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Unilateral 6-hydroxydopamine lesions of dopamine neurons produce bilateral self-stimulation deficits
Behavioural Brain Research, 6 (1982) 101-114
101
Elsevier Biomedical Press
Research Papers UNILATERAL 6-HYDROXYDOPAMINE LESIONS OF DOPAMINE NEURONS PRODUCE BILATERAL SELF-STIMULATION DEFICITS
ROBERT J. CAREY
Veterans Administration Medical Center and State University of New York, Upstate Medical Center, Syracuse, NY 13210 (U.S.A.) (Received January 29th, 1982) (Revised version received March 4th, 1982) (Accepted March 1 lth, 1982)
Key words: self-stimulation - 6-hydroxydopamine - hemispheric asymmetry - substantia nigra amphetamine - rat
SUMMARY
Sixteen rats, which had electrode implants in each hemisphere which generated comparable self-stimulation rate-intensity functions, were used in this study. Eight of the rats received unilateral 6-hydroxydopamine injections into the substantia nigra pars compacta, which produced severe unilateral lossesof dopamine and were effective in generating apomorphine-induced turning away from the injected hemisphere. Of the remaining 8 rats, 5 received unilateral 6-hydroxydopamine lesions aimed at the ventral tegmental area and 3 were given vehicle injections. The vehicle injections were without effect on self-stimulation and the ventral tegmental injections had an overall transient facilitative effect on selfstimulation. The 6-hydroxydopamine lesions of the pars compacta, however, had variable effects. In some rats there was a marked bilateral reduction in selfstimulation over 8 weeks; whereas, there was little, if any, effect in other rats. The rats which sustained the bilateral deficits also sustained the greatest unilateral loss of dopamine. The unilateral 6-hydroxydopamine lesions of the pars compacta consistently blocked the facilitative influence of 0.5 mg/kg of D-amphetamine on self-stimulation bilaterally, and this effect persisted over 8 weeks of postoperative testing. These results were considered supportive oi" a response rather than reinforcement role for dopamine in the mediation of self-stimulation behavior.
0166-4328/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
102 INTRODUCTION As pointed out by German and Bowden [9], evidence from a diverse array of experimental manipulations implicates brain catecholamines in the mediation of intracranial self-stimulation (ICS S). Although, initially, a pre-eminent role had been assigned to norepinephrine [7], other evidence has pointed to dopamine as possibly having the more critical role in the ICSS phenomenon [6]. While the importance of dopamine is unquestioned, the nature of the dopamine contribution remains controversial. The dilemma which arises when dopamine is considered as subserving a reinforcement function is that dopaminergic manipulations can profoundly alter operant behavior with dopaminergic stimulation producing hyperactivity and a dopamine deficiency or blockade inducing parkinsonian symptoms [3 ]. Since, by definition, a positive reinforcement is a behavioral contingency which enhances operant response rate [ 19], it becomes apparent that a dopamine manipulation will affect reinforced behavior by altering the operant response. The problem, therefore, is how to differentiate effects on the operant response from possible effects on the reinforcement. Recently, this issue has been approached by the use of unilateral lesions of the dopamine systems. With this paradigm, the effects of unilateral dopamine depletions on ICSS are compared in animals with electrodes either ipsilateral or contralateral to the depletion. Since the response requirement is the same to obtain reinforcement regardless of the brain hemisphere stimulated, it follows that an operant response deficit should not differentially alter responding generated by stimulation of the dopamine depleted versus the dopamine intact hemisphere. On the other hand, if the reinforcement is also mediated by the dopamine pathways, then stimulation of the dopamine depleted hemisphere should be less effective as a reinforcement than the dopamine-intact hemisphere. Overall, these studies have been inconclusive with reports of lateralized deficits in ICSS in the dopamine depleted hemisphere counterbalanced by reports of bilateral deficits in ICSS [5, 12, 16, 17]. The present experiment was undertaken in an attempt to resolve the experimental ambiguity in the dopamine contribution to ICSS with regard to responsereinforcement issue. While the unilateral dopamine depletion technique was employed it was used with a significant modification. Namely, instead of making ipsilateral versus contralateral ICSS comparisons between animals with unilateral electrode implants, the comparisons were made within the same animal by using animals with bilateral implants. While the advantages of making comparisons within the same animal are obvious, the test procedure was further refined by preoperatively determinirtg rate-intensity functions for each electrode and using only animals in which the functions were similar bilaterally. With this test preparation, the effects on ICSS of unilateral 6-hydroxydopamine injections aimed at the A9 region of the substantia nigra and A10 of the ventral medial tegmentum were studied. Additionally, the facilitative effect of D-amphetamine on ICSS [20] was studied to determine if this facilitation is lateralized.
103 MATERIALS AND METHODS
Subjects and surgicalprocedures Male Sprague-Dawley rats (400-450 g) were stereotaxicatly implanted bilaterally with bipolar platinum stimulating electrodes (diameter 0.25 mm) insulated except for the cut surface [4]. The stereotaxic coordinates were: 1 mm posterior to bregma, 3.1 mm lateral to the midline, and 8.0 mm below dura. Each electrode was angled 10 ° toward the midline and the incisor bar was fixed 3.2 mm above the interaurai line. Out of an original pool of 34 implanted rats 19 were obtained which satisfied the condition of having comparable bilateral selfstimulation rate-intensity functions. These 19 rats were subdivided into 3 groups, 2 experimental groups which received unilateral intracerebral 6-hydroxydopamine (6-OHDA) injections and a control group which received vehicle injections. Of the 16 rats which received 6-OHDA injections, 11 were given an 8/ag dose into the substantia nigra (SN) and 5 were given an 8 #g dose into the ventral tegmental area (VTA). The stereotaxic coordinates used were: 4.0 mm posterior to bregma, 8.0 mm below dura with lateral settings of 2.0 mm for the SN and 0.5 mm for the VTA. At these loci 2 #1 of a 4/ag/~tl solution of 6-OHDA-HBr (dosage expressed as the base) and dissolved in 0.15 M NaC1 containing 0.2 mg/ml ascorbic acid was injected. The 6-OHDA was injected through a 30-gauge cannula at the rate of 0.5/A/per min. In order to restrict the catecholamine lesions to the dopamine neurons, the animals were given intraperitoneal injections of a noradrenergic reuptake blocking agent of disipramine (DMI)-HCI (25 mg/kg). The 3 control rats received vehicle injections plus DMI pretreatment. The implantation surgery was conducted with the rats under Equithesin anesthesia (0.3 ml/100 g) and the injections were carried out with the rats under deep ether anesthesia. After each operation the rats were maintained on penicillin for several days.
Apparatus Two weeks after implantation the rats were trained to lever press in one of three operant conditioning chambers. Each chamber (Lehigh Valley Electronics 1417) had a 26 x 24 cm floor area with a lever (Ralph Gerbrands 6312) centered 3.5 cm above the grid floor which projected 1.5 cm into the chamber. The chambers were enclosed in sound attenuating enclosures equipped with a one-way observation mirror. The chamber was illuminated and white noise broadcast into the chamber but only when brain stimulation was available on the response lever. Relay circuits with pulse generators, electronic timers and digital counters were used to record responses and control stimulation (0.2 sec train of stimulation) and session duration. A Grass Brief Pulse Stimulator (Mdl. BPSI) provided pairs of 0.1 msec biphasic rectangular pulses with a 0.1 msec interpulse interval between positive and negative pulses at a frequency of 100 pulse pairs per second. Current was continuously monitored on one channel of dual beam oscilloscopes (Tex-
104 tronix 502A) connected across 1-kf~ resistors. The rats' electrodes were connected to the stimulator through mercury swivel commutators mounted in each chamber. The activity measurement which preceded ICSS tests were obtained using large photoactivity cages (Lehigh Valley Electronics 1497). These cages were cylindrical, 61.0 cm in diameter and 53 cm high with a metal mesh floor. Six infrared photocells placed 2.5 cm above the floor detected movements. The photobeam interruptions were recorded on two digital counters, with each counter recording from 3 photocells. Activity sessions lasted 15 min and the activity scores were recorded on separate counters in 3 successive 5 min intervals. Speakers located on the top of the activity chambers broadcast white noise throughout the activity sessions in order to mask external sounds. Although the test room was well illuminated, the chamber walls were black and the top was covered resulting in a dim illumination level (approximately 0.15 footcandles). D-Amphetamine-HCl (K and K Labs) was used in the drug testing phase. The amphetamine was calculated as the salt and dissolved in 0.99/0 sodium chloride. Apomorphine-HCl (Sigma Chemicals) was dissolved in 0.4 mg/ml solution of ascorbic acid.
Behavioral testing After the rat's response for ICSS was reliably performed, current intensity was manipulated in order to obtain the lowest current intensity which would generate an optimal rate of response for a particular animal. This current intensity was used to establish a reliable ICSS performance. After the response was well established, the animals were given a series of 10 min test sessions in which the current intensity was reduced in steps of 25 ~ of the preceding current level. On the basis of these results, 3 current intensities were selected which encompassed the range of the rate-intensity function (approximately 10, 50 and 100~ of optimal responding). Each rat then received 3 sessions with the 3 current intensities in an ascending sequence to establish reliable preoperative performance levels. In each session the tests at each current intensity were 10 min in duration and between tests the rats were returned to their home cage for 10-min breaks. The timing of the 10-min test sessions commenced after the current intensity was adjusted to the appropriate setting. Also, the test order of the two electrodes in each animal was always alternated between changes in current intensity. The current intensity settings for each animal were kept the same throughout testing and the applied voltage was recorded at each setting. Throughout the course of the study, there was never any change in the voltage required to generate a particular current level for any of the rats. Prior to ICSS testing for each of these rate-intensity determinations, each rat was given a 15-min activity test immediately preceding the ICSS testing. One day after completion of these 3 rate-intensity ICSS tests and activity measurements the rats were given the same rate-intensity ICSS and activity tests following an i.p. injection of 0.5 mg/kg D-amphetamine.
105 Activity testing commenced 5 min after the amphetamine injection and the ICSS rate-intensity procedure commenced immediately after completion of the activity determination. Within a few days of the completion of the amphetamine testing the rats were subjected to the unilateral 6-OHDA or vehicle injections. Postoperatively, starting one week after surgery, the rats were given the same rate-intensity and activity tests on two successive days per week. The first day was without drug treatment but the second day was with 0.5 mg/kg D-amphetamine. These weekly tests were continued for 8 weeks postoperation. In addition, after 4 weeks postoperation, all rats were tested for circling following injections of 0.5 mg/kg apomorphine. The circling was observed for 2 min on the laboratory floor, 10 min after the apomorphine injection. The number and direction of the 360 ° turns made in 2 min were recorded for each rat. Only those rats (n = 8) in the SN lesion group which exhibited turning contralateral to the injected hemisphere were included in the study. Catecholamine assay and histology At least one week after completion of testing the rats (under ether anesthesia) were placed in the stereotaxic instrument and their electrodes removed to permit electrode localization. Immediately after electrode removal, the rats were decapitated and their brains rapidly removed over ice. Each brain was then cut coronally at the ventral surface using the rhinal fissures as topographical guides to obtain forebraln tissue anterior to the electrodes for biochemical analysis. This anterior brain section was then divided sagitally in order to obtain comparable hemisphere sections. Each hemisphere was then dissected to remove striatal and limbic (olfactory lobe and accumbens) tissue samples. These tissue samples were then assayed for dopamine and norepinephrine using high performance liquid chromatography with electrochemical detection [ 1]. The LC-EC consisted of a Laboratory Data Control mini mump NS 1-33R, a cheminest CSU 20 injection valve and a glass column dry-packed with pellicular Vydac SC cation-exchange resin. A Model LC-4 electrochemical controller was used with a glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN, USA). The potential was set as + 0.5 with respect to a Ag/AgCI reference electrode. A citrate-acetate buffer solvent system was used. Briefly, the brain tissue samples were weighed to the nearest 0.1 mg and placed in polycarbonate centrifuge tubes containing 500 #1 of 0.1 M H C 1 0 4 (antioxidant) and 50/zl of 3 mg/ml dihydroxybenzylamine (DHBA) (internal standard). The tissue was then sonicated for two 30-sec periods using a Kontes Micro-Ultrasonic cell disrupter (Kontes, Vineland, NJ, USA). The samples were then centrifuged at 20,000 g for 10 min and the supernatant stored in a small polyethylene tube at - 2 0 °C. The amines were determined by injecting 20/~1 of supernatant into the LC-EC system. The norepinephrine (NE) and dopamine (DA) peak heights were ratioed to the DHBA peak height, and the concen-
106 trations (/~g/g brain) calculated knowing the relative response of standards, the amount of DHBA added and the brain sample weight. The remaining caudal brain section was placed in 10~o formalin for a 10-day fixation period. After this period of fixation, 3 mm thick sections containing the electrode or cannula tracts were embedded in paraffin and subsequently 10 ktm-thick sections were cut, mounted and stained with cresyl violet and Luxol fast blue [ 13]. All sections were studied microscopically to identify electrode and cannula tip locations. RESULTS
H istoh)gic al.[hTdings Microscopic examination of histologically prepared sections showed that the electrode tips were all located in the medial forebrain bundle area between the internal capsule laterally and the fornix and mammilo-thalamic tract medially at the level of the ventral medial nucleus pars medialis and between 0.5 and 1.0 mm above the base of the brain. Undoubtedly, the performance criterion used for selecting animals for inclusion in the experiment contributed to the observed homogeneity of electrode placements. Fig. 1 presents a photomicrograph of a representative set of electrode placements.
Fig. 1. Photomicrograph of Kluver-Berrera-stained section indicating location of bilateral electrode placements in the lateral hypothalamic area. The left hemisphere has been notched.
107
Examination of the cannula tracts showed that the needle tips for the substantia nigra injections were typically situated in the medial region of the pars compacta at the level of the interpeduncular nucleus. The more medial injections aimed at the ventral tegmental area were located in a similar coronal plane slightly medial to the most medial extension of the substantia nigra pars reticulata. Generally, the cannula tips were dorsal to the interpeduncular nucleus and slightly medial to the medial lemniscus.
Biochemicalfindings For all 6-OHDA-injected rats there was a marked disparity in dopamine levels in limbic and striatal areas between the injected and intact hemispheres. The overall results are summarized in Table I. As indicated in Table I, the SN lesion produced a severe reduction in striatal dopamilae but not norepinephrine. Limbic reductions were extensive but much more variable and generally less than the striatal deficiency. The rats subjected to 6-OHDA injections aimed at the VTA showed marked losses of dopamine but not norepinephrine in both striatal
TABLE I
Individual striatal and limbic norepinephrine and dopamine (levels expressed as lag/g of brain tissue) Group
Intact hemisphere Striatal
Injectea~hemisphere Limbic
DA
NE
DA
NE
SN 8
9.83 13.20 12.40 6.40 9.10 7.09 9.56 11.90
0.56 1.20 0.71 0.79 0.82 0.40 0.59 0.55
2.41 2.56 3.60 1.97 1.99 2.33 2.10 1.86
1.13 1.20 1.70 0.86 0.79 0.92 1.06 1.23
VTA l VTA 2 VTA3 VTA 4 VTA s
14.07 10.90 4.90 7.08 14.40
0.49 1.07 0.68 1.40 0.57
1.52 2.60 2.60 1.98 2.30
Vl V: V3
9.10 12.50 13.20
0.55 1.00 0.66
1.80 2.00 2.10
SN l SN 2
SN 3 SN 4
SN 5 SN 6 SN 7
Limbic
Striatal
DA
NE
DA
NE
0.07 0.19 0.16 0.37 0.74 0.84 1.02 2.39
0.46 0.41 1.20 0.57 0.78 0.34 0.50 0.38
0.23 0.17 0.27 0.66 0.50 i.45 0.86 1.46
1.30 1.40 1.20 0.61 0.89 0.82 0.77 1.44
0.78 0.98 1.10 1.13 1.07
3.58 2.68 2.70 3.50 8.90
0.27 1.27 0.62 0.66 0.33
0.56 1.29 0.59 1.00 0.94
0.75 0.91 0.86 1.10 1.02
1.10 1.50 1.20
7.50 11.00 12.10
0.40 1.80 0.57
2.00 1.83 2.06
0.97 1.38 0.88
108 and limbic tissue. In general, the limbic decreases were greater than the striatal, but the overall dopamine loss was less severe than that obtained for the SN injection.
Beha vioral findings Figs. 2 and 3 present the individual ICSS results for all animals both with and without the 0.5 mg/kg D-amphetamine treatment. In Fig. 2 the ICSS results are shown for the rats injected unilaterally with 6-OHDA into the pars compacta of the substantia nigra. In terms of the non-drug results, it is apparent that the effects of this lesion were quite .variable with several animals (SN~, SN2, SN3) severely impaired; whereas, others (SN7 and SN8)were only slightly affected. In contrast to this between-animal variability, the inter-hemispheric ICSS results were consistent. In each case when ICSS was reduced at the electrode site in the injected hemisphere, the.virtual same reduction occurred at electrode site located in the unoperated hemisphere. Similarly, when recovery occurred for an electrode INTACT 150 L M H
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HEMISPHERE
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Post Operative Week
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Fig. 2. Individual self-stimulation response rates of rats subjected to unilateral 6-hydroxydopamine injections into the substantia nigra pars compacta. Each rat bad self-stimulation generated from an electrode ipsilateral as well as eontralateral to the 6-hydroxydopamine-injected hemisphere. Three current intensities were used and separate test sessions were conducted both with and without 0.5 mg/kg D-amphetamine.
109 INTACT HEMISPHERE 200rLMH 150~ t. ~.
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3 4 5 6 7 8 Pre I 2 3 4 5 6 ? Post Operative Week Post Operative Week --Non Drug - - - 0 . 5 mg/kg, d-Amphetamine
8
Fig. 3. Individual self-stimulation response rates of rats subjected to unilateral 6-hydroxydopamine injections aimed at the ventral tegmental area and vehicle injections into the substantia nigra, self-stimulation ipsilateral and contralateral to the injected hemisphere is shown for each rat at each of 3 current intensities. Pre- and postoperative testing included separate sessions with and without 0.5 mg/kg D-amphetamine.
placement in the dopamine depleted hemisphere, there was a corresponding recovery in performance for the electrode situated in the intact hemisphere. Another important aspect of the findings presented in Fig. 2 are the effects of amphetamine on ICSS. For every animal, when amphetamine was given preoperatively, ICS S was facilitated at both electrode sites. Postoperatively, however, amphetamine had virtually no effect on self-stimulation generated from either the intact or the dopamine depleted hemisphere and this blockade of amphetamine facilitation of ICSS persisted throughout the 8 weeks of postoperative testing. Fig. 3 presents the ICSS results for the rats injected unilaterally with 6-OHDA into the VTA and for the vehicle-injected rats. In no instance did these injections impair ICSS. The performance of the vehicle-injected rats remained
110
essentially unaltered throughout the course of postoperative testing both with and without amphetamine. The unilateral 6-OHDA injections into the VTA were generally without effect although some transient facilitation of ICSS and attenuation of amphetamine enhancement of ICSS can be noted during the first few postoperative weeks. These modest changes in ICSS typically occurred bilaterally. Table II presents the mean locomotor activity results for the 3 treatment groups both with and without amphetamine. Preoperatively, the 3 groups were closely matched in terms of both non-drug and amphetamine activity levels. Postoperatively, the VTA and vehicle-injected groups remained at preoperative activity levels, but the pars compacta group no longer exhibited an increase in locomotor activity after the 0.5 mg/kg D-amphetamine treatment. In addition to this measure of general activity the spontaneous and apomorphine induced circling was observed. Over the first postoperative week all rats in the SN group exhibited circling toward the injected hemisphere which gradually subsided, although some animals (SN~, SN2, SN3) circled a few times when placed on the laboratory floor during the course of the study. None of the VTA or vehicleinjected rats exhibited spontaneous circling. Additionally, it should be noted that while all rats in the pars compacta substantia nigra 6-OHDA treatment group showed strong circling toward the intact hemisphere after the 0.5 mg/kg injections of apomorphine, none of the rats in the vehicle or VTA groups displayed any circling after this dose of apomorphine but, rather, exhibited the typical
TABLE lI
Mean 15 rain photoactivity counts with and without 0.5 mg/kg D-amphetamine for each treatment group Difference scores were estimated by the t-test. N D , non-drug; A, amphetamine.
Group
Preoperative score
Postoperativeweek I
2
3
4
5
6
7
8
SN ND A
347 450*
296 329
262 308
266 285
264 264
206 257
249 251
209 255
256 244
VTA ND A
310 495*
3"~0 542*
450 502*
434 566*
367 500*
421 596*
430 591'
338 474*
394 570*
Vehicle ND A
368 456
498 554
359 475
443 598
423 500
397 489
416 511
376 463
354 521
* P < 0.05.
111 behavioral stereotypy observed at this dose level of apomorphine. At the conclusion of the self-stimulation testing, the SN rats were again tested for apomorphine-induced circling, at 3 dose levels, 0.125, 0.25 and 0.5 mg/kg. Sensitivity to apomorphine-induced circling generally correlated with the degree of self-stimulation dysfunction with SN 1, SN:, and SN3, circling at 0.125, SN4_ 6 at 0.25 and SNT_8 at 0.5 mg/kg. As is evident from Table I, the lower .the threshold for apomorphine-induced circling, the lower the dopamine level in the injected hemisphere. DISCUSSION The present study was undertaken to determine the degree to which unilateral lesions of dopamine-containing neurons would have unilateral versus bilateral effects on ICSS in rats with bilateral electrode implants which independently generated comparable ICSS performance. Using this experimental paradigm, the present study showed that unilateral lesions of the pars compacta region can have very marked inhibitory effects on ICSS and that these effects are virtually bilaterally symmetrical. The occurrence of bilateral symmetry is entirely consistent with the initial report of Ornstein and Huston [ 16] that unilateral lesions of catecholaminergic neurons produce a profound impairment in ICSS performance. The bilateral symmetry of the performance deficit in the present study seems to be readily explicable as a motoric dysfunction and recovery occurs as the animal, either by learning or some other process, adapts to or spontaneously recovers from the motoric dysfunction and thereby becomes more efficient at obtaining ICS S. This bilateral symmetry in recovery of ICS S also implies that the reinforcement effect was similar for both hemispheres. These observations appear to contradict the earlier report of Koob et al. [ 12], in which self-stimulation was reduced in rats with unilateral electrode implants when the ipsilateral hemisphere was treated with 6-hydroxydopamine, but not when the contralateral hemisphere was injected with 6-hydroxydopamine. An important aspect of this study was that striatal dopamine was reduced to 0.08 #g/g by the ipsilateral injections; whereas the contrallateral injection reduced striatal dopamine to 0.73 #g/g. Since the persistent bilateral deficits in self-stimulation in the present study only occurred with the most severe dopamine reductions (0.4 #g/g or less), it would appear possible to account for the Koob et al. report of lateralization of self-stimulation deficits in terms of the differential severity of the dopamine depletions produced in ipsilateral and contralateral hemispheres. Seemingly, the electrode tract damage plus 6-hydroxydopamine injection in the ipsilateral hemisphere produced greater damage to dopamine neurons than the 6-hydroxydopamine injection alone into the contralateral hemisphere. Presumably, a small residual dopamine neuron population can have a very significant functional influence until reduced to some critical level. This finding appears consistent with the report of Lashley [ 14] on
112 the sparing of visual function in rats which had only small remnants of the visual cortex remaining after visual cortex extirpation. In this context, it is not too surprising that the 6-hydroxydopamine injections aimed at the A10 area in the present study did not have detrimental effects on self-stimulation. These injections produced substantial but only incomplete decreases in limbic dopamine and striatal dopamine. Furthermore, no motoric deficits were apparent including the absence of spontaneous and apomorphineinduced turning behavior. The amphetamine findings, however, are not entirely consistent with the non-drug self-stimulation results in that the pars compacta lesions reliably bilaterally blocked the facilitation of self-stimulation by amphetamine; whereas, the pars compacta lesions produced non-drug self-stimulation deficits only in the cases of the most severe dopamine depletions. It should be noted that the 0.5 mg/kg dose level of amphetamine used in this study induced neither circling nor hyperactivity. Possibly at this dose level the approximately 50 ~ reduction in total brain striatal dopamine by the pars compacta lesion functionally reduced the amphetamine dose to 0.25 mg/kg which was subthreshold for provoking hyperactivity. Seemingly then, the most prosaic explanation for the inconsistency between the non-drug and amphetamine-induced changes in self-stimulation is that the amphetamine-induced behavioral effects are more related to changes in total brain dopamine; whereas the severity of the interhemispheric dopamine asymmetry is more critical for the non-drug behavioral effects to become manifest. In conclusion, the results of the present study show that unilateral injury to the pars compacta of the substantia nigra can have marked bilateral effects on ICCS. The bilateral symmetry of these effects on ICSS seems most easily explicable as a motoric impairment which interferes with ICSS performance although bilateral dysfunction of reinforcement efficacy cannot be ruled out solely on the basis of the present study. More generally, however, brain stimulation reinforcement test procedures can be arranged to make performance easily disrupted by using low current intensities and complex motoric response requirements or difficult to impair by employing high current intensities and very simple response requirements. The use of paradigms which represent these extremes as well as numerous intermediate variations greatly complicate efforts to decipher performance from reinforcement deficits in the effects of experimental manipulations on self-stimulation. In addition, treatments which produce no apparent motoric dysfunction yet impair self-stimulation might create gastrointestinal distress, nausea or some untoward effect without conspicuous symptoms in animals that might otherwise make it readily explicable why an animal failed to perform for the brain stimulation reinforcement. Certainly, studies [10, 22] which have shown that self-stimulation can survive radical removals of brain tissue, have made it apparent that no specific telencephalic structure it necessary for self-stimulation. On the other hand, pharmacological studies suggest transmitter specificity in that
113
cholinergic agonists [8] on catecholaminergic antagonists [23] block self-stimulation in conventional paradigms. While it is obvious that these drug treatments which block self-stimulation also typically induce parkinsonism symptomatology, there is also the more general issue of brain dysfunction produced by transmitter imbalance. In the case of severe motoric disorders such as chorea and parkinsonism, in which specific transmitter systems are affected by pathological processes, the symptoms can be alleviated by blocking other remaining intact transmitter symptoms. Thus, transmitter imbalance can be more disruptive to brain function than a more extensive interference with neuronal activity that does not create transmitter imbalances. Extensive brain lesions which presumably leave residual tissue in a state of approximate transmitter balance, therefore, may be the most appropriate methodology for determining the neural systems necessary for brain stimulation reinforcement. ACKNOWLEDGEMENTS
This work was supported by VA Medical Research Service Funds and NIMH Grant MH 33959. REFERENCES 1 Anderson, G.M., Batter, D.K., Young, J.G., Shaywitz, B.A. and Cohen, D.J., Simplified liquid chromatographic-electrochemical determination of norepinephrine and dopamine in rat brain, J. Chromatogr., 181 (1980)453--455. 2 Belluzzi, J.J., Ritter, S., Wise, C.D. and Stein, L., Substantia nigra self-stimulation: dependence on noradrenergic reward pathways, Behav. Biol., 13 (1975) 103-111. 3 Bernheimer, H., Birkmayer, W., Honykiewicz, O., Jellinger, K. and Seitelberger, F., Brain dopamine and the syndromes of Parkinson and Huntington, J. neurol. Sci., 20 (1973) 415--455. 4 Carey, R., Goodall, E. and Lorens, S., Differential effects of amphetamine and food deprivation of self-stimulation of the lateral hypothalamus and medial frontal cortex, J. comp. physiol. Psychol., 88 (1975) 224. 5 Carey, R.J., Anticholinergic drugs promote recovery from self-stimulation deficits produced by bilateral but not unilateral dopamine lesions, Neurosci. Abstr., 6 (1980) 368. 6 Cooper, B.R., Cott, J.M. and Breese, G.R., Effects of catecholamine-depleting drugs and amphetamine on self-stimulation of the brain following various 6-hydroxydopamine treatments, Psychopharrnacologica, 37 (1974) 235-248. 7 Crow, T.J., Spear, P.J. and Arbuthnott, G.W., Intracranial self-stimulation with electrodes in the region of the locus coeruleus, Brain Res., 36 (1972) 275-287. 8 Domino, E.F. and Olds, M.E., Cholinergic inhibition of self-stimulation behavior, J. Pharmacol. exp. Ther., 164 (1968) 202-211. 9 German, D.C. and Bowden, D.M., Catecholamine systems as the neural substrate for intracranial self-stimulation: an hypothesis, Brain Res., 73 (1974) 381-419. 10 Huston, J.P. and Borbely, A.A., Operant conditioning in forebrain ablated rats by use of rewarding hypothalamic stimulation, Brain Res., 50 (1973) 467-472. 11 Kadzielawa, K., Dopamine receptor in the reward system of the rat, Arch. int. Pharmacodyn., 209 (1974) 214-226.
114 12 Koob, G.F., Fray, P.J. and Iversen, S.D., Self-stimulation at the lateral hypothalamus and locus coeruleus after specific unilateral lesions of the dopamine system, Brain Res., 146 (1978) 123-140. 13 Kluver, H. and Barrera, E., Method for combined staining of cells and fibers in the nervous system, J. Neuropath. exp. Neurol., 12 (1953) 400-403. 14 Lashley, K.S., The functioning of small remnants of the visual cortex, J. comp. Neurol., 70 (1939) 45-67. 15 Olds, J. and Milner, P., Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain, J. comp. physiol. Psychol., 47 (1954) 419-427. 16 Ornstein, K. and Huston, J.P., Influence of 6-hydroxydopamine injections in the substantia nigra on lateral hypothalamic reinforcement, Neurosci. Lett., 1 (1975) 339-342. 17 Phillips, A.G., Carter, D.A. and Fibiger, H.C., Dopaminergic substrates of intracranial selfstimulation in the caudate-putamen, Brain Res., 104 (1976) 222-232. 18 Prado-Alcala, R.A., Kent, E.W. and Reid, L.D., Intracranial self-stimulation effects along the route of the nigro-striatal bundle, Brain Research, 84 (1975) 531-540. 19 Skinner, B.F., The Behavior of Organisms, Appleton-Century-Crofts, New York, 1938. 20 Stein, L. and Wise, D., Amphetamine and noradrenergic reward pathways. E. Usdin and S. Snyder (Eds.), In Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 963-968. 21 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta physiol, scand., Suppl. 367 (1971) 69-93. 22 Valenstein, E.S., The anatomical locus of reinforcement. In E. Stellar and J. Sprague (Eds.), Progress in Physiological Psychology, Vol. 1, Academic Press, New York, 1966, pp. 149-190. 23 Wauquier, A. and Niemegeers, C.J.E., Restoration of self-stimulation inhibited by neuroleptics, Europ. J. Pharrnacol., 40 (1976) 191-194.
101
Elsevier Biomedical Press
Research Papers UNILATERAL 6-HYDROXYDOPAMINE LESIONS OF DOPAMINE NEURONS PRODUCE BILATERAL SELF-STIMULATION DEFICITS
ROBERT J. CAREY
Veterans Administration Medical Center and State University of New York, Upstate Medical Center, Syracuse, NY 13210 (U.S.A.) (Received January 29th, 1982) (Revised version received March 4th, 1982) (Accepted March 1 lth, 1982)
Key words: self-stimulation - 6-hydroxydopamine - hemispheric asymmetry - substantia nigra amphetamine - rat
SUMMARY
Sixteen rats, which had electrode implants in each hemisphere which generated comparable self-stimulation rate-intensity functions, were used in this study. Eight of the rats received unilateral 6-hydroxydopamine injections into the substantia nigra pars compacta, which produced severe unilateral lossesof dopamine and were effective in generating apomorphine-induced turning away from the injected hemisphere. Of the remaining 8 rats, 5 received unilateral 6-hydroxydopamine lesions aimed at the ventral tegmental area and 3 were given vehicle injections. The vehicle injections were without effect on self-stimulation and the ventral tegmental injections had an overall transient facilitative effect on selfstimulation. The 6-hydroxydopamine lesions of the pars compacta, however, had variable effects. In some rats there was a marked bilateral reduction in selfstimulation over 8 weeks; whereas, there was little, if any, effect in other rats. The rats which sustained the bilateral deficits also sustained the greatest unilateral loss of dopamine. The unilateral 6-hydroxydopamine lesions of the pars compacta consistently blocked the facilitative influence of 0.5 mg/kg of D-amphetamine on self-stimulation bilaterally, and this effect persisted over 8 weeks of postoperative testing. These results were considered supportive oi" a response rather than reinforcement role for dopamine in the mediation of self-stimulation behavior.
0166-4328/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
102 INTRODUCTION As pointed out by German and Bowden [9], evidence from a diverse array of experimental manipulations implicates brain catecholamines in the mediation of intracranial self-stimulation (ICS S). Although, initially, a pre-eminent role had been assigned to norepinephrine [7], other evidence has pointed to dopamine as possibly having the more critical role in the ICSS phenomenon [6]. While the importance of dopamine is unquestioned, the nature of the dopamine contribution remains controversial. The dilemma which arises when dopamine is considered as subserving a reinforcement function is that dopaminergic manipulations can profoundly alter operant behavior with dopaminergic stimulation producing hyperactivity and a dopamine deficiency or blockade inducing parkinsonian symptoms [3 ]. Since, by definition, a positive reinforcement is a behavioral contingency which enhances operant response rate [ 19], it becomes apparent that a dopamine manipulation will affect reinforced behavior by altering the operant response. The problem, therefore, is how to differentiate effects on the operant response from possible effects on the reinforcement. Recently, this issue has been approached by the use of unilateral lesions of the dopamine systems. With this paradigm, the effects of unilateral dopamine depletions on ICSS are compared in animals with electrodes either ipsilateral or contralateral to the depletion. Since the response requirement is the same to obtain reinforcement regardless of the brain hemisphere stimulated, it follows that an operant response deficit should not differentially alter responding generated by stimulation of the dopamine depleted versus the dopamine intact hemisphere. On the other hand, if the reinforcement is also mediated by the dopamine pathways, then stimulation of the dopamine depleted hemisphere should be less effective as a reinforcement than the dopamine-intact hemisphere. Overall, these studies have been inconclusive with reports of lateralized deficits in ICSS in the dopamine depleted hemisphere counterbalanced by reports of bilateral deficits in ICSS [5, 12, 16, 17]. The present experiment was undertaken in an attempt to resolve the experimental ambiguity in the dopamine contribution to ICSS with regard to responsereinforcement issue. While the unilateral dopamine depletion technique was employed it was used with a significant modification. Namely, instead of making ipsilateral versus contralateral ICSS comparisons between animals with unilateral electrode implants, the comparisons were made within the same animal by using animals with bilateral implants. While the advantages of making comparisons within the same animal are obvious, the test procedure was further refined by preoperatively determinirtg rate-intensity functions for each electrode and using only animals in which the functions were similar bilaterally. With this test preparation, the effects on ICSS of unilateral 6-hydroxydopamine injections aimed at the A9 region of the substantia nigra and A10 of the ventral medial tegmentum were studied. Additionally, the facilitative effect of D-amphetamine on ICSS [20] was studied to determine if this facilitation is lateralized.
103 MATERIALS AND METHODS
Subjects and surgicalprocedures Male Sprague-Dawley rats (400-450 g) were stereotaxicatly implanted bilaterally with bipolar platinum stimulating electrodes (diameter 0.25 mm) insulated except for the cut surface [4]. The stereotaxic coordinates were: 1 mm posterior to bregma, 3.1 mm lateral to the midline, and 8.0 mm below dura. Each electrode was angled 10 ° toward the midline and the incisor bar was fixed 3.2 mm above the interaurai line. Out of an original pool of 34 implanted rats 19 were obtained which satisfied the condition of having comparable bilateral selfstimulation rate-intensity functions. These 19 rats were subdivided into 3 groups, 2 experimental groups which received unilateral intracerebral 6-hydroxydopamine (6-OHDA) injections and a control group which received vehicle injections. Of the 16 rats which received 6-OHDA injections, 11 were given an 8/ag dose into the substantia nigra (SN) and 5 were given an 8 #g dose into the ventral tegmental area (VTA). The stereotaxic coordinates used were: 4.0 mm posterior to bregma, 8.0 mm below dura with lateral settings of 2.0 mm for the SN and 0.5 mm for the VTA. At these loci 2 #1 of a 4/ag/~tl solution of 6-OHDA-HBr (dosage expressed as the base) and dissolved in 0.15 M NaC1 containing 0.2 mg/ml ascorbic acid was injected. The 6-OHDA was injected through a 30-gauge cannula at the rate of 0.5/A/per min. In order to restrict the catecholamine lesions to the dopamine neurons, the animals were given intraperitoneal injections of a noradrenergic reuptake blocking agent of disipramine (DMI)-HCI (25 mg/kg). The 3 control rats received vehicle injections plus DMI pretreatment. The implantation surgery was conducted with the rats under Equithesin anesthesia (0.3 ml/100 g) and the injections were carried out with the rats under deep ether anesthesia. After each operation the rats were maintained on penicillin for several days.
Apparatus Two weeks after implantation the rats were trained to lever press in one of three operant conditioning chambers. Each chamber (Lehigh Valley Electronics 1417) had a 26 x 24 cm floor area with a lever (Ralph Gerbrands 6312) centered 3.5 cm above the grid floor which projected 1.5 cm into the chamber. The chambers were enclosed in sound attenuating enclosures equipped with a one-way observation mirror. The chamber was illuminated and white noise broadcast into the chamber but only when brain stimulation was available on the response lever. Relay circuits with pulse generators, electronic timers and digital counters were used to record responses and control stimulation (0.2 sec train of stimulation) and session duration. A Grass Brief Pulse Stimulator (Mdl. BPSI) provided pairs of 0.1 msec biphasic rectangular pulses with a 0.1 msec interpulse interval between positive and negative pulses at a frequency of 100 pulse pairs per second. Current was continuously monitored on one channel of dual beam oscilloscopes (Tex-
104 tronix 502A) connected across 1-kf~ resistors. The rats' electrodes were connected to the stimulator through mercury swivel commutators mounted in each chamber. The activity measurement which preceded ICSS tests were obtained using large photoactivity cages (Lehigh Valley Electronics 1497). These cages were cylindrical, 61.0 cm in diameter and 53 cm high with a metal mesh floor. Six infrared photocells placed 2.5 cm above the floor detected movements. The photobeam interruptions were recorded on two digital counters, with each counter recording from 3 photocells. Activity sessions lasted 15 min and the activity scores were recorded on separate counters in 3 successive 5 min intervals. Speakers located on the top of the activity chambers broadcast white noise throughout the activity sessions in order to mask external sounds. Although the test room was well illuminated, the chamber walls were black and the top was covered resulting in a dim illumination level (approximately 0.15 footcandles). D-Amphetamine-HCl (K and K Labs) was used in the drug testing phase. The amphetamine was calculated as the salt and dissolved in 0.99/0 sodium chloride. Apomorphine-HCl (Sigma Chemicals) was dissolved in 0.4 mg/ml solution of ascorbic acid.
Behavioral testing After the rat's response for ICSS was reliably performed, current intensity was manipulated in order to obtain the lowest current intensity which would generate an optimal rate of response for a particular animal. This current intensity was used to establish a reliable ICSS performance. After the response was well established, the animals were given a series of 10 min test sessions in which the current intensity was reduced in steps of 25 ~ of the preceding current level. On the basis of these results, 3 current intensities were selected which encompassed the range of the rate-intensity function (approximately 10, 50 and 100~ of optimal responding). Each rat then received 3 sessions with the 3 current intensities in an ascending sequence to establish reliable preoperative performance levels. In each session the tests at each current intensity were 10 min in duration and between tests the rats were returned to their home cage for 10-min breaks. The timing of the 10-min test sessions commenced after the current intensity was adjusted to the appropriate setting. Also, the test order of the two electrodes in each animal was always alternated between changes in current intensity. The current intensity settings for each animal were kept the same throughout testing and the applied voltage was recorded at each setting. Throughout the course of the study, there was never any change in the voltage required to generate a particular current level for any of the rats. Prior to ICSS testing for each of these rate-intensity determinations, each rat was given a 15-min activity test immediately preceding the ICSS testing. One day after completion of these 3 rate-intensity ICSS tests and activity measurements the rats were given the same rate-intensity ICSS and activity tests following an i.p. injection of 0.5 mg/kg D-amphetamine.
105 Activity testing commenced 5 min after the amphetamine injection and the ICSS rate-intensity procedure commenced immediately after completion of the activity determination. Within a few days of the completion of the amphetamine testing the rats were subjected to the unilateral 6-OHDA or vehicle injections. Postoperatively, starting one week after surgery, the rats were given the same rate-intensity and activity tests on two successive days per week. The first day was without drug treatment but the second day was with 0.5 mg/kg D-amphetamine. These weekly tests were continued for 8 weeks postoperation. In addition, after 4 weeks postoperation, all rats were tested for circling following injections of 0.5 mg/kg apomorphine. The circling was observed for 2 min on the laboratory floor, 10 min after the apomorphine injection. The number and direction of the 360 ° turns made in 2 min were recorded for each rat. Only those rats (n = 8) in the SN lesion group which exhibited turning contralateral to the injected hemisphere were included in the study. Catecholamine assay and histology At least one week after completion of testing the rats (under ether anesthesia) were placed in the stereotaxic instrument and their electrodes removed to permit electrode localization. Immediately after electrode removal, the rats were decapitated and their brains rapidly removed over ice. Each brain was then cut coronally at the ventral surface using the rhinal fissures as topographical guides to obtain forebraln tissue anterior to the electrodes for biochemical analysis. This anterior brain section was then divided sagitally in order to obtain comparable hemisphere sections. Each hemisphere was then dissected to remove striatal and limbic (olfactory lobe and accumbens) tissue samples. These tissue samples were then assayed for dopamine and norepinephrine using high performance liquid chromatography with electrochemical detection [ 1]. The LC-EC consisted of a Laboratory Data Control mini mump NS 1-33R, a cheminest CSU 20 injection valve and a glass column dry-packed with pellicular Vydac SC cation-exchange resin. A Model LC-4 electrochemical controller was used with a glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN, USA). The potential was set as + 0.5 with respect to a Ag/AgCI reference electrode. A citrate-acetate buffer solvent system was used. Briefly, the brain tissue samples were weighed to the nearest 0.1 mg and placed in polycarbonate centrifuge tubes containing 500 #1 of 0.1 M H C 1 0 4 (antioxidant) and 50/zl of 3 mg/ml dihydroxybenzylamine (DHBA) (internal standard). The tissue was then sonicated for two 30-sec periods using a Kontes Micro-Ultrasonic cell disrupter (Kontes, Vineland, NJ, USA). The samples were then centrifuged at 20,000 g for 10 min and the supernatant stored in a small polyethylene tube at - 2 0 °C. The amines were determined by injecting 20/~1 of supernatant into the LC-EC system. The norepinephrine (NE) and dopamine (DA) peak heights were ratioed to the DHBA peak height, and the concen-
106 trations (/~g/g brain) calculated knowing the relative response of standards, the amount of DHBA added and the brain sample weight. The remaining caudal brain section was placed in 10~o formalin for a 10-day fixation period. After this period of fixation, 3 mm thick sections containing the electrode or cannula tracts were embedded in paraffin and subsequently 10 ktm-thick sections were cut, mounted and stained with cresyl violet and Luxol fast blue [ 13]. All sections were studied microscopically to identify electrode and cannula tip locations. RESULTS
H istoh)gic al.[hTdings Microscopic examination of histologically prepared sections showed that the electrode tips were all located in the medial forebrain bundle area between the internal capsule laterally and the fornix and mammilo-thalamic tract medially at the level of the ventral medial nucleus pars medialis and between 0.5 and 1.0 mm above the base of the brain. Undoubtedly, the performance criterion used for selecting animals for inclusion in the experiment contributed to the observed homogeneity of electrode placements. Fig. 1 presents a photomicrograph of a representative set of electrode placements.
Fig. 1. Photomicrograph of Kluver-Berrera-stained section indicating location of bilateral electrode placements in the lateral hypothalamic area. The left hemisphere has been notched.
107
Examination of the cannula tracts showed that the needle tips for the substantia nigra injections were typically situated in the medial region of the pars compacta at the level of the interpeduncular nucleus. The more medial injections aimed at the ventral tegmental area were located in a similar coronal plane slightly medial to the most medial extension of the substantia nigra pars reticulata. Generally, the cannula tips were dorsal to the interpeduncular nucleus and slightly medial to the medial lemniscus.
Biochemicalfindings For all 6-OHDA-injected rats there was a marked disparity in dopamine levels in limbic and striatal areas between the injected and intact hemispheres. The overall results are summarized in Table I. As indicated in Table I, the SN lesion produced a severe reduction in striatal dopamilae but not norepinephrine. Limbic reductions were extensive but much more variable and generally less than the striatal deficiency. The rats subjected to 6-OHDA injections aimed at the VTA showed marked losses of dopamine but not norepinephrine in both striatal
TABLE I
Individual striatal and limbic norepinephrine and dopamine (levels expressed as lag/g of brain tissue) Group
Intact hemisphere Striatal
Injectea~hemisphere Limbic
DA
NE
DA
NE
SN 8
9.83 13.20 12.40 6.40 9.10 7.09 9.56 11.90
0.56 1.20 0.71 0.79 0.82 0.40 0.59 0.55
2.41 2.56 3.60 1.97 1.99 2.33 2.10 1.86
1.13 1.20 1.70 0.86 0.79 0.92 1.06 1.23
VTA l VTA 2 VTA3 VTA 4 VTA s
14.07 10.90 4.90 7.08 14.40
0.49 1.07 0.68 1.40 0.57
1.52 2.60 2.60 1.98 2.30
Vl V: V3
9.10 12.50 13.20
0.55 1.00 0.66
1.80 2.00 2.10
SN l SN 2
SN 3 SN 4
SN 5 SN 6 SN 7
Limbic
Striatal
DA
NE
DA
NE
0.07 0.19 0.16 0.37 0.74 0.84 1.02 2.39
0.46 0.41 1.20 0.57 0.78 0.34 0.50 0.38
0.23 0.17 0.27 0.66 0.50 i.45 0.86 1.46
1.30 1.40 1.20 0.61 0.89 0.82 0.77 1.44
0.78 0.98 1.10 1.13 1.07
3.58 2.68 2.70 3.50 8.90
0.27 1.27 0.62 0.66 0.33
0.56 1.29 0.59 1.00 0.94
0.75 0.91 0.86 1.10 1.02
1.10 1.50 1.20
7.50 11.00 12.10
0.40 1.80 0.57
2.00 1.83 2.06
0.97 1.38 0.88
108 and limbic tissue. In general, the limbic decreases were greater than the striatal, but the overall dopamine loss was less severe than that obtained for the SN injection.
Beha vioral findings Figs. 2 and 3 present the individual ICSS results for all animals both with and without the 0.5 mg/kg D-amphetamine treatment. In Fig. 2 the ICSS results are shown for the rats injected unilaterally with 6-OHDA into the pars compacta of the substantia nigra. In terms of the non-drug results, it is apparent that the effects of this lesion were quite .variable with several animals (SN~, SN2, SN3) severely impaired; whereas, others (SN7 and SN8)were only slightly affected. In contrast to this between-animal variability, the inter-hemispheric ICSS results were consistent. In each case when ICSS was reduced at the electrode site in the injected hemisphere, the.virtual same reduction occurred at electrode site located in the unoperated hemisphere. Similarly, when recovery occurred for an electrode INTACT 150 L M H
Current
HEMISPHERE
6-OHDA LMH ,,"
Intensities
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oJ-
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INJECTED HEMISPHERE - - N o n Drug - - - 0 . S m g / k g , d-Amphetomine
.
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I
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2
5
4
5
6
Post Operative Week
7
8
Pre
I
2
5
4
5
6
7
Post Operative Week
I 8
Fig. 2. Individual self-stimulation response rates of rats subjected to unilateral 6-hydroxydopamine injections into the substantia nigra pars compacta. Each rat bad self-stimulation generated from an electrode ipsilateral as well as eontralateral to the 6-hydroxydopamine-injected hemisphere. Three current intensities were used and separate test sessions were conducted both with and without 0.5 mg/kg D-amphetamine.
109 INTACT HEMISPHERE 200rLMH 150~ t. ~.
,oo~ i/ 150" I00 5O
Current
f//liJ/i
i5°: •~ 100
;.l/ii/iii
5O 15 ° :
/.A,i/
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a: I00 g 5O o 200:
E 150
,~ ioo ,u ffl
6-OHDA INJECTED HEMISPHERE
Intensities
50 O,
~////
/ /
7717-'1 777 1 7/ /77 i'?/:f?
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.
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.
I
.
2
,,Z/71/Z
.
3 4 5 6 7 8 Pre I 2 3 4 5 6 ? Post Operative Week Post Operative Week --Non Drug - - - 0 . 5 mg/kg, d-Amphetamine
8
Fig. 3. Individual self-stimulation response rates of rats subjected to unilateral 6-hydroxydopamine injections aimed at the ventral tegmental area and vehicle injections into the substantia nigra, self-stimulation ipsilateral and contralateral to the injected hemisphere is shown for each rat at each of 3 current intensities. Pre- and postoperative testing included separate sessions with and without 0.5 mg/kg D-amphetamine.
placement in the dopamine depleted hemisphere, there was a corresponding recovery in performance for the electrode situated in the intact hemisphere. Another important aspect of the findings presented in Fig. 2 are the effects of amphetamine on ICSS. For every animal, when amphetamine was given preoperatively, ICS S was facilitated at both electrode sites. Postoperatively, however, amphetamine had virtually no effect on self-stimulation generated from either the intact or the dopamine depleted hemisphere and this blockade of amphetamine facilitation of ICSS persisted throughout the 8 weeks of postoperative testing. Fig. 3 presents the ICSS results for the rats injected unilaterally with 6-OHDA into the VTA and for the vehicle-injected rats. In no instance did these injections impair ICSS. The performance of the vehicle-injected rats remained
110
essentially unaltered throughout the course of postoperative testing both with and without amphetamine. The unilateral 6-OHDA injections into the VTA were generally without effect although some transient facilitation of ICSS and attenuation of amphetamine enhancement of ICSS can be noted during the first few postoperative weeks. These modest changes in ICSS typically occurred bilaterally. Table II presents the mean locomotor activity results for the 3 treatment groups both with and without amphetamine. Preoperatively, the 3 groups were closely matched in terms of both non-drug and amphetamine activity levels. Postoperatively, the VTA and vehicle-injected groups remained at preoperative activity levels, but the pars compacta group no longer exhibited an increase in locomotor activity after the 0.5 mg/kg D-amphetamine treatment. In addition to this measure of general activity the spontaneous and apomorphine induced circling was observed. Over the first postoperative week all rats in the SN group exhibited circling toward the injected hemisphere which gradually subsided, although some animals (SN~, SN2, SN3) circled a few times when placed on the laboratory floor during the course of the study. None of the VTA or vehicleinjected rats exhibited spontaneous circling. Additionally, it should be noted that while all rats in the pars compacta substantia nigra 6-OHDA treatment group showed strong circling toward the intact hemisphere after the 0.5 mg/kg injections of apomorphine, none of the rats in the vehicle or VTA groups displayed any circling after this dose of apomorphine but, rather, exhibited the typical
TABLE lI
Mean 15 rain photoactivity counts with and without 0.5 mg/kg D-amphetamine for each treatment group Difference scores were estimated by the t-test. N D , non-drug; A, amphetamine.
Group
Preoperative score
Postoperativeweek I
2
3
4
5
6
7
8
SN ND A
347 450*
296 329
262 308
266 285
264 264
206 257
249 251
209 255
256 244
VTA ND A
310 495*
3"~0 542*
450 502*
434 566*
367 500*
421 596*
430 591'
338 474*
394 570*
Vehicle ND A
368 456
498 554
359 475
443 598
423 500
397 489
416 511
376 463
354 521
* P < 0.05.
111 behavioral stereotypy observed at this dose level of apomorphine. At the conclusion of the self-stimulation testing, the SN rats were again tested for apomorphine-induced circling, at 3 dose levels, 0.125, 0.25 and 0.5 mg/kg. Sensitivity to apomorphine-induced circling generally correlated with the degree of self-stimulation dysfunction with SN 1, SN:, and SN3, circling at 0.125, SN4_ 6 at 0.25 and SNT_8 at 0.5 mg/kg. As is evident from Table I, the lower .the threshold for apomorphine-induced circling, the lower the dopamine level in the injected hemisphere. DISCUSSION The present study was undertaken to determine the degree to which unilateral lesions of dopamine-containing neurons would have unilateral versus bilateral effects on ICSS in rats with bilateral electrode implants which independently generated comparable ICSS performance. Using this experimental paradigm, the present study showed that unilateral lesions of the pars compacta region can have very marked inhibitory effects on ICSS and that these effects are virtually bilaterally symmetrical. The occurrence of bilateral symmetry is entirely consistent with the initial report of Ornstein and Huston [ 16] that unilateral lesions of catecholaminergic neurons produce a profound impairment in ICSS performance. The bilateral symmetry of the performance deficit in the present study seems to be readily explicable as a motoric dysfunction and recovery occurs as the animal, either by learning or some other process, adapts to or spontaneously recovers from the motoric dysfunction and thereby becomes more efficient at obtaining ICS S. This bilateral symmetry in recovery of ICS S also implies that the reinforcement effect was similar for both hemispheres. These observations appear to contradict the earlier report of Koob et al. [ 12], in which self-stimulation was reduced in rats with unilateral electrode implants when the ipsilateral hemisphere was treated with 6-hydroxydopamine, but not when the contralateral hemisphere was injected with 6-hydroxydopamine. An important aspect of this study was that striatal dopamine was reduced to 0.08 #g/g by the ipsilateral injections; whereas the contrallateral injection reduced striatal dopamine to 0.73 #g/g. Since the persistent bilateral deficits in self-stimulation in the present study only occurred with the most severe dopamine reductions (0.4 #g/g or less), it would appear possible to account for the Koob et al. report of lateralization of self-stimulation deficits in terms of the differential severity of the dopamine depletions produced in ipsilateral and contralateral hemispheres. Seemingly, the electrode tract damage plus 6-hydroxydopamine injection in the ipsilateral hemisphere produced greater damage to dopamine neurons than the 6-hydroxydopamine injection alone into the contralateral hemisphere. Presumably, a small residual dopamine neuron population can have a very significant functional influence until reduced to some critical level. This finding appears consistent with the report of Lashley [ 14] on
112 the sparing of visual function in rats which had only small remnants of the visual cortex remaining after visual cortex extirpation. In this context, it is not too surprising that the 6-hydroxydopamine injections aimed at the A10 area in the present study did not have detrimental effects on self-stimulation. These injections produced substantial but only incomplete decreases in limbic dopamine and striatal dopamine. Furthermore, no motoric deficits were apparent including the absence of spontaneous and apomorphineinduced turning behavior. The amphetamine findings, however, are not entirely consistent with the non-drug self-stimulation results in that the pars compacta lesions reliably bilaterally blocked the facilitation of self-stimulation by amphetamine; whereas, the pars compacta lesions produced non-drug self-stimulation deficits only in the cases of the most severe dopamine depletions. It should be noted that the 0.5 mg/kg dose level of amphetamine used in this study induced neither circling nor hyperactivity. Possibly at this dose level the approximately 50 ~ reduction in total brain striatal dopamine by the pars compacta lesion functionally reduced the amphetamine dose to 0.25 mg/kg which was subthreshold for provoking hyperactivity. Seemingly then, the most prosaic explanation for the inconsistency between the non-drug and amphetamine-induced changes in self-stimulation is that the amphetamine-induced behavioral effects are more related to changes in total brain dopamine; whereas the severity of the interhemispheric dopamine asymmetry is more critical for the non-drug behavioral effects to become manifest. In conclusion, the results of the present study show that unilateral injury to the pars compacta of the substantia nigra can have marked bilateral effects on ICCS. The bilateral symmetry of these effects on ICSS seems most easily explicable as a motoric impairment which interferes with ICSS performance although bilateral dysfunction of reinforcement efficacy cannot be ruled out solely on the basis of the present study. More generally, however, brain stimulation reinforcement test procedures can be arranged to make performance easily disrupted by using low current intensities and complex motoric response requirements or difficult to impair by employing high current intensities and very simple response requirements. The use of paradigms which represent these extremes as well as numerous intermediate variations greatly complicate efforts to decipher performance from reinforcement deficits in the effects of experimental manipulations on self-stimulation. In addition, treatments which produce no apparent motoric dysfunction yet impair self-stimulation might create gastrointestinal distress, nausea or some untoward effect without conspicuous symptoms in animals that might otherwise make it readily explicable why an animal failed to perform for the brain stimulation reinforcement. Certainly, studies [10, 22] which have shown that self-stimulation can survive radical removals of brain tissue, have made it apparent that no specific telencephalic structure it necessary for self-stimulation. On the other hand, pharmacological studies suggest transmitter specificity in that
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cholinergic agonists [8] on catecholaminergic antagonists [23] block self-stimulation in conventional paradigms. While it is obvious that these drug treatments which block self-stimulation also typically induce parkinsonism symptomatology, there is also the more general issue of brain dysfunction produced by transmitter imbalance. In the case of severe motoric disorders such as chorea and parkinsonism, in which specific transmitter systems are affected by pathological processes, the symptoms can be alleviated by blocking other remaining intact transmitter symptoms. Thus, transmitter imbalance can be more disruptive to brain function than a more extensive interference with neuronal activity that does not create transmitter imbalances. Extensive brain lesions which presumably leave residual tissue in a state of approximate transmitter balance, therefore, may be the most appropriate methodology for determining the neural systems necessary for brain stimulation reinforcement. ACKNOWLEDGEMENTS
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