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Amphetamine-like stimulus properties produced by electrical stimulation of reward sites in the ventral tegmental area
175
Behavioural Brain Research, 38 (1990) 175-184 Elsevier BBR 01051
Amphetamine-like stimulus properties produced by electrical stimulation of reward sites in the ventral tegmental area J.P. D r u h a n
1,*,
H . C . Fibiger 2 a n d A . G . Phillips 1
Department of Psychology, and 2Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B. C. (Canada) (Received 15 August 1989) (Revised version received 25 September 1989) (Accepted 5 January 1990)
Key words: Stimulus property; D-Amphetamine; Electrical brain stimulation; Ventral tegmental area; Drug discrimination; Stimulus generalization; Intracranial self stimulation; Rat
The present study examined whether the stimulus properties of D-amphetamine could be mimicked by electrical stimulation of the ventral tegmental area (VTA). Rats trained to discriminate 1.0 mg/kg D-amphetamine from saline were given generalization tests with a range of D-amphetamine doses administered either alone or in combination with VTA stimulation. The results suggested that the VTA stimulation could enhance the cueing effects of D-amphetamine, as levels of responding on the D-amphetamine-appropriate lever during stimulation trials were increased relative to tests without stimulation. Individual differences were observed in the amount of drug-lever responding elicited by the VTA stimulation during drug-free substitution tests, and the different levels of drug-lever responding correlated positively with the response rates obtained from these rats during subsequent intracranial self-stimulation tests. These findings suggest that VTA stimulation can have D-amphetamine-like stimulus properties and such stimulus properties may be related to the rewarding effects of the brain stimulation.
INTRODUCTION
Numerous studies have demonstrated that rats can discriminate between the presence of the psychomotor stimulant, D-amphetamine, and a n o n - d r u g c o n d i t i o n 16,17,26,3°. The neural processes responsible for the transduction of D-amphetamine's actions into stimulus properties appear to involve dopamine-containing neurons. For example, the D-amphetamine stimulus can be
* Present address: Center for Studies in Behavioral Neurobiology, Concordia University, 1455 De Maissoneuve West, Montreal, Quebec, Canada H3A 2J3. Correspondence: A.G. Phillips, Department of Psychology, University of British Columbia, Vancouver, B.C., Canada V6T IY7.
mimicked by dopamine (DA) receptor agonists and by drugs which increase the release or block the uptake of this transmitter 7"~4,~5,28.Conversely, D-amphetamine stimuli can be attenuated by DA receptor antagonists or synthesis inhibitors 13'25'29. Such results are not obtained with drugs that specifically affect noradrenergic or serotonergic neurotransmission 13,29,3°. Recent studies have revealed an important role for the mesolimbic DA projection in transducing D-amphetamine stimuli. For example, the stimulus properties of D-amphetamine were antagonized by atypical neurolepticsz5 which are thought to preferentially block DA receptors in the mesolimbic DA system. In a subsequent study26, rats trained to discriminate the stimulus properties of systemic D-amphetamine emitted drug-appro-
0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 priate responses when D-amphetamine was injected directly into the nucleus accumbens. This effect was blocked by co-injections of the DA receptor antagonist sulpiride. If the stimulus properties of D-amphetamine are related to the facilitatory actions of this drug on mesolimbic DA neurotransmission, these stimulus properties may be mimicked by electrical stimulation ofmesolimbic DA neurons. The present study assesses whether electrical stimulation of mesolimbic DA neurons within the ventral tegmental area (VTA) can augment the stimulus properties of low D-amphetamine doses in rats trained to discriminate 1.0 mg/kg D-amphetamine from saline. Recent in vivo electrochemical and ex vivo chromatographic measures indicate that VTA stimulation can induce the release of DA in the ipsilateral nucleus accumbens 4'1''27. Psychopharmacological studies also suggest that VTA stimulation can produce stimuli that are mediated by DA-containing neurons 9. The present study also investigated the relationship between the rewarding effect of VTA stimulation and the production of D-amphetamine-like stimulus properties. Specifically, it was hypothesized that because the rewarding effects of D-amphetamine and VTA stimulation appear to be mediated by DA projections to the nucleus accumbensl.5,6,11,19,23,27.31 a common dopaminergic substrate may mediate their rewarding and stimulus properties. To examine this, the correlation between the level of D-amphetamineappropriate responding produced by the VTA stimulation, and measures of the rewarding effects of the same VTA stimulation were determined.
GENERAL METHODS
Subjects Male hooded rats (Charles River, Long-Evans strain) served as subjects. All rats were housed individually in stainless steel cages with tap water available adlibitum. Access to food was restricted, and 22-24 g of standard rat chow was provided once each day. The rats were tested towards the end of the 12-h light phase of a 24-h light-dark cycle.
Surgery and histology Prior to discrimination training, each rat was anesthetized with 65 mg/kg sodium pentobarbital and a bipolar electrode (Plastic Products MS303/2) was implanted into the VTA. The coordinates for stereotaxic implantation were: 3.1 mm anterior and 1.7 mm dorsal to the interaural line, and 0.6 mm lateral to the midline (in the left hemisphere). The incisor bar was set 3.2 mm below the interaural line. Electrodes were anchored chronically to the skull with jeweller's screws and dental cement. Upon completion of the experiments, the rats were given an overdose of sodium pentobarbital and perfused transcardially with 0.9~o saline followed by 10~o formol-saline. The brains were removed, sliced in 30-/~m frozen sections, and stained with Cresyl violet for microscopic verification of the electrode placements.
Apparatus The discrimination experiments were conducted in 4 test chambers (32 x 32 x 40 cm), constructed with one aluminum and three Plexiglas walls, a Plexiglas ceiling and a wire grid floor. A food hopper was positioned 8 cm above the floor in the middle of the aluminum wall, with a 28-V house-light located 8 cm directly above. A retractable lever (Coulbourn Instruments, model E23-05, 4 × 4 cm) was stationed 6.5 cm on either side of each food hopper (8 cm above the floor). Each chamber was contained within a soundattenuating outer enclosure (55 × 55 x 60 cm), and ventilation fans masked extraneous noise. Constant current brain-stimulation was delivered from a 6-channel programmable, sine-wave stimulator via electrode leads (Plastic Products, 303-302) that were suspended from Mercotac commutators and passed through openings in the chamber ceilings. An INI computer with MANX software was used to control the experimental events and record responses made by the rats. Stimulation currents were monitored continuously on a Tektronix T912 storage oscilloscope. The self-stimulation tests in Expt. 2 were conducted in 5 chambers (23 × 29 × 46 cm), each having Plexiglas walls and a wire grid floor. A single lever (5 x 8 cm) was mounted on the
177 middle of one wall 2.5 cm above the floor. Constant-current brain stimulation was delivered from 5 independent sine-wave stimulators via electrode leads that were suspended from Mercotac commutators located above the chambers. Responses were recorded on mechanical counters.
out of 10 consecutive sessions. A correct response was defined as the completion of the first FR-32 requirement within a session on the lever appropriate for the injection received on that trial.
Drug discrimination training
Me~o~
Prior to the discrimination training, the rats were given five 30-min intracranial self-stimulation (ICSS) sessions in which they could leverpress for 200-ms trains of 20 #A, 60 Hz sine wave stimulation on a continuous reinforcement C R F schedule. This initial screening ensured that the brain stimulation was capable of supporting ICSS. The rats were then trained to lever-press for food (45 mg Bioserve pellets) on a C R F schedule during four 30-min drug-free sessions. Subsequently, the rats were given daily 30-min discrimination trials, with either 1.0mg/kg D-amphetamine sulphate (Smith, Kline and French; dissolved in 0.9~o saline to a concentration of 1.0 mg/ml) or 1.0 mg/kg saline injected intraperitoneally immediately prior to each session. During the initial 15 min of the session the house-light was turned off, the levers were retracted, and the pellet dispenser was inactivated. Subsequently, the house-light was turned on and the levers inserted for the remaining 15min, and the rats could obtain food by responding selectively on one of the two levers following injections of D-amphetamine or on the alternative lever after saline injections. The lever appropriate for each injection was counterbalanced among rats and responses on the inappropriate lever were recorded but had no programmed consequences. Throughout training the D-amphetamine and saline trials were presented randomly, but neither solution was administered on more than two consecutive sessions. During the first 4 discrimination sessions, each response on the appropriate lever resulted in the delivery of a food pellet. Subsequently, the number of responses required for each food pellet was doubled after every fourth session, until a fixedratio-32 (FR-32) reinforcement schedule was in effect. Training then continued with the FR-32 schedule until the rats responded correctly on 8
Fifteen rats that acquired the D-amphetamine discrimination were given generalization tests with saline or a range of D-amphetamine doses (0.125, 0.25, 0.5 and 1.0 mg/kg)given either alone or in combination with intermittent VTA stimulation. At the start of the brain stimulation trials, the rats were injected with one of the solutions and then immediately attached to electrode leads and placed in the testing chambers. The intermittent delivery of electrical stimulation then began 3 min later and continued throughout the test session. The stimulation was maintained at a constant intensity within each test (15 or 20/~A), and presented every 10 s. Each presentation consisted of four 200-ms trains of 60 Hz sine-wave stimulation delivered 200 ms apart. During generalization tests with D-amphetamine alone, stimulation was not delivered. The house-light was turned on after 15 min and the responses on both levers were recorded. The lever appropriate for responding during generalization tests could not be determined a priori, thus the lever providing reinforcement during these sessions was determined by the rat's initial lever selection. Specifically, the first lever on which 32 responses were made was designated as the 'selected lever', regardless of whether additional responses were made on the alternative lever prior to the completion of the first FR-32 requirement. Reinforcement was delivered for the first and each subsequent completion of an FR-32 on this lever. After the selected lever had been determined, responses on the alternative ('non-selected') lever had no programmed consequence. The tests were spaced at least two days apart, with saline and D-amphetamine baseline trials occurring on the intervening days. The order in which D-amphetamine doses and stimulation currents were administered was counterbalanced for all rats.
EXPERIMENT
1
178 The percentage of responses emitted prior to the first reinforcement that occurred on the D-amphetamine-appropriate lever was determined for each test session. These percentages were averaged across rats and dose-response functions were plotted to indicate the amount of responding on the D-amphetamine-appropriate lever at each dose as a function of the stimulation current. These data were then analyzed using a two-way repeated measures analysis of variance (ANOVA) with the current intensity as one factor and the D-amphetamine dose as the second factor. Additional two-way, repeated measures ANOVA's were performed both on the total number of responses emitted during the last 15 min of the generalization sessions and on the percentages of the responses occurring after the first reinforcement that were emitted on the initially selected (i.e. reinforced) lever. These latter analyses might reveal possible disruptive influences of the VTA stimulation on operant performance. When significant main effects were found with any of the above ANOVA's, post hoc analyses were performed using Newman-Keuls test. Differences revealed with both the ANOVA's and Newman-Keuls tests were considered significant when the probability level was tess than 0.05. Results
The electrode placements for the 15 rats employed in the present experiment are included in Fig. 2. These rats reached the criterion of 8 correct responses in 10 trials within 20-30 training sessions. Subsequent tests with a range of D-amphetamine doses resulted in orderly stimulus generalization gradients, with the amount of responding on the D-amphetamine-appropriate lever increasing monotonically as the D-amphetamine dose was increased from 0.0 mg/kg (i.e. saline) to 1.0 mg/kg (see Fig. 1). An ANOVA performed on the D-amphetamine dosage effects confirmed the significance of this trend (F4.56 = 25.93; P < 0.0001). The presentation of VTA stimulation during trials with saline or low-dose D-amphetamine injections resulted in increased levels of responding on the D-amphetamine-appropriate lever, so
A. AVERAGE
~
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g
C. CONTRALATERAL
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10
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AM~AW~ OOSe~/k~ Fig. 1. Effects of electrical stimulation of the VTA on amphetamine stimulus generalization functions. Rats were tested for generalization to amphetamine in the absence of VTA stimulation (O), with the delivery of VTA stimulation at a constant intensity of 15 y A (O), or with VTA stimulation delivered at a constant intensity of 20 #A (I-q), A: generalization functions averaged across all rats. B: generalization functions obtained from rats for which the drug-lever was ipsilateral to the stimulating electrode. C: generalization functions obtained from rats for which the drug-lever was contralateral to the stimulating electrode. Significant increases in drug-lever responding were observed under all 3 conditions, causing the generalization functions to be shifted relative to the control curves.
that the D-amphetamine stimulus generalization functions were shifted relative to the control curve (see Fig. 1A). Statistical analysis of the current intensity variable confirmed the significance of this effect (F2,28 = 5.34; P < 0.025), and post hoc analyses indicated that both intensities elicited significantly more drug-appropriate responses relative to tests in the absence of the stimulation. Analyses of the interaction between the current intensity and D-amphetamine dosage variables also revealed significant effects (F8,1o4 = 5.84; P < 0.0001), which were due to the selective increases in drug-lever responses when each stimulation intensity was delivered in combination with either saline or the lower doses of D-amphetamine (0.125 and 0.25 mg/kg). An asymmetrical bias in sensorimotor function associated with unilateral stimulation of the VTA could have influenced lever selection. Therefore the data were re-analyzed according to whether the drug-lever was ipsilateral or contralateral to the stimulating electrode. These analyses indicated that responses on the D-amphetamine appropriate lever during brain stimulation trials were increased relative to trials conducted in the
179 TABLE I
Total number of responses during tests with VTA stimulation Intensity level
No stimulation 15 ~A 20 ~A
Amphetamine dose (mg/kg) 0.0
0.125
0.25
0.5
1.0
1978 1709 1257
1660 1406 1071
1788 1548 1235
1978 1318 939
1498 859 643
ability to produce rewarding effects. This was achieved by correlating the extent of generalization between the stimulus properties of D-amphetamine and those of VTA stimulation, with the self-stimulation rates obtained from the same electrodes. The subjects employed for this experiment included the 15 rats employed in Expt. 1 and 5 additional male hooded rats that were experimentally naive.
*Data shown are the mean values of all subjects tested.
Me~o~
absence of stimulation, for both the ipsilateral group (Fig. 1B; intensity × D-amphetamine dosage interaction: F8,43 = 4.74; P < 0.0005) and the contralateral group (Fig. 1C; intensity effect: F2,z8 = 5.34; P < 0.025; intensity × D-amphetamine dosage interaction: F8,53= 2.81 ; P < 0.01). These results confirmed that VTA stimulation could increase responding on the drug-appropriate lever, regardless of whether the lever was ipsilateral or contralateral to the stimulating electrode. The VTA stimulation also influenced the rate of operant responding during generalization trials (Table 1; F2,28 = 26.31; P < 0.0001). Posthoc analyses of this effect revealed that responses were reduced significantly at both stimulation intensities relative to tests without stimulation, with significantly fewer responses being emitted at the higher intensity (20 #A) relative to tests with the lower intensity (15/~A). In contrast to these effects on response rates, the stimulation did not affect the consistency of responding on the lever initially selected. In general, the rats continued to respond on the lever selected initially, regardless of whether VTA stimulation was present or absent (percentage of selected-lever responses: range of means = 91 to 99~o). Therefore the stimulation did not significantly affect the control of discriminative responding by the reinforcing stimulus.
All subjects were first trained to discriminate 1.0mg/kg D-amphetamine from saline, as described in Expt. 1. The rats were then given 6 drug-free generalization tests with VTA stimulation. The parameters of VTA stimulation were varied with respect to current intensity (15-20/~A) and interstimulation interval (10 s [0.1 Hz], 5 s [0.2 Hz], 2.5 s [0.4 Hz]), to determine the optimal conditions for generalization. Each rat was given a saline injection and placed in the chamber with the electrode leads attached. As in Expt. 1, the intermittent presentations of VTA stimulation began 3 rain after the start of the half-hour session and continued throughout. Each presentation consisted of four 200-msec trains of 60 Hz sine-wave stimulation, delivered 200 ms apart. Two days after the final generalization test, each rat was placed in a separate self-stimulation chamber and given 10 free stimulations (200-ms trains of 60 Hz sine-wave spaced 1 s apart) of 15-#A current intensity. The rats could then leverpress during a 5-min period to receive single trains of VTA stimulation on a CRF reinforcement schedule. The total number of responses emitted during this period were recorded on mechanical counters. Subsequently, the current intensity was increased to 20 #A and responses were recorded for a further 5 rain. Rats that were not already lever-pressing at the end of the first 5 min were given 10 free stimulations at the higher intensity.
EXPERIMENT2
Results This experiment assessed whether the capacity of VTA stimulation to produce D-amphetaminelike stimulus properties might be related to its
An examination of the degree of responding on the drug-appropriate lever during trials with
180 PLATE #
45b
46b
47b
48b
49b
Fig. 2. Relationship between the electrode placements and stimulus generalization with amphetamine for the 20 rats employed in Expt. 2. The different symbols reflect the levels of drug-appropriate responding (V, > 67 % ; I I , 33-67 % ; O, < 33 %) when each electrode site was stimulated at a current intensity of either 15/~A (left sides of the sections) or 20/~A (right sides of the sections). The response percentages for each group of rats were obtained by averaging over the tests with different stimulus presentation rates. Plate numbers to the right of the diagrams correspond to the coronal sections from the brain atlas of K~nig and KlippeP 8.
brain-stimulation revealed pronounced individual differences within the group. Eight of the rats responded primarily on the drug lever ( > 67 % of pre-reinforcement responses) across all tests with VTA stimulation, while 6 rats responded primarily on the saline lever ( < 33 % of pre-reinforcement responses on the drug-lever). Fig. 2 indicates the electrode placements for the rats employed in this experiment. Inspection of the figure reveals that moderate to high levels of drug-appropriate responding could be obtained from stimulation of regions surrounding the anterior portion of the interpeduncular nucleus (Plates 47b and 48b) and ventromedial placements at a level just anterior to this nucleus (Plate 46b). Placements that produced the least amount of drug-appropriate responding were located pri-
marily in the dorsal part of the region just anterior to the interpeduncular nucleus (Plates 45b and 46b). Two additional placements associated with low levels of drug-appropriate responding were situated at dorsal and ventral extremities in the posterior VTA (Plate 49b). Statistical analyses of the effects of different stimulation parameters on the mean levels of drug-lever responding did not reveal any significant differences related to either the current intensity or the stimulus presentation rate (range of means = 48-64~o). However, a significant interaction between these variables was obtained (F2.34 = 6.75; P < 0.005). The 20-/2A current intensity elicited significantly more drug-lever responding (64 % ) than the 15-/~A intensity (48 % ) when the stimulation was presented at 0.4 Hz, but not when it was delivered at 0.1 or 0.2 Hz. During subsequent self-stimulation tests all of the rats responded for the VTA stimulation. However, as illustrated in Fig. 3, there were substantial individual differences in the ICSS rates among rats. These differences did not result from differences in the maximum lever-pressing rates of the rats. In fact, each rat showed an increase in response rate when the current intensity was increased from 15 to 20 #A (mean rates = 290 and 429 presses/5 min, respectively; two-tailed tl9 = 7.77; P < 0.0001), indicating that the rats were responding at sub-asymptotic levels for the lower current intensity. It is likely that the individual response rates reflected differences in the degree to which processes mediating the rewarding effects of VTA stimulation could be activated by each electrode. To determine whether the individual response rates correlated positively with the percentages of drug-lever responses elicited by the stimulation, the ICSS and stimulus generalization data obtained at each intensity were analyzed separately using a Pearson's correlation coefficient. The drug-lever response levels employed for this analysis were obtained by averaging across the 3 presentation frequencies for each intensity. These analyses revealed significant positive correlations between drug-lever responses and ICSS rates for tests with both the low current intensity (15/~A, Fig. 3a; r = 0.46; d.f.-- 19; P < 0.05) and the
181 R 2CX~Ir E .7~
i 10080-
f
a
2b ,'o ab 8b ~o i~0
; 2'o ,'0 8b 8b ,~o ~0
Fig. 3. Scattergrams showing the correlations between stimulus generalization with amphetamine and ICSS rates obtained with VTA stimulation. The data are expressed as the percent of responses emitted on the drug-lever during generalization tests (Y-axis) relative to the response rates for the VTA stimulation (X-axis; average responses per min) obtained during ICSS tests at: A: 15 #A (Pearson's r = 0.46; P < 0.05); and B: 2 0 / t A (Pearson's r = 0.71; P < 0.01). The generalization data represent the averaged scores for the 3 tests at the different rates of stimulus presentation. The solid lines through the scattergrams represent the lines of best fit obtained from the regression equations.
higher current intensity (20 #A, Fig. 3b; r = 0.71; d.f. = 18; P < 0.01). As might be expected from the behavioral correlations described above, the relationship between ICSS rates and electrode placements for each rat was similar to the relationship observed between the elicitation of drug-lever responding and the electrode placements. Moderate to high self-stimulation rates were obtained from all but 6 sites when the current intensity was set at 15 #A. Four of these 6 sites were situated at the extremities of the VTA and correspond to the sites which were also associated with low levels of drug-appropriate responding during stimulus generalization tests. All of the sites yielded moderate to high levels of ICSS when the current intensity was set at 20 #A. Many of the sites that yielded high self-stimulation rates also were associated with high levels of D-amphetamine-appropriate responding during generalization tests, whereas sites that yielded only moderate ICSS rates tended to produce only moderate to low levels of drug-appropriate responding.
DISCUSSION
The present study examined whether the stimulus properties produced by 1.0mg/kg D-amphetamine could be mimicked by electrical stimulation of the VTA in rats. The first experiment assessed the effects of unilateral VTA stimulation on D-amphetamine stimulus generalization functions. VTA stimulation (15 and 20#A) increased drug-appropriate responses elicited during saline and low-dose D-amphetamine trials, so that the D-amphetamine stimulus generalization functions were shifted relative to the control curves. Therefore, VTA brain stimulation appears both to substitute partially for the stimulus properties of D-amphetamine, and to augment the cueing effects of low D-amphetamine doses. Although unilateral stimulation of regions containing DA neurons can produce both contralateral circling behavior 12 and greater responsiveness to stimuli in the contralateral sensory f i e l d 2'3'24, such sensorimotor effects did not appear to be responsible for the increased responding on the drug-lever. The rats increased their responses on the drug-lever during stimulation trials regardless of whether this lever was ipsilateral or contralateral to the stimulating electrode. The effects of the stimulation on D-amphetamine generalization functions were stronger when the drug-lever was contralateral to the electrode. Therefore, the sensorimotor effects may have exaggerated the response biases of these rats. However, the significant elevation observed with the ipsilateral group suggests that the main effect of the stimulation was to produce stimulus properties which could interact in an additive manner with the cueing effects of D-amphetamine. The summation observed between the stimulus properties of VTA stimulation a n d D-amphetamine in Expt. 1 contrasts with the findings of a previous study 8 in which VTA stimulation appeared to have only disruptive effects on discriminated responses to a D-amphetamine stimulus. However, the stimulation parameters employed in that study consisted of trains with short-duration (i.e. 0.2 ms) square-wave pulses. Recent in vivo electrochemical measurements have indicated that such parameters may be insuf-
182 ficient to activate mesotelencephalic DA neurons 2~. In the present investigation, 60-Hz sine-wave currents were used to stimulate the VTA. This type of stimulation appears to be adequate for activating DA neurons, as indicated by the increases in DA release and turnover within the nucleus accumbens of rats following sinewave stimulation of the V T A 4A1'27. Conceivably, this capacity to activate mesoaccumbens DA neurons could have accounted for the summation between the stimulus properties of the brain stimulation and those of D-amphetamine in the present study. The results of Expt. 2 again demonstrated that VTA stimulation could generalize with the stimulus properties of D-amphetamine. Comparisons of the data obtained during generalization and ICS S tests for each rat revealed a significant positive correlation between the amount of drugappropriate responding elicited by the stimulation and the rate of responding for the brain stimulation reward. Thus, stimulus generalization with D-amphetamine was strongest when the VTA stimulation produced high rates of responding. Although under certain circumstances response rates may not be the most precise index of brain stimulation r e w a r d 2°'33'34'35, individual differences in this measure can provide an estimate of the relative efficacy with which the stimulating currents activate reward processes within separate animals. Rats responding at higher rates for a moderate current intensity of VTA stimulation often will have both lower self-stimulation thresholds and rate-intensity curves that are displaced leftward relative to rats that respond at low rates for the same intensity*. Given this relationship between response rates and other measures of the * The relationship between rate and threshold measures of brain stimulation reward was confirmed by analyses of baseline random rate-intensity functions obtained from an independent group of 25 rats with VTA electrodes. Significant negative correlations were obtained between current thresholds for each rat and response rates measured at current intensities ranging from 10 to 20 #A (i.e. rats with higher rates had lower thresholds; the range of Pearsons correlation coefficients were r = -0.40 to r = - 0 . 8 9 ; df = 24, P < 0.05). This relationship is not maintained when rates are at asymptotic levels2°'33'34; however the rates measured in Expt. 2 were below asymptotic levels.
rewarding efficacy of brain stimulation, the results of Expt. 2 suggest that the ability of the VTA stimulation to generalize with D-amphetamine may be related to its capacity to produce rewarding effects. In particular, the generalization may have been due to rewarding effects that arise from the activation of the mesolimbic DA pathw a y s 11'22. The stimulus generalization with D-amphetamine was strongest when the electrodes were located within regions of the VTA that contain high densities of DA perikarya "~. Furthermore, VTA stimulation did not generalize or summate with the stimulus properties of D-amphetamine in a previous study8 that employed stimulation parameters that were capable of producing rewarding effects, but which were inappropriate for activating DA neurons. The apparent hedonic basis for stimulus generalization between D-amphetamine and VTA stimulation observed in the present study suggests that the stimulus properties of D-amphetamine may be related to the positive affective properties of the drug. These positive affective properties appear to be responsible for the abuse potential of D-amphetamine and D-amphetamine-like compounds in humans 32. Accordingly, studies of the stimulus properties of D-amphetamine may provide important insights into the neuropharmacological basis of drug abuse. ACKNOWLEDGEMENTS
The research was supported by Grant PG-23 from the Medical Research Council of Canada. J.P.D. was supported by a Macmillan Family Graduate Fellowship. The authors would like to thank Chris Yamakura for her technical assistance. REFERENCES l Aulisi, E.F. and Hoebel, B.G., Rewarding effects of amphetamine and cocaine in the nucleus accumbens and block by alpha-flupenthixol, Soc. Neurosci. Abstr., 9 (1983) 121. 2 Bandler, R. and Flynn, J.P., Visual patterned reflex present during hypothalamically elicited attack, Science, 171 (1971) 817-818. 3 Beagley, W.K. and Holley, T.L., Hypothalamic stimula-
183 tion facilitates contralateral visual control of a learned response, Science, 196 (1977) 321-322. 4 Blaha, C.D., Phillips, A.G. and Fibiger, H.C., Self-stimulation of the ventral tegrnentum and concurrent release of dopamine measured by in vivo electrochemistry, Soc. Neurosci. Abstr., 14 (1988) 742. 5 Carr, G.D. and White, N.M., Conditioned place preference from intra-accumbensbut not intra-caudate amphetamine injections, Life Sci., 33 (1983) 2551-2557. 6 Carr, G.D. and White, N.M., Anatomical disassociation of amphetamine's rewarding and aversive effects: an intracranial microinjection study, Psychopharmacology, 89 (1986) 340-346. 7 Colpaert, F.C., Niemegeers, C.J.E. and Janssen, P.A.J., Discriminative stimulus properties of cocaine and D-amphetamine, and antagonism by haloperidol: a comparative study, Neuropharmacology, 17 (1978) 937-942. 8 D'Mello, G.D., A comparison of some behavioral effects of amphetamine and electrical brain stimulation of the mesolimbic dopamine system in rats, Psychopharmacology, 75 (1981) 184-192. 9 Druhan, J.P., Martin-Iverson, M.T., Wilkie, D.M., Fibiger, H.C. and Phillips, A.G., Dissociation of dopaminergic and non-dopaminergicsubstrates for cues produced by electrical stimulation of the ventral tegmental area, Pharmacol. Biochem. Behav., 28 (1987) 251-259. 10 Fallon, J.H. and Moore, R.Y., Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum, J. Comp. Neurol., 180 (1978) 545-580. 11 Fibiger, H.C., LePiane, F.G., Jakubovic, A. and Phillips, A.G., The role of dopamine in intracranial self-stimulation of the ventral tegmental area, J. Neurosci., 7 (1987) 3888-3896. 12 Gratton, A. and Wise, R.A., Mapping of contraversive and ipsiversive circling responses to ventral tegmental and substantia nigra electrical stimulation, Physiol. Behav., 35 (1985) 61-65. 13 Ho, B.T. and Huang, J.T., Role ofdopamine in D-amphetamine-induced discriminative responding, Pharmacol. Biochem. Behav., 3 (1975) 1085-1092. 14 Ho, B.T. and McKenna, M.L., Discriminative stimulus properties of central stimulants. In B.T. Ho, D.W. Richards III and D.L. Chute (Eds.), Drug Discrimination and State-Dependent Learning, Academic Press, New York, 1978, pp. 67-77. 15 Huang, J.T. and Ho, B.T., Discriminative stimulus properties of D-amphetamine and related compounds in rats, Pharmacol. Biochem. Behav., 2 (1974) 669-673. 16 Jarbe, T.U.C., Discriminative stimulus properties of D-amphetamine in pigeons, Pharmacol. Biochem. Behav., 17 (1982) 671-675. 17 Jones, C.N., Hill, H.F. and Harris, R.T., Discriminative response control by D-amphetamine and related compounds in the rat, Psychopharmacologia, 36 (1974) 347-356. 18 K~nig, J.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas, Williams and Wilkins, Baltimore 1963.
19 Lyness, W.H., Friedle, N.M. and Moore, K.E., Destruction of dopaminergic nerve terminals in the nucleus accumbens: effect on D-amphetamine self-administration, Pharmacol. Biochem. Behav., 11 (1979) 553-556. 20 Miliaressis, E. and Malette, J., Summation and saturation properties in the rewarding effect of brain stimulation, Physiol. Behav., 41 (1987) 595-604. 21 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the median forebrain bundle, Eur. J. Pharmacol., 109 (1985) 341-348. 22 Mogenson, G.J., Takigawa, M., Robertson, A. and Wu, M., Self-stimulation of the nucleus accumbens and ventral tegmental area of Tsai attenuated by microinjections of spiroperidol into the nucleus accumbens, Brain Res., 171 (1979) 247-259. 23 Monaco, A.P., Hernandez, L. and Hoebel, B.G., Nucleus accumbens: site of amphetamine injection: comparison with the lateral ventricle. In R.B. Chronister and J.F. DeFrance (Eds.), The Neurobiology of the Nucleus Accumbens, Haer Institute, New Brunswick, 1980, pp. 338-342. 24 Nakahara, D. and Ikeda, T., Differential behavioral responsiveness to ipsilateral and contralateral visual stimuli produced by unilateral rewarding hypothalamic stimulation in the rat, Physiol. Behav., 32 (1984) 1005-1010. 25 Nielsen, E.B. and Jepsen, S.A., Antagonism of the amphetamine cue by both classical and atypical antipsychotic drugs, Eur. J. Pharmacol., 111 (1985) 167-176. 26 Nielsen, E.B. and Scheel-Kruger, J., Cueing effects of amphetamine and LSD: elicitation by direct microinjection of the drugs into the nucleus accumbens, Eur. J. Pharmacol., 125 (1986) 85-92. 27 Phillips, A.G., Blaha, C.D. and Fibiger, H.C., Neurochemical correlates of brain stimulation reward measured by ex vivo and in vivo analyses, Neurosci. Biobehav. Rev., 13 (1989) 99-104. 28 Schecter, M.D., Amphetamine discrimination as a test for anti-Parkinsonism drugs, Eur. J. Pharmacol., 44 (1977) 51-56. 29 Schecter, M.D. and Cook, P.G., Dopaminergic mediation of the interoceptive cue produced by D-amphetamine in rats, Psychopharmacologia, 42 (1975) 185-193. 30 Silverman, P.B. and Ho, B.T., Characterization of discriminative response control by psychomotor stimulants, in H. Lal (Ed.), Discriminative Stimulus Properties of Drugs, Plenum, New York, 1977, pp. 107-119. 31 Spyraki, C., Fibiger, H.C. and Phillips, A.G., Dopaminergic substrates of amphetamine-induced place preference conditioning, Brain Res., 253 (1982) 185-193. 32 Stewart, J., De Wit, H. and Eikelboom, R., Role of unconditioned and conditioned drug effects in the self-administration of opiates and stimulants, Psychol. Rev., 91 (1984) 211-227. 33 Valenstein, E., Problems of measurement and inter-
184 pretation with reinforcing brain stimulation, Psychol.
Rev., 71 (1964) 415-437. 34 Waraczynski, M., Stellar, J.R. and GaUistel, C.R., Reward summation in medial forebrain bundle self-
stimulation, Physiol. Behav., 41 (1987) 585-593. 35 Yeomans, J.S., Quantitative measurement of neural poststimulation excitability with behavioral methods, Physiol. Behav., 15 (1975) 593-602.
Behavioural Brain Research, 38 (1990) 175-184 Elsevier BBR 01051
Amphetamine-like stimulus properties produced by electrical stimulation of reward sites in the ventral tegmental area J.P. D r u h a n
1,*,
H . C . Fibiger 2 a n d A . G . Phillips 1
Department of Psychology, and 2Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B. C. (Canada) (Received 15 August 1989) (Revised version received 25 September 1989) (Accepted 5 January 1990)
Key words: Stimulus property; D-Amphetamine; Electrical brain stimulation; Ventral tegmental area; Drug discrimination; Stimulus generalization; Intracranial self stimulation; Rat
The present study examined whether the stimulus properties of D-amphetamine could be mimicked by electrical stimulation of the ventral tegmental area (VTA). Rats trained to discriminate 1.0 mg/kg D-amphetamine from saline were given generalization tests with a range of D-amphetamine doses administered either alone or in combination with VTA stimulation. The results suggested that the VTA stimulation could enhance the cueing effects of D-amphetamine, as levels of responding on the D-amphetamine-appropriate lever during stimulation trials were increased relative to tests without stimulation. Individual differences were observed in the amount of drug-lever responding elicited by the VTA stimulation during drug-free substitution tests, and the different levels of drug-lever responding correlated positively with the response rates obtained from these rats during subsequent intracranial self-stimulation tests. These findings suggest that VTA stimulation can have D-amphetamine-like stimulus properties and such stimulus properties may be related to the rewarding effects of the brain stimulation.
INTRODUCTION
Numerous studies have demonstrated that rats can discriminate between the presence of the psychomotor stimulant, D-amphetamine, and a n o n - d r u g c o n d i t i o n 16,17,26,3°. The neural processes responsible for the transduction of D-amphetamine's actions into stimulus properties appear to involve dopamine-containing neurons. For example, the D-amphetamine stimulus can be
* Present address: Center for Studies in Behavioral Neurobiology, Concordia University, 1455 De Maissoneuve West, Montreal, Quebec, Canada H3A 2J3. Correspondence: A.G. Phillips, Department of Psychology, University of British Columbia, Vancouver, B.C., Canada V6T IY7.
mimicked by dopamine (DA) receptor agonists and by drugs which increase the release or block the uptake of this transmitter 7"~4,~5,28.Conversely, D-amphetamine stimuli can be attenuated by DA receptor antagonists or synthesis inhibitors 13'25'29. Such results are not obtained with drugs that specifically affect noradrenergic or serotonergic neurotransmission 13,29,3°. Recent studies have revealed an important role for the mesolimbic DA projection in transducing D-amphetamine stimuli. For example, the stimulus properties of D-amphetamine were antagonized by atypical neurolepticsz5 which are thought to preferentially block DA receptors in the mesolimbic DA system. In a subsequent study26, rats trained to discriminate the stimulus properties of systemic D-amphetamine emitted drug-appro-
0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 priate responses when D-amphetamine was injected directly into the nucleus accumbens. This effect was blocked by co-injections of the DA receptor antagonist sulpiride. If the stimulus properties of D-amphetamine are related to the facilitatory actions of this drug on mesolimbic DA neurotransmission, these stimulus properties may be mimicked by electrical stimulation ofmesolimbic DA neurons. The present study assesses whether electrical stimulation of mesolimbic DA neurons within the ventral tegmental area (VTA) can augment the stimulus properties of low D-amphetamine doses in rats trained to discriminate 1.0 mg/kg D-amphetamine from saline. Recent in vivo electrochemical and ex vivo chromatographic measures indicate that VTA stimulation can induce the release of DA in the ipsilateral nucleus accumbens 4'1''27. Psychopharmacological studies also suggest that VTA stimulation can produce stimuli that are mediated by DA-containing neurons 9. The present study also investigated the relationship between the rewarding effect of VTA stimulation and the production of D-amphetamine-like stimulus properties. Specifically, it was hypothesized that because the rewarding effects of D-amphetamine and VTA stimulation appear to be mediated by DA projections to the nucleus accumbensl.5,6,11,19,23,27.31 a common dopaminergic substrate may mediate their rewarding and stimulus properties. To examine this, the correlation between the level of D-amphetamineappropriate responding produced by the VTA stimulation, and measures of the rewarding effects of the same VTA stimulation were determined.
GENERAL METHODS
Subjects Male hooded rats (Charles River, Long-Evans strain) served as subjects. All rats were housed individually in stainless steel cages with tap water available adlibitum. Access to food was restricted, and 22-24 g of standard rat chow was provided once each day. The rats were tested towards the end of the 12-h light phase of a 24-h light-dark cycle.
Surgery and histology Prior to discrimination training, each rat was anesthetized with 65 mg/kg sodium pentobarbital and a bipolar electrode (Plastic Products MS303/2) was implanted into the VTA. The coordinates for stereotaxic implantation were: 3.1 mm anterior and 1.7 mm dorsal to the interaural line, and 0.6 mm lateral to the midline (in the left hemisphere). The incisor bar was set 3.2 mm below the interaural line. Electrodes were anchored chronically to the skull with jeweller's screws and dental cement. Upon completion of the experiments, the rats were given an overdose of sodium pentobarbital and perfused transcardially with 0.9~o saline followed by 10~o formol-saline. The brains were removed, sliced in 30-/~m frozen sections, and stained with Cresyl violet for microscopic verification of the electrode placements.
Apparatus The discrimination experiments were conducted in 4 test chambers (32 x 32 x 40 cm), constructed with one aluminum and three Plexiglas walls, a Plexiglas ceiling and a wire grid floor. A food hopper was positioned 8 cm above the floor in the middle of the aluminum wall, with a 28-V house-light located 8 cm directly above. A retractable lever (Coulbourn Instruments, model E23-05, 4 × 4 cm) was stationed 6.5 cm on either side of each food hopper (8 cm above the floor). Each chamber was contained within a soundattenuating outer enclosure (55 × 55 x 60 cm), and ventilation fans masked extraneous noise. Constant current brain-stimulation was delivered from a 6-channel programmable, sine-wave stimulator via electrode leads (Plastic Products, 303-302) that were suspended from Mercotac commutators and passed through openings in the chamber ceilings. An INI computer with MANX software was used to control the experimental events and record responses made by the rats. Stimulation currents were monitored continuously on a Tektronix T912 storage oscilloscope. The self-stimulation tests in Expt. 2 were conducted in 5 chambers (23 × 29 × 46 cm), each having Plexiglas walls and a wire grid floor. A single lever (5 x 8 cm) was mounted on the
177 middle of one wall 2.5 cm above the floor. Constant-current brain stimulation was delivered from 5 independent sine-wave stimulators via electrode leads that were suspended from Mercotac commutators located above the chambers. Responses were recorded on mechanical counters.
out of 10 consecutive sessions. A correct response was defined as the completion of the first FR-32 requirement within a session on the lever appropriate for the injection received on that trial.
Drug discrimination training
Me~o~
Prior to the discrimination training, the rats were given five 30-min intracranial self-stimulation (ICSS) sessions in which they could leverpress for 200-ms trains of 20 #A, 60 Hz sine wave stimulation on a continuous reinforcement C R F schedule. This initial screening ensured that the brain stimulation was capable of supporting ICSS. The rats were then trained to lever-press for food (45 mg Bioserve pellets) on a C R F schedule during four 30-min drug-free sessions. Subsequently, the rats were given daily 30-min discrimination trials, with either 1.0mg/kg D-amphetamine sulphate (Smith, Kline and French; dissolved in 0.9~o saline to a concentration of 1.0 mg/ml) or 1.0 mg/kg saline injected intraperitoneally immediately prior to each session. During the initial 15 min of the session the house-light was turned off, the levers were retracted, and the pellet dispenser was inactivated. Subsequently, the house-light was turned on and the levers inserted for the remaining 15min, and the rats could obtain food by responding selectively on one of the two levers following injections of D-amphetamine or on the alternative lever after saline injections. The lever appropriate for each injection was counterbalanced among rats and responses on the inappropriate lever were recorded but had no programmed consequences. Throughout training the D-amphetamine and saline trials were presented randomly, but neither solution was administered on more than two consecutive sessions. During the first 4 discrimination sessions, each response on the appropriate lever resulted in the delivery of a food pellet. Subsequently, the number of responses required for each food pellet was doubled after every fourth session, until a fixedratio-32 (FR-32) reinforcement schedule was in effect. Training then continued with the FR-32 schedule until the rats responded correctly on 8
Fifteen rats that acquired the D-amphetamine discrimination were given generalization tests with saline or a range of D-amphetamine doses (0.125, 0.25, 0.5 and 1.0 mg/kg)given either alone or in combination with intermittent VTA stimulation. At the start of the brain stimulation trials, the rats were injected with one of the solutions and then immediately attached to electrode leads and placed in the testing chambers. The intermittent delivery of electrical stimulation then began 3 min later and continued throughout the test session. The stimulation was maintained at a constant intensity within each test (15 or 20/~A), and presented every 10 s. Each presentation consisted of four 200-ms trains of 60 Hz sine-wave stimulation delivered 200 ms apart. During generalization tests with D-amphetamine alone, stimulation was not delivered. The house-light was turned on after 15 min and the responses on both levers were recorded. The lever appropriate for responding during generalization tests could not be determined a priori, thus the lever providing reinforcement during these sessions was determined by the rat's initial lever selection. Specifically, the first lever on which 32 responses were made was designated as the 'selected lever', regardless of whether additional responses were made on the alternative lever prior to the completion of the first FR-32 requirement. Reinforcement was delivered for the first and each subsequent completion of an FR-32 on this lever. After the selected lever had been determined, responses on the alternative ('non-selected') lever had no programmed consequence. The tests were spaced at least two days apart, with saline and D-amphetamine baseline trials occurring on the intervening days. The order in which D-amphetamine doses and stimulation currents were administered was counterbalanced for all rats.
EXPERIMENT
1
178 The percentage of responses emitted prior to the first reinforcement that occurred on the D-amphetamine-appropriate lever was determined for each test session. These percentages were averaged across rats and dose-response functions were plotted to indicate the amount of responding on the D-amphetamine-appropriate lever at each dose as a function of the stimulation current. These data were then analyzed using a two-way repeated measures analysis of variance (ANOVA) with the current intensity as one factor and the D-amphetamine dose as the second factor. Additional two-way, repeated measures ANOVA's were performed both on the total number of responses emitted during the last 15 min of the generalization sessions and on the percentages of the responses occurring after the first reinforcement that were emitted on the initially selected (i.e. reinforced) lever. These latter analyses might reveal possible disruptive influences of the VTA stimulation on operant performance. When significant main effects were found with any of the above ANOVA's, post hoc analyses were performed using Newman-Keuls test. Differences revealed with both the ANOVA's and Newman-Keuls tests were considered significant when the probability level was tess than 0.05. Results
The electrode placements for the 15 rats employed in the present experiment are included in Fig. 2. These rats reached the criterion of 8 correct responses in 10 trials within 20-30 training sessions. Subsequent tests with a range of D-amphetamine doses resulted in orderly stimulus generalization gradients, with the amount of responding on the D-amphetamine-appropriate lever increasing monotonically as the D-amphetamine dose was increased from 0.0 mg/kg (i.e. saline) to 1.0 mg/kg (see Fig. 1). An ANOVA performed on the D-amphetamine dosage effects confirmed the significance of this trend (F4.56 = 25.93; P < 0.0001). The presentation of VTA stimulation during trials with saline or low-dose D-amphetamine injections resulted in increased levels of responding on the D-amphetamine-appropriate lever, so
A. AVERAGE
~
B.I~kATI~:tAk
60-
/
_1
g
C. CONTRALATERAL
,__._.---,
1oo
10
,j
/ 6 .~5 ~b
/
r 1.6
AM~AW~ OOSe~/k~ Fig. 1. Effects of electrical stimulation of the VTA on amphetamine stimulus generalization functions. Rats were tested for generalization to amphetamine in the absence of VTA stimulation (O), with the delivery of VTA stimulation at a constant intensity of 15 y A (O), or with VTA stimulation delivered at a constant intensity of 20 #A (I-q), A: generalization functions averaged across all rats. B: generalization functions obtained from rats for which the drug-lever was ipsilateral to the stimulating electrode. C: generalization functions obtained from rats for which the drug-lever was contralateral to the stimulating electrode. Significant increases in drug-lever responding were observed under all 3 conditions, causing the generalization functions to be shifted relative to the control curves.
that the D-amphetamine stimulus generalization functions were shifted relative to the control curve (see Fig. 1A). Statistical analysis of the current intensity variable confirmed the significance of this effect (F2,28 = 5.34; P < 0.025), and post hoc analyses indicated that both intensities elicited significantly more drug-appropriate responses relative to tests in the absence of the stimulation. Analyses of the interaction between the current intensity and D-amphetamine dosage variables also revealed significant effects (F8,1o4 = 5.84; P < 0.0001), which were due to the selective increases in drug-lever responses when each stimulation intensity was delivered in combination with either saline or the lower doses of D-amphetamine (0.125 and 0.25 mg/kg). An asymmetrical bias in sensorimotor function associated with unilateral stimulation of the VTA could have influenced lever selection. Therefore the data were re-analyzed according to whether the drug-lever was ipsilateral or contralateral to the stimulating electrode. These analyses indicated that responses on the D-amphetamine appropriate lever during brain stimulation trials were increased relative to trials conducted in the
179 TABLE I
Total number of responses during tests with VTA stimulation Intensity level
No stimulation 15 ~A 20 ~A
Amphetamine dose (mg/kg) 0.0
0.125
0.25
0.5
1.0
1978 1709 1257
1660 1406 1071
1788 1548 1235
1978 1318 939
1498 859 643
ability to produce rewarding effects. This was achieved by correlating the extent of generalization between the stimulus properties of D-amphetamine and those of VTA stimulation, with the self-stimulation rates obtained from the same electrodes. The subjects employed for this experiment included the 15 rats employed in Expt. 1 and 5 additional male hooded rats that were experimentally naive.
*Data shown are the mean values of all subjects tested.
Me~o~
absence of stimulation, for both the ipsilateral group (Fig. 1B; intensity × D-amphetamine dosage interaction: F8,43 = 4.74; P < 0.0005) and the contralateral group (Fig. 1C; intensity effect: F2,z8 = 5.34; P < 0.025; intensity × D-amphetamine dosage interaction: F8,53= 2.81 ; P < 0.01). These results confirmed that VTA stimulation could increase responding on the drug-appropriate lever, regardless of whether the lever was ipsilateral or contralateral to the stimulating electrode. The VTA stimulation also influenced the rate of operant responding during generalization trials (Table 1; F2,28 = 26.31; P < 0.0001). Posthoc analyses of this effect revealed that responses were reduced significantly at both stimulation intensities relative to tests without stimulation, with significantly fewer responses being emitted at the higher intensity (20 #A) relative to tests with the lower intensity (15/~A). In contrast to these effects on response rates, the stimulation did not affect the consistency of responding on the lever initially selected. In general, the rats continued to respond on the lever selected initially, regardless of whether VTA stimulation was present or absent (percentage of selected-lever responses: range of means = 91 to 99~o). Therefore the stimulation did not significantly affect the control of discriminative responding by the reinforcing stimulus.
All subjects were first trained to discriminate 1.0mg/kg D-amphetamine from saline, as described in Expt. 1. The rats were then given 6 drug-free generalization tests with VTA stimulation. The parameters of VTA stimulation were varied with respect to current intensity (15-20/~A) and interstimulation interval (10 s [0.1 Hz], 5 s [0.2 Hz], 2.5 s [0.4 Hz]), to determine the optimal conditions for generalization. Each rat was given a saline injection and placed in the chamber with the electrode leads attached. As in Expt. 1, the intermittent presentations of VTA stimulation began 3 rain after the start of the half-hour session and continued throughout. Each presentation consisted of four 200-msec trains of 60 Hz sine-wave stimulation, delivered 200 ms apart. Two days after the final generalization test, each rat was placed in a separate self-stimulation chamber and given 10 free stimulations (200-ms trains of 60 Hz sine-wave spaced 1 s apart) of 15-#A current intensity. The rats could then leverpress during a 5-min period to receive single trains of VTA stimulation on a CRF reinforcement schedule. The total number of responses emitted during this period were recorded on mechanical counters. Subsequently, the current intensity was increased to 20 #A and responses were recorded for a further 5 rain. Rats that were not already lever-pressing at the end of the first 5 min were given 10 free stimulations at the higher intensity.
EXPERIMENT2
Results This experiment assessed whether the capacity of VTA stimulation to produce D-amphetaminelike stimulus properties might be related to its
An examination of the degree of responding on the drug-appropriate lever during trials with
180 PLATE #
45b
46b
47b
48b
49b
Fig. 2. Relationship between the electrode placements and stimulus generalization with amphetamine for the 20 rats employed in Expt. 2. The different symbols reflect the levels of drug-appropriate responding (V, > 67 % ; I I , 33-67 % ; O, < 33 %) when each electrode site was stimulated at a current intensity of either 15/~A (left sides of the sections) or 20/~A (right sides of the sections). The response percentages for each group of rats were obtained by averaging over the tests with different stimulus presentation rates. Plate numbers to the right of the diagrams correspond to the coronal sections from the brain atlas of K~nig and KlippeP 8.
brain-stimulation revealed pronounced individual differences within the group. Eight of the rats responded primarily on the drug lever ( > 67 % of pre-reinforcement responses) across all tests with VTA stimulation, while 6 rats responded primarily on the saline lever ( < 33 % of pre-reinforcement responses on the drug-lever). Fig. 2 indicates the electrode placements for the rats employed in this experiment. Inspection of the figure reveals that moderate to high levels of drug-appropriate responding could be obtained from stimulation of regions surrounding the anterior portion of the interpeduncular nucleus (Plates 47b and 48b) and ventromedial placements at a level just anterior to this nucleus (Plate 46b). Placements that produced the least amount of drug-appropriate responding were located pri-
marily in the dorsal part of the region just anterior to the interpeduncular nucleus (Plates 45b and 46b). Two additional placements associated with low levels of drug-appropriate responding were situated at dorsal and ventral extremities in the posterior VTA (Plate 49b). Statistical analyses of the effects of different stimulation parameters on the mean levels of drug-lever responding did not reveal any significant differences related to either the current intensity or the stimulus presentation rate (range of means = 48-64~o). However, a significant interaction between these variables was obtained (F2.34 = 6.75; P < 0.005). The 20-/2A current intensity elicited significantly more drug-lever responding (64 % ) than the 15-/~A intensity (48 % ) when the stimulation was presented at 0.4 Hz, but not when it was delivered at 0.1 or 0.2 Hz. During subsequent self-stimulation tests all of the rats responded for the VTA stimulation. However, as illustrated in Fig. 3, there were substantial individual differences in the ICSS rates among rats. These differences did not result from differences in the maximum lever-pressing rates of the rats. In fact, each rat showed an increase in response rate when the current intensity was increased from 15 to 20 #A (mean rates = 290 and 429 presses/5 min, respectively; two-tailed tl9 = 7.77; P < 0.0001), indicating that the rats were responding at sub-asymptotic levels for the lower current intensity. It is likely that the individual response rates reflected differences in the degree to which processes mediating the rewarding effects of VTA stimulation could be activated by each electrode. To determine whether the individual response rates correlated positively with the percentages of drug-lever responses elicited by the stimulation, the ICSS and stimulus generalization data obtained at each intensity were analyzed separately using a Pearson's correlation coefficient. The drug-lever response levels employed for this analysis were obtained by averaging across the 3 presentation frequencies for each intensity. These analyses revealed significant positive correlations between drug-lever responses and ICSS rates for tests with both the low current intensity (15/~A, Fig. 3a; r = 0.46; d.f.-- 19; P < 0.05) and the
181 R 2CX~Ir E .7~
i 10080-
f
a
2b ,'o ab 8b ~o i~0
; 2'o ,'0 8b 8b ,~o ~0
Fig. 3. Scattergrams showing the correlations between stimulus generalization with amphetamine and ICSS rates obtained with VTA stimulation. The data are expressed as the percent of responses emitted on the drug-lever during generalization tests (Y-axis) relative to the response rates for the VTA stimulation (X-axis; average responses per min) obtained during ICSS tests at: A: 15 #A (Pearson's r = 0.46; P < 0.05); and B: 2 0 / t A (Pearson's r = 0.71; P < 0.01). The generalization data represent the averaged scores for the 3 tests at the different rates of stimulus presentation. The solid lines through the scattergrams represent the lines of best fit obtained from the regression equations.
higher current intensity (20 #A, Fig. 3b; r = 0.71; d.f. = 18; P < 0.01). As might be expected from the behavioral correlations described above, the relationship between ICSS rates and electrode placements for each rat was similar to the relationship observed between the elicitation of drug-lever responding and the electrode placements. Moderate to high self-stimulation rates were obtained from all but 6 sites when the current intensity was set at 15 #A. Four of these 6 sites were situated at the extremities of the VTA and correspond to the sites which were also associated with low levels of drug-appropriate responding during stimulus generalization tests. All of the sites yielded moderate to high levels of ICSS when the current intensity was set at 20 #A. Many of the sites that yielded high self-stimulation rates also were associated with high levels of D-amphetamine-appropriate responding during generalization tests, whereas sites that yielded only moderate ICSS rates tended to produce only moderate to low levels of drug-appropriate responding.
DISCUSSION
The present study examined whether the stimulus properties produced by 1.0mg/kg D-amphetamine could be mimicked by electrical stimulation of the VTA in rats. The first experiment assessed the effects of unilateral VTA stimulation on D-amphetamine stimulus generalization functions. VTA stimulation (15 and 20#A) increased drug-appropriate responses elicited during saline and low-dose D-amphetamine trials, so that the D-amphetamine stimulus generalization functions were shifted relative to the control curves. Therefore, VTA brain stimulation appears both to substitute partially for the stimulus properties of D-amphetamine, and to augment the cueing effects of low D-amphetamine doses. Although unilateral stimulation of regions containing DA neurons can produce both contralateral circling behavior 12 and greater responsiveness to stimuli in the contralateral sensory f i e l d 2'3'24, such sensorimotor effects did not appear to be responsible for the increased responding on the drug-lever. The rats increased their responses on the drug-lever during stimulation trials regardless of whether this lever was ipsilateral or contralateral to the stimulating electrode. The effects of the stimulation on D-amphetamine generalization functions were stronger when the drug-lever was contralateral to the electrode. Therefore, the sensorimotor effects may have exaggerated the response biases of these rats. However, the significant elevation observed with the ipsilateral group suggests that the main effect of the stimulation was to produce stimulus properties which could interact in an additive manner with the cueing effects of D-amphetamine. The summation observed between the stimulus properties of VTA stimulation a n d D-amphetamine in Expt. 1 contrasts with the findings of a previous study 8 in which VTA stimulation appeared to have only disruptive effects on discriminated responses to a D-amphetamine stimulus. However, the stimulation parameters employed in that study consisted of trains with short-duration (i.e. 0.2 ms) square-wave pulses. Recent in vivo electrochemical measurements have indicated that such parameters may be insuf-
182 ficient to activate mesotelencephalic DA neurons 2~. In the present investigation, 60-Hz sine-wave currents were used to stimulate the VTA. This type of stimulation appears to be adequate for activating DA neurons, as indicated by the increases in DA release and turnover within the nucleus accumbens of rats following sinewave stimulation of the V T A 4A1'27. Conceivably, this capacity to activate mesoaccumbens DA neurons could have accounted for the summation between the stimulus properties of the brain stimulation and those of D-amphetamine in the present study. The results of Expt. 2 again demonstrated that VTA stimulation could generalize with the stimulus properties of D-amphetamine. Comparisons of the data obtained during generalization and ICS S tests for each rat revealed a significant positive correlation between the amount of drugappropriate responding elicited by the stimulation and the rate of responding for the brain stimulation reward. Thus, stimulus generalization with D-amphetamine was strongest when the VTA stimulation produced high rates of responding. Although under certain circumstances response rates may not be the most precise index of brain stimulation r e w a r d 2°'33'34'35, individual differences in this measure can provide an estimate of the relative efficacy with which the stimulating currents activate reward processes within separate animals. Rats responding at higher rates for a moderate current intensity of VTA stimulation often will have both lower self-stimulation thresholds and rate-intensity curves that are displaced leftward relative to rats that respond at low rates for the same intensity*. Given this relationship between response rates and other measures of the * The relationship between rate and threshold measures of brain stimulation reward was confirmed by analyses of baseline random rate-intensity functions obtained from an independent group of 25 rats with VTA electrodes. Significant negative correlations were obtained between current thresholds for each rat and response rates measured at current intensities ranging from 10 to 20 #A (i.e. rats with higher rates had lower thresholds; the range of Pearsons correlation coefficients were r = -0.40 to r = - 0 . 8 9 ; df = 24, P < 0.05). This relationship is not maintained when rates are at asymptotic levels2°'33'34; however the rates measured in Expt. 2 were below asymptotic levels.
rewarding efficacy of brain stimulation, the results of Expt. 2 suggest that the ability of the VTA stimulation to generalize with D-amphetamine may be related to its capacity to produce rewarding effects. In particular, the generalization may have been due to rewarding effects that arise from the activation of the mesolimbic DA pathw a y s 11'22. The stimulus generalization with D-amphetamine was strongest when the electrodes were located within regions of the VTA that contain high densities of DA perikarya "~. Furthermore, VTA stimulation did not generalize or summate with the stimulus properties of D-amphetamine in a previous study8 that employed stimulation parameters that were capable of producing rewarding effects, but which were inappropriate for activating DA neurons. The apparent hedonic basis for stimulus generalization between D-amphetamine and VTA stimulation observed in the present study suggests that the stimulus properties of D-amphetamine may be related to the positive affective properties of the drug. These positive affective properties appear to be responsible for the abuse potential of D-amphetamine and D-amphetamine-like compounds in humans 32. Accordingly, studies of the stimulus properties of D-amphetamine may provide important insights into the neuropharmacological basis of drug abuse. ACKNOWLEDGEMENTS
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