. TROWlLL. Modulation of intracranial reward-punishment interaction by scopolamine. PHYSIOL. BEHAV. 23(2)223--228, 1979.--Rats were tested in a passive avoidance paradigm involving the alternation of punished and unpunished schedules of reinforcement. The rate of self-stimulating for 60 Hz sine wave current (15-130 p.a) to the lateral hypothalamus (LH) was lowered to 18% and 20% when response contingent aversive electrical stimulation to the medial hypothalamus (MH) (15--60 /za) and dorsal midbrain (DM) (25-90 p,a), respectively, was introduced. Scopolamine (1 mg/kg, IP) was found to significantly disinhibit the suppression of responding produced by the MH stimulation to 67%, but had no significant effect on suppression of responding produced by DM stimulation. Since approximately 50% of the placements in the MH were found to be positively reinforcing, a group of rats was run such that the rate of self-stimulation to the LH was increased to at least 130%when the positive MH stimulation was made contingent upon LH self-stimulation. Scopolamine had no effect on this response enhancement. The passive avoidance results demonstrating scopolamine's disinhibitory effect on suppression produced by MH stimulation support the hypothesis that cholinergic synapses within the MH form part of a periventricular system mediating the effects of punishment on "behavior. Self-stimulation
Passiveavoidance
Scopolamine
SEVERAL investigators have dealt with the competing effects of reward and punishment upon behavior, and the effects of drugs on this competition. Punishment, which occurs when a response produces an aversive stimulus, has the general effect of suppressing the response which produced the aversive stimulus. Miller [12] dealt with approach avoidance conflict between food rewards and punishing foot shock, such that two incompatible response tendencies occurred simultaneously, i.e., approaching the food and avoiding the shock. Food reward could not be obtained without receiving shocks, and thus approach behavior was suppressed. In several different paradigms, the conflict reducing effects of meprobamate and amobarbital were demonstrated. Geller and Seifter [7] also examined the effects of drugs upon a food shock conflict using a passive avoidance paradigm. In this paradigm, an unpunished schedule of reinforcement in which responding was rewarded with milk, was alternated with a punished schedule in which each response produced both a milk reward and a foot shock punisher. Responding was suppressed during the punished schedule. Drugs such as meprobamate and barbiturates were found to restore responding that had been suppressed by punishment, while amphetamine and promazine were found to further suppress responding during the punished interval. Margales and Stein [7], using a similar passive avoidance paradigm, examined the effects of direct MH chemical stimulation on shock induced suppression of responding for milk rewards. Cholinergic agonists such as carbachol, physostigmine, and musearine were found to enhance the suppressant effects of punishment, while cholinergic antagonists such as atropine and scopolamine had a disinhibitory effect
Aversivestimulation
on the suppressed behavior. They thus suggested that the MH forms part of a periventricular system involved in the mediation of behavioral suppression by punishment. Their findings stressed the importance of cholinergic synapses in the MH for the mediation of the behavioral suppression. Several investigators [4, 11, 15] have adopted the view that a cholinergic pathway suppressed behavior by antagonizing an adrenergic system for activation. According to this view, anticholinergics disinhibit behavior that has been suppressed by habituation, non-reward, and punishment by releasing an adrenergic system from inhibition. An increase in rate of LH self-stimulation has been demonstrated with anticholinergics in rats [13], as well as a dramatic decrease in self-stimulation rate with cholinergic agonists in rats [8] and dogs [20]. This rate decrease with cholinergic agonists has also been shown to be antagonized by scopolamine [15]. A decrement in LH self-stimulation with direct application of the cholinergic agonist carbachol to the DM has also been demonstrated in rats [18]. Thus evidence for a central cholinergic system involved in behavioral inhibition has been accumulating. The present study focuses upon the effects of the anticholinergic scopolamine on the punishment induced suppression of LH self-stimulation. Aversive stimulation to two different brain areas, the MH and the DM, was compared. A passive avoidance paradigm similar to that of Geller and Seifter [7] was used to measure any disinhibitory effects of scopolamine on the behavioral suppression. Since stimulation at many MH sites is positively reinforcing [1, 2, 3, 17], enhancement of LH self-stimulation, and scopolamine's effect upon it, were also examined.
Copyright © 1979 Brain Research Publications Inc.~0031-9384/79/080223-06501.10/0
224
HOFFMAN AND TROWILL METHOD
Animals and Surgery. Thirty-nine male albino Sprague-Dawley rats weighing approximately 330 g at the time of surgery were chronically implanted with monopolar electrodes constructed from No. 00 insect pins (Clay Adams). Electrodes were insulated with Insulex except for 0.5 mm at the tip. All rats were implanted with one electrode aimed at the LH, and a second electrode aimed at either the DM or MH. Coordinates for the LH were A. - 1 . 4 mm: L. 1.6 ram; H - 9 . 1 . For the DM, coordinates were A. - 4 . 4 mm: L. 0.5 ram: H. - 5 . 9 mm. Coordinates for the MH were varied between A. 1.0 to - 1.0: L. 0.6 ram: H. - 1 0 . 0 . All coordinates were in relation to bregma, midline, and skull top with the incisor bar 5.0 mm above the intraaural line. Ground electrodes consisted of 0--80× 1/8 in. machine screws (Small Parts Inc.) attached to the skull. Rats were implanted under sodium pentobarbital anaesthesia (50 mg/kg).
Histology At the completion of the study, each rat was sacrificed for determination of the location of the stimulating electrode tips. Rats were perfused with 10% Formalin solution under sodium pentobarbital anaesthesia. Brains were frozen sectioned at 48 p., mounted on glass slides, and stained with cresyl violet.
Apparatus A shuttle box (1.0×0.5x0.5 m) was used for screening and running of all animals. A central fulcrum created two compartments within the shuttle box. When the animal crossed from one side to the other, the shift in its weight activated microswitches which operated electromechanical relay equipment. The relay equipment was programmed to deliver intracranial stimulation either continuously or in 0.5 sec trains on a continuous reinforcement (CRF) or a variable ratio two (VR2) schedule in response to shuttle crossings. A commutator connecting the electrodes to the stimulator allowed for free movement in the shuttle box. Stimulation current was 60 Hz sine wave. The shuttle box was housed in a room separate from the relay equipment. Overhead lighting was provided by a 30 W red bulb.
Procedure Following seven days recovery from surgery, the rats were screened for aversiveness on the DM and MH electrodes. The screening procedure involved placing the rat in the shuttle box with the on-side set to deliver a continuous train of current to either the DM or MH. If the rat failed to make a response of crossing to the other side of the shuttle box within one minute the on and off sides were automatically switched, forcing the rat to escape the stimulation. Current level w a s i n c r e a s e d in 5 p.a steps until the animal would reliably (four out of five trials) escape the stimulation within 30 sec of its onset. The range of current intensities tested was 5-100/~a. The DM or MH placement was rated as aversive if during a 10 minute test the rat performed at least five consecutive escapes separated by an inter-trial interval of 1 rain, with escape latencies less than 30 sec. Thus the rats with aversive placements performed five consecutive e s c a p e s which were not interrupted by self-initiations of the stimula-
tion. If the criterion for aversiveness was not met, higher intensities were tested. Rats failing to meet the criterion due to continued reinitiation of the stimulation were rated as positive. All others were rated as neutral. Following the DM and MH screening, the rats were screened for positivity on the LH electrode. This involved allowing the rat to autoshape to 0.5 sec trains of LH stimulation of a CRF schedule such that each shuttle crossing produced a stimulus. Following shaping, current was adjusted until the rats reached a criterion of 80 responses/5 min. Rats were given at least three days, 1.5 hr/day to autoshape. Current intensities from five to 100 ~a were tested. Rats which reached criterion were rated positive while all others were rated neutral. On the basis of the two screening procedures, groups were formed for further testing. Rats with a positive LH electrode and either an aversive DM or aversive MH electrode were tested for suppression of LH self-stimulation by aversive stimulation. These rats were first trained to perform LH self-stimulation on a VR2 schedule such that one 0.5 sec train of brain stimulation was delivered for two shuttle crossings on the average. Training required a minimum of three daily 90 min sessions. When responding on the VR2 schedule stabilized, LH current intensity was adjusted to maintain response rate between 80 and 160 responses/5 min for a 45 min period. Testing for suppression was then begun. Daily suppression testing involved allowing the rats to self-stimulate on a VR2 schedule for a five minute warmup period for response stabilization, followed by a 5 min baseline rate measure (VR2). Then the first 5 rain suppression test followed in which 0.5 sec trains of current to the aversive placement were made contingent upon the responses that were not reinforced by the LH stimulation in the VR2 schedule. Thus the suppression tests were actually CRF schedules with one half of the responses reinforced with LH stimulation and the other half punished with either MH or DM stimulation. Following the first suppression test, a 5 min recovery period followed in which the rat could be primed (given free non-contingent LH stimulation) to reinstate responding. The recovery was followed by a second 5 min baseline (VR2) and a second 5 min suppression test. A third recovery, baseline, and suppression test (5 min each) was also run during the screening for operant suppression. For the suppression tests, current to the aversive DM or MH placement was increased in 5/za steps until an intensity was found which would reduce responding to less than 50% of the baseline rate for three consectuve suppression tests. Animals in which DM or MH stimulation failed to produce sufficient suppression were not tested further. Once current intensities for adequate suppression were established, five daily sessions were run to examine the effect of scopolamine (1 mg/kg, IP) upon the operant suppression by DM and MH stimulation. Daily sessions consisted of a 5 rain warmup (VR2), a 5 min baseline (VR2), a 5 rain suppression test, a 5 min recovery, a second baseline, a second suppression test, a second recovery, and a 5 min extinction test in which no brain stimulation was delivered. Thus daily sessions were 40 min. Two consecutive days of testing were conducted under equivolume saline injections (0.2 cc, IP). On the third day rats received scopolamine hydrochloride (1 mg/kg). On the fourth and fifth days saline injections were continued. All injections were given I hr prior to testing. Response rates were measured for baseline, suppression, and extinction intervals, and analyzed for drug ef-
INTRACRANIAL REWARD-PUNISHMENT INTERACTION fects with an ANOVA repeated measures design employing specific planned comparisons of drug and control sessions. In the second phase of the study, which was undertaken as a result of the high frequency of MH placements that were found to be positive, it was decided to determine the effect of the positive MH stimulation when made contingent upon operant responding for LH stimulation, as well as to examine the effect of scopolamine on this interaction. Thus rats with both a positive LH and a positive MH placement were tested in the same manner as the rats which were tested for operant suppression with aversive DM and MH placements. The second group was trained and screened in the same way as the first, except that instead of screening for suppression, they were screened for enhancement of LH selfstimulation with contingent delivery of positive MH stimulation. A reliable increase was defined as at least 130% of baseline on three enhancement tests/day for two consecutive days. The MH current intensity was increased in 5 p,a steps until the criterion was met, or until the current intensity reached I00/~a. Following the determination of the appropriate MH and LH current intensities, daily saline and scopolamine sessions were run exactly as with the first group to determine the effect of scopolamine on the enhancement of responding. Response rates during baseline, enhancement, recovery, and extinction were measured and analyzed for drug effects within an ANOVA repeated measures design utilizing gpecific planned comparisons.
225
RESULTS Out of 29 rats with MH placements, eight were classified as aversive, 16 as positive, and five as neutral. There were no apparent histological differences between the positive and aversive MH placements as there was considerable overlap between them. All placements were located medial to the fornix in the ventral hypothalamus (Fig. 1). Three of the rats with aversJve MH placements could not be used in the operant suppression paradigm (one lost electrode assembly while two had neutral LH placements). Thus five rats with aversive MH placements were tested for the effect of scopolamine on the operant suppression of LH selfstimulation. Out of 19 rats with DM placements, 16 were classified as aversive, one as positive, and two as neutral. Out of the 16 classified as aversive, 10 could not be used in the operant suppression paradigm (three showed no suppression, two had neutral LH placements, while five lost electrode assemblies). Thus six rats with aversive DM placements were tested for the effect of scopolamine on the operant suppression. The three rats with aversive DM placements which failed to produce operant suppression differed histologically from the six rats demonstrating suppression. Placements producing escape but no suppression of self-stimulation were found to lie in the superior colliculus, while those producing escape as well as suppression were more ventral in the dorsal lateral
FIG. 1. Electrode placements in the DM (above) and MH (below) classified as aversive 0, positive &, and aversive but failing to suppress LH selfstimulation • (from Pelligrino and Cushman [16]).
226
H O F F M A N AND TROWILL
TABLE 1 MEAN RATES OF RESPONDING PER FIVE M1N FOR MH AND DM ANIMALS ON BASELINE(B), SUPPRESSION(S), AND EXTINCTION (E) TESTS FOLLOWINGSALINE OR SCOPOLAMINE(SCOP) ADMINISTRATION. ± SEM IS INDICATED Saline
Scop 132 --- 16 152 _+ 17
B
MH DM
129 -+ 14 137 - 11
S
MH DM
32 -+ 12 35 -+ 9
87 - 13f 35 _+ 9
E
MH DM
24 _+ 2 19_+ 2
55 -+ 8T 21 _+ 6"
1.()()
I
I
I
t
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/ 1
I
TI ME
I
!
1/
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findicates p <0.01. central gray (Fig. 1). Placements in the central gray had much lower thresholds and elicited a more rapid escape than those in the superior coUiculus. Specific planned comparisons were conducted to contrast responding on the scopolamine day with responding on the four saline days. For baseline response rate there was no significant effect of scopolamine for either the MH, F(1.16)=0.15, p>0.05, or DM animals, F(1.20)=0.26, p>0.05 (Table 1). For responding during the suppression interval, MH rats showed a highly significant disinhibition as a function of scopolamine, F(1.16)=39.13, p <0.00! (Table 1, Fig. 2). In contrast, the DM rats showed no significant disinhibition of responding during suppression tests as a function of scopolamine, F(1.20)=0.01, p>0.05 (Table 1). For the four saline days, the MH and DM groups did not differ significantly from each other on baseline responding, F(1.9)=1.5, p>0.05. The MH group was suppressed to an average of 17.9% of baseline while the DM group was suppressed to 19.7% on the two saline test days preceeding scopolamine injections. The median current intensity delivered to the MH placements was 36/~a while for the DM placements the median intensity was 33/za. On the extinction tests, MH rats showed a significant resistance to extinction as a function of scopolamine, F(1.16)=24.9, p<0.001, while the DM rats showed no significant change as a function of scopolamine, F(1.20)=0.17, p>0.05 (Table 1). In the second phase of the study, eight rats which had both positive MH and LH placements were screened for response enhancement. Four rats met the criterion for consistent enhancement of operant rate with contingent MH stimulation. When tested under scopolamine, these four rats showed no significant changes in baseline rate, F(1.12)= 1.0, p >0.05, or rate during enhancement intervals, F(1.12)=0.4, p>0.05, as a function of scopolamine (Table 2). Resistance to extinction was significantly increased by scopolamine, F(1.12) = 11.1, p <0.01 (Table 2). In addition, rates during the recovery period following the enhancement were significantly greater under scopolamine as compared to saline days, F(1.12)=40.5, p<0.001 (Table 2). Of the four rats that did not show enhancement of responding with MH stimulation, two showed some suppression while two showed no effect of the contingent MH stimulation. Scopolamine disinhibited the suppressed responding but data were not analyzed for significance due to low number of subjects.
/
25
I
15(min )
|
•
30
FIG. 2. Representative cumulative response record from a rat with an aversive MH placement in the passive avoidance paradigm under conditions of saline (A) and scopolamine (B) i~ections demonstrating disinhibitionof suppressed responding with scopotamine~Region between pen offset marks indicates five rain suppression interval. Number above suppression interval indicates responses made during that interval.
TABLE 2 MEAN RATESOF RESPONDINGPER FIVEMIN FORANIMALSWITH POSITIVE MH PLACEMENTSON B A S E ~ (BASE), ENHANCEMENT (Enh), RECOVERY (Rec), AND EXTINCTION(Ext) TESTS FOLLOWINGSALINEOR SCOPOLAMINE(Scop) ADMINISTRATION: ± SEM IS INDICATED
Base Enh Rec Ext *indicates p<0.01. 1"indicatesp <0.001.
Saline
Scop
84 -+ 30 131 -+ 21 78 -+ 21 36 -+ l0
92 -+ 26 137 _+ 23 1tl +_ 26f 60 -+ 25*
INTRACRANIAL REWARD-PUNISHMENT INTERACTION
DISCUSSION The present findings demonstrate that electrical brain stimulation can serve as the rewarding and punishing stimuli in a passive avoidance paradigm. Response contingent electrical stimulation to two different brain areas, the MH and DM produced a suppression of LH self-stimulation to less than 20% of baseline rats. The finding that scopolamine at 1 mg/kg significantly disinhibited response suppression produced by MH but not DM stimulation lends support to the notion that cholinergic synapses within the MH play a role in response suppression [11]. These preliminary findings also suggest that response suppression due to punishing DM stimulation may have a different neurochemical substrate than suppression due to punishing MH stimulation, but larger doses of scopolamine as well as other neuropharmacological agents need to be tested before a more conclusive statement can be made. It is possible that the density of cholinergic cells in the DM is higher than those in the MH necessitating larger doses of scopolamine to produce disinhibition. Another possibility is that the DM stimulation activated a non-cholinergic efferent to the MH. Thus if cholinergic synapses within the MH are important for integrating positive and aversive stimuli and modulating behavioral inhibition [14] via the efferent fibers of the dorsal longitudinal fasciculus [5,21], then electrical stimulation of the DM could bypass any cholinergic integration in the MH. A serotonergic mediation of aversive stimuli in the midbrain has been suggested [9]. The serotonergic synthesis inhibitor pCPA significantly elevated aversive thresholds in a decremental bar press paradigm while the catecholamine synthesis inhibitor aMPT was without effect. One important difference found in the present study was that 80% of the DM placements were classified as aversive compared to 30% for the MH placements. The majority of the rats with MH placements failed to meet the criterion for aversiveness due to repeated initiation of the stimulation. Although many rats with MH placements consistently selfstimulated, their self-stimulation seemed ambivalent when compared to LH self-stimulation, as it was characterized by what seemed to be longer inter-stimulus intervals and shorter preferred durations of the stimulation when the animals were allowed to self select stimulation duration during the screening procedure. Rats with DM placements rarely selfstimulated and thus easily met the criterion for aversiveness. The rats with MH placements that did meet the criterion give support to other reports [6, 10, 11] which consider the MH as part of the neural substrate mediating responses to aversive stimuli. Thus qualitative differences may exist within what
227
has been described as a unified periventricular punishment system [11]. In the second portion of the study it was of interest to demonstrate a consistent enhancement of responding with the addition of contingent positive MH stimulation. This provided additional evidence for the positivity of this stimulation. When the positive MH stimulation was removed during the recovery intervals following the enhancement tests, a rapid rate decrease toward baseline normally occurred. Interestingly, under scopolamine the rats did not return as rapidly to baseline rates during the recovery and thus recovery rates under scopolamine were significantly greater than those under saline. This effect could also be interpreted as resulting from a reduced efficiency of a cholinergic system that inhibits behavior when reward is reduced or removed. Response rates during baseline and enhancement intervals were not significantly increased with scopolamine presumably because little or no inhibition was operative during these periods. It was also of interest to demonstrate a consistent suppression of responding with MH stimulation in two rats which were initially classified as positive at the MH placement. This indicated that these particular placements were more aversive than those resulting in enhancement of LH self-stimulation. Scopolamine's disinhibitory effect on responding during the suppression interval for these two rats is consistent with the present cholinergic hypothesis. With respect to the extinction tests, the significant resistance to extinction for the rats with aversive MH placements as well as those with positive MH placements is also in agreement with Carlton's model [4] of an inhibitory cholinergic system. But the lack of a significant resistance to extinction for the rats with the DM placements cannot be readily explained. It is possible that the aversive DM stimulation from the suppression interval had a carryover effect to the extinction test, thus counteracting any general disinhibitory effect of scopolamine. In conclusion, passive avoidance behavior has been demonstrated using intracranial reward and punishment. Scopolamine's disinhibitory effect on responding suppressed by MH stimulation has provided additional support for the notion that cholinergic synapses in the MH play a role in the suppression of operant behavior. In addition, the sensitivity of the passive avoidance paradigm to pharmacological manipulation has been demonstrated. The present study suggests the advantages of using pharmacological agents in exploring the neurochemical basis of aversive brain stimulation. The interrelationships of the several brain loci mediating aversive brain stimulation deserve further study.
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228 10. Krasne, F. B. General disruption resulting from stimulation of the ventromedial hypothalamus. Science 138: 822-823, 1962. 11. Margules, D. L. and L. Stein. Cholinergic synapses of a periventricular punishment system in the medial hypothalamus. Am. J. Physiol. 217: 475-480, 1969. 12. Miller, N. E. Some recent studies of conflict behavior and drugs. Am. Psychol. 16: 12-24, 1960. 13. Olds, M. E. Comparative effects of amphetamine, scopolamine, chlordiazepoxide, and diphenylhydantoin on operant and extinction behavior with brain stimulation and food reward. Neuropharmacology 9- 519-532, 1970. 14. Olds, M. E. and J. Olds. Approach-avoidance analysis of rat diencephalon. J. comp. Neurol. 120: 259-295, 1%3. 15. Olds, M. E. and E. F. Domino. Differential effects of cholinergic agonists on self-stimulation and escape behavior. J. Pharmac. exp. Ther, 170: 157-167, 1969.
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