On the role of ascending catecholaminergic projections in intracranial self-stimulation of the substantia nigra

Brain Research, 131 (1977) 271-286 © Elsevier/North-Holland Biomedical Press

271

ON T H E ROLE OF A S C E N D I N G C A T E C H O L A M I N E R G I C PROJECTIONS IN I N T R A C R A N I A L S E L F - S T I M U L A T I O N OF T H E SUBSTANTIA N I G R A

RONALD M. CLAVIER* and HANS C. FIBIGER** Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T1W5 (Canada)

(Accepted November 1lth, 1976)

SUMMARY The role of ascending dopaminergic (DA) and noradrenergic (NA) neurons in the rewarding properties of intracranial self-stimulation (ICSS) obtained from electrodes in the zona compacta of the substantia nigra (SNC) was investigated. Rats were trained on a bar-press response to obtain ICSS and then received 6-hydroxydopamine (6-OHDA) lesions of the DA nigrostriatal bundle (NSB) either ipsilateral or contralateral to the electrodes. Lesions ipsilateral to the ICSS electrode, which reduced striatal DA levels to less than 3 ~ of control values, resulted in profound but temporary reductions in ICSS which returned to preoperative levels 8-10 days after the lesions. Identical effects were obtained in animals subjected to NSB lesions contralateral to the electrodes. Alpha-methyl-p-tyrosine at a dose (25 mg/kg) which did not affect responding in unlesioned control animals decreased responding in the ipsilateral and contralateral lesioned groups to a similar extent. The facilitation of responding for ICSS produced by D-amphetamine (1.0 mg/kg) was blocked by the ipsilateral but not by the contralateral NSB lesions. Lesions of ascending NA axons produced by stereotaxic injections of 6-OHDA caudal to the SNC failed to impair ICSS. It is concluded that the deficits in responding for ICSS produced by unilateral 6O H D A lesions of the NSB can be accounted for in terms of transient motor deficits produced by these lesions. The data indicate that the NSB is not the critical neuronal substrate which mediates the rewarding properties of nigral ICSS. Rather, this system appears to serve a non-essential, modulatory function and is involved in the rate-increasing effects of D-amphetamine on nigral ICSS. No evidence for a role of ascending NA systems in nigral ICSS was obtained.

* Present address: Department of Anatomy, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, Iil. 60201, U.S.A. ** To whom correspondence and reprint requests should be sent.

272 INTRODUCTION The demonstration of intracranial self-stimulation (ICSS) from electrodes in the substantia nigra pars compacts (SNC) a0 has led to the hypothesis 9 that stimulation of the A9 dopaminergic (DA) cell group 10 was responsible for the behavior. Pharmacological 24 and histochemical 5 results are consistent with dopaminergic mediation of nigral 1CSS. Dopaminergic mediation has also been hypothesized to account for ICSS in cases where DA neurons are not stimulated directly7,16,1s,2°,22- Cooper and Breese v attenuated ICSS from both the dorsal pontine region of the locus coeruleus and the lateral hypothalamus, as well as the SNC, by depleting brain dopamine, but not brain noradrenaline (NA). It has also been shown that injections of DA blocking agents such as pimozide attenuate ICSS from non-DA brain regions such as the periaqueductal gray 16 and the mesencephalic tegmental area '~'~. Because lesions or pharmacological blockade of ascending DA systems have been shown to disrupt operant responding markedly 11,12,26, it may be questioned whether the reductions of ICSS (above) were truly the consequence of'reward' deficits, or simply the result of performance incapacitation. That DA blockade may, in fact, nullify the rewarding properties of ICSS is suggested by the recent finding by Fouriezos and Wise 1~, who showed that with lateral hypothalamic placements, systemic injections of pimozide resulted in response patterns that were comparable to those seen during extinction consequent to removal of stimulation current. It has also been shown that intracerebral injections of DA blocking agents attenuate ICSS from regions such as the nucleus accumbens, in which DA elements are stimulated, but not from regions such as that of the locus coeruleus, in which DA neurons are not stimulated directly 20. Finally, Phillips et al. 23 have shown that ICSS from the neostriatum is permanently reduced after unilateral 6-hydroxydopamine (6-OHDA) lesions of the ipsilateral A9 cell group, but is only temporarily attenuated after identical lesions of the contralateral A9 group. Thus, at least for certain 1CSS loci, DA neurons may play a critical role in brain-stimulation reward. It is noteworthy, however, that the studies that demonstrated an involvement of DA concerned themselves with ICSS placements other than in the vicinity of DA perikarya. With respect to ICSS from the regions of the DA cell groups themselves, several different studies seem to contradict the dopaminergic hypothesis. Clavier and Corcoran 4 have shown that electrolytic lesions of the sulcal prefrontal cortex result in marked attenuation of nigral ICSS, which is paralleled by heavy orthograde degeneration extending from the lesions to the region of the ICSS electrodes. This indicates that non-DA systems may mediate ICSS obtained from electrodes in SNC. Indications of NA involvement in ICSS from DA cell groups are found in the data of Belluzzi et al. z. These authors showed that knife cuts or 6-OHDA injections caudal to nigral ICSS electrodes temporarily interrupted responding. This interruption was then correlated with depleted levels of NA in the forebrain. In the same study, systemic administration of the dopamine-fl-hydroxylase inhibitor diethyldithiocarbamate (DDC) blocked nigral ICSS, and this was reinstated with intraventricular injections of NA. These data

273 therefore provide evidence of a noradrenergic mediation of nigral ICSS. The present experiments were aimed at determining the relative roles of DA and NA neurons in the nigral ICSS. In the first of two studies, 6-OHDA was injected into the nigrostriatal bundle (NSB) either ipsilateral or contralateral to nigral ICSS electrodes. In the second experiment, 6-OHDA was injected bilaterally caudal to nigral ICSS electrodes; the injections were aimed at the trajectories of ascending NA axons that pass in the vicinity of the SNC, and were administered under conditions that minimize non-specific damage. Neither experiment provided evidence for an exclusive role of ascending catecholaminergic systems in the rewarding properties of nigral ICSS. METHODS

Experiment I Unilateral NSB 6-OHDA: effects on nigral 1CSS Twenty-six male albino rats of the Wistar strain (300-350 g) served as subjects in this experiment. Each rat had a single bipolar nichrome electrode (Plastic Products Co., MS-303-.018-.312-0-0.005 in.) implanted while under pentobarbital anesthesia. Electrodes were aimed at the left ( n = 14) or the right (n-- 12) SNC using the following stereotaxic coordinates: 3.1 mm anterior, 2.1 mm lateral, and 2.1 mm dorsal to the interaural line (Kopf). Electrodes were implanted normal to the horizontal plane, with the stereotaxic incisor bar placed 4.2 mm ventral to that plane. A 7-day recovery period preceded behavioral testing. Testing for ICSS was conducted with the rats in operant conditioning chambers, in which each depression of a lever resulted in a 0.5-sec pulse of 60 Hz sine wave brain stimulation ranging from 25 to 50 #A (rms), depending on the animal. The number of responses in daily 15-min test sessions was counted as the animals' ICSS score. Once optimal performance was obtained at a specific current level, this was not altered for the duration of the experiment. This procedure resulted in stable performance baselines in all animals. At least 5 days after stable baselines were established, the duration of the test session was extended from 15 min to 1 h for one session, and then returned to 15 min the next day. At least 3 days after the 1-h sessions, the animals were given intraperitoneal injections of D-amphetamine sulfate (1.0 mg/kg) 15 min prior to the ICSS session. This was followed by at least 3 non-drug ICSS sessions, after which, unilateral 6-OHDA lesions were administered. In order to restrict the lesions to DA neurons, the animals were given intraperitoneal injections of desipramine HC! (25 mg/kg). This procedure blocks uptake of 6O H D A by NA neurons 2s. After 30 min, each animal was anesthetized (Halothane) and had a 34-gauge cannula inserted into the left NSB at the level of the lateral hypothalamus. Fourteen of the lesions were ipsilateral to nigral electrodes, and 12 were contralateral to those electrodes. Lesion coordinates, with the incisor bar at 4.2 mm below the interaural line, were: 5.9 mm anterior, 2.3 mm lateral, and 1.9 mm dorsal to the interaural line. With the cannula in place, 4/~g of 6-OHDA hydrobromide (dosage

274 expressed as the base and dissolved in 0.15 M NaC1 containing 0.2 mg/ml ascorbic acid) was injected in a volume of 2 #1 over a period of 10 min. The animals were all tested for nigral ICSS 24 h after the lesions, and for at least the next 21 days. At least 16 days after the lesions, a single 1-h test session as well as an intraperitoneal injection of o-amphetamine sulfate (1.0 mg/kg) were conducted in the same manner as prior to the lesions. In addition, intraperitoneal injections of alphamethyl-p-tyrosine (AMPT) (25 mg/kg) were given 4 h prior to the next-to-final ICSS session. A similar dose of A M P T was given to 5 non-lesioned animals with nigral ICSS electrodes as a control. A minimum of 48 h was allowed between the final test session and the sacrificing of the animals. Sacrificing was accomplished by means of cervical fracture; the forebrains were then removed, dissected over ice, and analyzed for striatal DA content on the lesioned and non-lesioned sides 19. The mid-brains were immersed in 10~o formalin-saline for at least 1 week, after which frozen sections (30 #m) were taken in the frontal plane and stained with cresyl violet for histological examination. Experiment H NA system 6-OHDA: effects on nigral I C S S Six male albino (Wistar) rats served as subjects in this experiment. Each animal had a bipolar ICSS electrode implanted in the region of the SNC in the same manner as described in Experiment I. Testing for ICSS, including the establishment of current levels and stable performance baselines were also similar to Experiment I. After stable baselines were achieved, the animals were anesthetized (Halothane) and given bilateral injections of 6-OHDA (4/~g/2 #1) as in Experiment I, except that the cannulae were aimed at the caudal mesencephalic trajectories of ascending NA fibers in the central tegmental tract 17, formerly referred to as the ventral NE bundle 35. Stereotaxic coordinates for these injections were: 0.9 mm anterior, 1.2 mm lateral, and 2.0 mm dorsal to the interaural line, with the incisor bar set at 4.2 mm below that line. These animals were tested for ICSS on each of 15 postlesion days. They were then killed by means of cervical fracture, and their brains were removed and dissected. Cortical, hypothalamic, and hippocampal NA and striatal DA were then measured. As in Experiment I, the midbrains were kept in 1 0 ~ formalin-saline to permit histological verification of the nigral ICSS placements. RESULTS Experiment I Unilateral N S B 6-OHDA: effects on nigral I C S S Biochemistry. Twenty of the lesions in the this study resulted in extensive depletions of DA in the ipsilateral striatum. These results are summarized in Table I. In 10 of the animals, lesions were ipsilateral to nigral ICSS electrodes; the average residual DA level in the 'ipsilateral' group was 1.2 ~ . In 5 of these cases the residual

275 TABLE I

Effects of unilateral 6-OHDA lesions of the left nigrostriatal projection on ipsilateral and contralateral striatal dopamine*

lpsilateral (n = 10) Contralateral (n = 10) Partial lesions (n -- 6)

Left

Right

Left/Right (%)

0.14 :k 0.07 0.25 :t: 0.07 8.00 :k 0.19

11.24 -4- 0.49 10.43 ± 0.33 11.80 ± 1.59

1.2 2.4 67.8

* Data expressed as the mean 4- S.E.M.

IPSh

CONTRA.

Fig. 1. Schematic summary of ICSS sites in rats with extensive 6-OHDA lesions of the left NSB (Experiment I). Most of the placements in the ipsilateral and contralateral groups corresponded to the medial half of the SNC, although some of the sites were in the SNR. A, B, C, refer to the placements in the corresponding photomicrographs in Fig. 2.

276

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£iiii¸i~ii~

i i iii:!ili ii!! ;5

Fig. 2. Photomicrographs of ICSS regions in 3 animals with extensive ipsilateral depletions of striatal DA (Experiment 1). Note that two of the electrode tips (small arrows, A, C) contacted the region of the SNC, while that in case B was slightly ventral to the SNC, in the SNR. Inserts 1-4 compare the regions of the A9 and A10 DA cells groups on the lesioned and unlesioned sides. In all 3 cases, the areas of both the ipsilateral A9 and AI0 cells (inserts 1 and 3, respectively) were devoid of the moderately stained, 15-20/~m cell bodies (large arrows) that characterized those two regions on the unlesioned side of the brain (inserts 2 and 4, respectively). Chromatolysis in the A9 region corresponded with the average 98.2 % depletion of striatal DA. Comparable chromatolysis in the A 10 region indicated severe damage of the mesolimbic DA system; however, no corresponding measure of DA depletion was obtained to verify the degree of damage to that system. Magnification: A, B, C, x 40; inserts, :x 100. Abbreviations: Ill, oculomotor nerve; ML, medial lemniscus; E, electrode tip. D A levels gave blank readings. The assay technique used is sensitive to 5 ng. I n the other 10 animals with extensive lesions, the damaged NSB was contralateral to the nigral electrodes; the average residual level of D A in this 'contralateral' group was o/ 2.4/o. Two of the animals in this group had b l a n k levels of DA. Of the 6 rats that did not show extensive depletions of DA, 4 had ipsilateral electrodes a n d 2 had contralateral electrodes. These 6 animals were grouped u n d e r the heading 'partial lesions' in order to compare their behavioral data with animals that received extensive depletions. The average residual level of D A on the side of their lesions was 67.8 %~. I C S S sites. Microscopic examination of the ICSS sites indicated that most of the electrode tips contacted the SN just dorsal to the pars compacta. Fig. 1 summarizes these placements in both the ipsilateral and contralateral groups. E x a m i n a t i o n of the region s u r r o u n d i n g the electrode sites in the ipsilateral g r o u p revealed an interesting pattern of chromatolysis, which is demonstrated in Fig. 2. The low-power micrographs

277 <~ 150 uJ :>
~10C LU

---:-

L ............

,'-'-'~r--

: -

........

u. 0 N

5C

o"

/

/

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--LESIONS

~CONTRALATERA" NSB LESIONS

o

5432 DAYS BEFORE NSB 6-OHDA

3

5

7 9 11 13 15 17 19 DAYS AFTER NSB 6-OHDA

21

Fig. 3. Recovery of nigral 1CSS after 6-OHDA-induced lesions of either the ipsilateral (filled triangles) or the contralateral (open triangles) nigrostriatal projection (Experiment I). That the 8-10-day recovery period of the two groups was specific to extensive NSB damage is evidenced by the less severe disruption at 24-48 h, and the faster recovery to prelesion rates in a group of animals with only an average 32700 damage of the NSB (open circles). Prelesion rates were: ipsilateral 469 i 56; contralateral 419 ± 60; and partial NSB lesions 532 ± 109 bar-presses/15 min. Data expressed as the daily group mean ~ of prelesion average scores ± S.E.M.

(Fig. 2 A, B, C) show the nigral ICSS sites in three of the rats with extensive ipsilateral NSB 6-OHDA lesions. The inserts numbered 1 and 2 show the region of the SNC on the ipsilateral and contralateral sides, respectively. In all cases, the ipsilateral side (1) appeared to be devoid of the moderately stained, 15-20 /~m cell bodies that characterized the SNC on the unlesioned side (2). It is presumed that the chromatolyzed cells constituted the A9 DA cell group, in view of the extensive depletions of DA in the ipsilateral striatum. Of equal interest is that the region medial to the SN, which contains the A10 DA cell group, also showed severe chromatolysis on the side of the lesions. The A10 regions are shown in inserts 3 and 4 of Fig. 2. Note that compared to the unlesioned side (4), moderately stained cell bodies of the order of 15-20 #m are virtually absent among the longitudinally sectioned fibers of the third cranial nerve (HI) on the lesioned side (3). Thus, the AI0 cell group of the me~olimbic DA system a~ appears to have been extensively damaged by the 6-OHDA lesions in this study; however, biochemical or histochemical data with which to quantify the degree of mesolimbic loss were not obtained. Effects on nigral ICSS. The effects of extensive NSB lesions on nigral ICSS are presented in Fig. 3. The graphs in Fig. 3 show that ICSS was virtually abolished in both the ipsilateral and contralateral groups for 48 h after the lesions. ICSS recovered over the next few days, however, and the contralateral group reached 100~o of prelesion scores on the 8th postlesion day while the ipsilateral group reached its prelesion level on the 10lh postlesion day. Both groups then stabilized at approximately prelesion levels for the remainder of the experiment. There was no difference in the rate of recovery of ICSS between the ipsilateral and contralateral lesioned groups with extensive NSB damage (F=0.036, N.S.). The group with partial depletions of striatal

278 ETAMINE ,g/kg)

*30 c~ Z

~ATMENT

0 o. * 2 0 co uJ B~

"AN

cO +10 69 0 Z

~ ~10 -to -20

CONTRA IPSI PRELESION

CONTRA IPSl POSTLESION

Fig. 4. Effects of D-amphetamine sulphate (1 mg/kg, i.p.) on nigral ICSS. Prior to unilateral NSB lesions, all animals showed significant increases in ICSS to about 140% o f their predrug averages. Similar elevations were seen after extensive NSB lesions in the contralateral group. However, rats with extensive NSB lesions ipsilateral to their nigral ICSS electrodes showed a significant group average reduction in performance rates to about 69 % of their predrug values. Data are expressed as the group mean deviation in ICSS scores ( ± S.E.M.). * Significantly different from control rate, P < 0.05.

DA responded at over 5 0 ~ of its prelesion rate 24 h after the lesion surgery, and recovered to 100% of those levels on the 5th postlesion day; after this, they too stabilized at prelesion levels. From these data it appears that extensive unilateral lesions of the NSB retard the recovery of nigral ICSS, when compared with less severe lesions of that system. But, complete recovery of the behavior does occur within 3-10 days of extensive unilateral NSB lesions, regardless of whether the nigral ICSS electrode is ipsilateral or contralateral to those lesions. D-Amphetamine. Prior to lesions of the NSB, intraperitoneal injections of oamphetamine enhanced nigral ICSS significantly in both groups of animals (t ipsilateral = 2.08, P < 0.05, t contralateral ~ 3.12, P < 0.02). These results are summarized in Fig. 4. In this figure, the data for each treatment are expressed as the resultant deviation from the mean of the 3 daily scores preceding that treatment. The same procedure was used in subsequent figures that deal with AMPT and extended duration treatments. As Fig. 4 shows, o-amphetamine prior to the lesions elevated ICSS by approximately 200 responses in both groups. This corresponded to about 140 % of predrug values in both the ipsilateral and the contralateral groups. After extensive NSB lesions, the 10 animals with contralateral electrodes responded to D-amphetamine in virtually the same manner as they had before the lesions. The elevations were again significant (t=2.78; P ~ 0.05), and averaged approximately 201 responses above predrug rates, which corresponded to an increase to about 169 % of predrug rates. However, the animals with electrodes ipsilateral to extensive NSB lesions showed a group average reduction of ICSS after injections of Damphetamine. This reduction was significant ( t = 2.74; P -< 0.05), and averaged about 145 responses, which corresponded to 69% of predrug values. Thus, extensive 6OHDA lesions of the NSB ipsilaterak but not contralateral, to nigral electrodes not

279 [~] ¢0140C

PRELESION ICSS/lhr

I POSTLESION

120(

ICSS/lhr

z100( nl 80C

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6OC

z°

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IPSI

Fig. 5. Effects of extending ICSS sessions from 15 min to 1 h. Data are expressed as the group average deviation (± S.E.M.) from the mean of the three 15-min sessions prior to the 1-h session ( ± S.E.M.). Prior to the lesions (shaded bars), the contralateral and ipsilateral groups showed ICSS elevations to 355 ~ and 400 ~ of their 15-min averages, respectively. After extensive NSB lesions (hatched bars) the elevation in the contralateral group (to 303 ~) and the ipsilateral group to (326 ~) tended to be less, but not significantly so, than those seen before the lesions.

I I'--IPREDRUQ

A M P T (25 mg/kg)

~, 7 +1oo

z

-loc

uJ o z
IPSI

CONTRA

Fig. 6. Effects of alpha-methyl-p-tyrosine (AMPT; 25 mg/kg, i.p.) on nigral ICSS after unilateral 6-OHDA lesions of the NSB. Injections were administered 4 h prior to the ICSS sessions. Unoperated control animals were not significantly affected by the AMPT. The lesioned animals showed approximately 30-40~ reductions from their predrug averages after AMPT injections. These reductions did not differ significantly, however, between the ipsilateral and the contralateral groups. Data are expressed as the absolute deviation in ICSS scores from the mean of the 3 predrug test sessions for each group -~- S.E.M. * Significantly different from control rate, P < 0.05. only attenuate b u t in fact reverse the a m p h e t a m i n e - m e d i a t e d facilitation of nigral ICSS.

Extended duration. E x t e n d i n g the test d u r a t i o n from 15 m i n to 1 h resulted in a 3-4-fold elevation in ICSS scores. These results are shown in Fig. 5. Specifically, the ipsilateral group average rose from a b o u t 468 to 1874, or 400 ~ before the lesions, a n d from a b o u t 457 to 1492, or 326 ~ after the lesions. The slight r e d u c t i o n i n the elevation of ICSS over extended d u r a t i o n s after the NSB lesions was n o t significant (t=0.81; P > 0.05). Similar results characterized the contralateral group. I n this group, the average ICSS score rose from a b o u t 351 to 1245, a n increase of 355 ~ . After NSB

280 TABLE II Effects of bilateral 6-OHDA lesions of ascending NA pathways on NA levels in the hippocampus and cerebral cortex and on DA levels in the striatum* Group

Striatal DA (Izg/g)

6-OHDA lesions (n -- 6) 7.66 ± 0.60 Control (n 3) 10.04 ± 0.38

% of control

Hypothalamic % of NA (t~g/g) control

Hippocampus- % of cortex NA control (~g/g)

76.3

0.44 :k 0.07

20.0

0.022 ± 0.002

100.0

2.19 ± 0.14

100.0

5.4

0.396 ± 0.030 100.0

* Data expressed as the mean zk S.E.M. lesions, the group average rose from approximately 314 to 951, or about 303 ~o. Thus, in this group too there was an overall reduction in extended responding after extensive contralateral NSB lesions; but, as in the group with ipsilateral lesions, this reduction was not statistically significant (t=0.055, P > 0.05). Alpha-methyl-p-tyrosine. The effects of A M P T (25 mg/kg) on nigral ICSS are presented in Fig. 6. The average score of the 5 unlesioned control animals rose insignificantly from about 632 to about 638 bar-presses after AMPT. However, both groups with unilateral NSB lesions did show significant reductions of ICSS after A M P T injections. The ipsilateral group was reduced from about 507 responses to about 350 responses, or to approximately 6 9 ~ of predrug rates (t=2.01, P < 0.05). The contralateral group was reduced from about 322 responses to about 197 responses, or to about 61 ~ of predrug rates (t=5.59; P < 0.001). Thus, unilateral NSB lesions augment the inhibitory action of A M P T on nigral ICSS; but, as was the case in the extended duration treatment, this effect was the same whether the lesions were ipsilateral or contralateral to the nigral ICSS electrodes. Experiment H N A system 6 - O H D A : effects on nigral I C S S Biochemistry and histology. The biochemical effects of bilateral 6-OHDA lesions

caudal to nigral ICSS electrodes are presented in Table II. All 6 lesioned animals had severe depletions of hippocampal and cortical NA. The residual N A levels in these regions was 5.4 ~ , compared with the same regions in 3 unimplanted, unlesioned control brains. Hypothalamic NA was also extensively reduced ; residual NA levels in that region averaged 2 0 . 0 ~ of control values. The nigrostriatal system was only slightly damaged, as evidenced by the 76.3 ~o residual striatal DA levels compared to controls. Histological analysis indicated that all 6 ICSS electrodes contacted the SNC, which was not in a chromatolyzed state. Nor was the ventral tegmental area, in which the A10 DA cell group is found, in a chromatolyzed condition.

281

1°°f tr W I.U > rr Z 0 O9 W or O. u_ 0 N

2

4 DAYS

6 AFTER

8

10

6-OHDA

12

14

LESIONS

Fig. 7. Nigral ICSS in animals after extensivebilateral 6-OHDA lesions of ascending NA systems. The average telencephalic depletion of NA in these animals was 94.6~; that of hypothalamic NA was 80.0 % The lesions failed to significantlyinfluence nigral ICSS.

Effects on nigral ICSS. Fig. 7 shows that extensive damage of ascending NA systems did not disrupt nigral ICSS. Responding was at prelesion levels 24 h after the lesions, and did not differ significantly from the preoperative rate at any time during the 15-day postoperative testing period. DISCUSSION On the basis of several studies utilizing neuroleptics and 6-OHDA it has been proposed that rather than disrupting central reinforcement mechanisms; these treatments decrease operant responding for ICSS primarily by impairing the function of DA systems critically involved in the initiation or maintenance of operant behaviorll, 22. The present observations are entirely consistent with this hypothesis. Unilateral lesions of the NSB, which resulted in a near complete loss of striatal DA, resulted in a profound but temporary reduction in ICSS obtained from electrodes in the SNC. More importantly, however, the reduction and recovery of ICSS did not differ between groups of animals that received lesions either ipsilateral or contralateral to the SNC electrodes. Clearly therefore the transient reduction in ICSS was not the result of the destruction of a neuronal substrate critically involved in the rewarding properties of nigral ICSS. Had this been the case, lesions ipsilateral to the SNC electrodes would, at a minimum, have been expected to result in more severe reductions in and a slower rate of recovery of ICSS. Rather, the data indicate that unilateral lesions of the NSB result in transient deficits in operant behavior and that these deficits can account completely for the disruption of responding for ICSS. Ornstein and Huston 21 recently reached identical conclusions regarding ICSS in the lateral hypothalamus. They found that unilateral injections of 6O H D A into the substantia nigra disrupted ICSS equally in both the ipsilateral and contralateral lateral hypothalamus. Furthermore, bilateral 6-OHDA injections abol-

282 ished ICSS when lever-pressing was the criterion but not when more simple motor responses were used as operants. In order to evaluate more thoroughly the possible role of the dopaminergic NSB in nigral ICSS additional experiments were conducted. One alternative that was considered was that the few remaining DA terminals in the striatum may have been sufficient to maintain ICSS responding after the NSB lesions. This was examined first by extending the ICSS session from 15 min to 1 h and secondly by determining the effects of a low dose of A M P T on responding for ICSS. In the first instance it was hypothesized that stimulation of the remaining 2 ~ of the NSB may have been sufficient to support the behavior for short test sessions but might not support the behavior throughout extended sessions. In the second instance, advantage was taken of the finding by Cooper et al. 8 that animals pretreated with intraventricular injections of 6-OHDA are more sensitive to the tyrosine hydroxylase inhibitor AMPT. It was reasoned that if the remaining stores of striatal DA were responsible for the rewarding properties of nigral ICSS after ipsilateral NSB lesions, then the group with ipsilateral electrodes should be particularly susceptible to the rate-decreasing effects of AMPT. On the other hand, if AMPT was equally effective in modifying ICSS in the ipsilateral and contralateral groups then this would be consistent with the motor deficit hypothesis. As is evident in Figs. 5 and 6, the results clearly support the latter hypothesis. In both tests the contralateral group showed effects which were indistinguishable from the ipsilateral group. The data obtained with AMPT also suggest that the recovery from performance deficits, observed over 8-10 days in both lesioned groups, was mediated by the remaining striatal DA stores in combination with the development of postjunctional supersensitivity2~, 36 and are consistent with previous reports of increased sensitivity to AMPT in animals with 6-OHDA-induced lesions 8, 33 It is well established that D-amphetamine can increase the rate of responding for ICSS~2, 31. Inasmuch as D-amphetamine increases the release and decreases the reuptake of catecholamines 34,37, it has been hypothesized that the facilitation of ICSS by D-amphetamine is related to the combined effects of the drug and electrical stimulation on central CA neurons. The present experiments attempted to test this hypothesis directly. Before the NSB lesions, o-amphetamine (1.0 mg/kg) significantly increased ICSS in ipsilateral and contralateral groups to a similar extent. After the NSB lesions the drug continued to facilitate responding in the group with lesions contralateral to the nigral electrodes. In animals with lesions on the same side as the ICSS electrodes, however, D-amphetamine failed to enhance responding for brain stimulation. In fact, after the lesions D-amphetamine produced a significant decrease in the ipsilateral group. These data provide direct evidence that ascending dopaminergic neurons play a critical role in the rate-increasing effects of D-amphetamine on nigral ICSS. Cooper et al. 8 reached similar conclusions regarding ICSS in the lateral hypothalamus after observing that selective depletion of brain DA by intraventricular 6-OHDA injections significantly reduced the rate-increasing effect of D-amphetamine (0.25 mg/kg). The present results clearly suggest that facilitation of responding for nigral ICSS by D-amphetamine is due to the additive effect of electrical stimulation and D-amphetamine on the release of DA in the terminal regions of ascending DA systems.

283 That the ipsilateral NSB lesions not only abolished amphetamine-induced facilitation of nigral ICSS but in fact resulted in a significant inhibition of responding requires further comment. This observation may have implications for the finding that D-amphetamine is much more effective at increasing ICSS from some sites in the brain than others 22,24. The relatively small facilitation of nigral ICSS produced by D-amphetamine ~4 may be due to the drug having two opposing actions on the systems mediating this behavior. On the one hand, the present findings indicate that the facilitatory effects of D-amphetamine on nigral ICSS are mediated by the dopaminergic NSB. On the other hand, it appears that the post-lesion ICSS behavior, which is supported primarily by the non-dopaminergic systems in the vicinity of the SNC, is inhibited by D-amphetamine. In this regard, it is of interest that we have occasionally observed unlesioned animals with electrodes in the substantia nigra in which ICSS is consistently decreased by D-amphetamine. Presumably in these cases, the electrical stimulation predominantly influences the non-dopaminergic neural elements in the substantia nigra. When taken together, these findings indicate that the dopaminergic NSB is not the critical neural substrate for the reinforcing properties of ICSS maintained by electrodes in the SNC. On the other hand, the results obtained with D-amphetamine indicate that it would be incorrect to conclude that this projection is irrelevant to the phenomenon. Indeed, the latter observations clearly suggest that this system serves a modulatory or ancillary role. Whether the dopaminergic NSB would be sufficient to maintain nigral ICSS after lesions of the non-dopaminergic neural elements is not presently known. These considerations raise questions regarding the nature of the system(s) in the vicinity of the nigral electrodes which supported ICSS after the 6OHDA lesions. One possibility is that there was sufficient current spread to stimulate the A10 neurons in the ventral tegmental area (VTA) and that the dopaminergic mesolimbic system which originates in this region maintained the ICSS behavior. Although DA concentrations in the terminal regions of this system were not measured in the present experiments, this alternative appears unlikely for several reasons. First, as was the case in the zona compacta, there was extensive chromatolysis of cells in the A10 region. Secondly, we have found recently that lesions identical to those utilized in the present experiments reduce dopamine in the terminal regions of the mesolimbicmesocortical projection by approximately 90 ~ (Fibiger, in preparation). Inasmuch as some of the remaining dopamine in these regions represents precursor in noradrenergic terminals, these 'NSB' lesions also clearly produce very extensive damage to the mesolimbic-mesocortical dopaminergic projections. Finally, the D-amphetamine data show that the dopaminergic substrate, be it A9 or A10, which mediates the druginduced facilitation of responding was destroyed by the 6-OHDA lesions. Belluzzi et al. 2 have suggested that ascending noradrenergic axons in the vicinity of the SNC 17 mediate the rewarding properties of nigral ICSS. The present experiments failed to support this hypothesis. 6-OHDA lesions of the ascending NA axons, which resulted in equal or greater depletions of hypothalamic and forebrain NA than were obtained by Belluzzi et al. 2, did not attenuate nigral ICSS at any time after the lesions. However, the present experiments utilized considerably lower concentrations

284 and amounts of 6-OHDA and indicate therefore that the transient reductions in nigral ICSS observed by Belluzzi et al. 2 were probably due to non-specific effects of the lesions, unrelated to destruction of ascending NA systems 1. With regard to the observation that the DDC-induced attenuation of nigral ICSS can be reinstated with intraventricular injections of NA 2, these experiments failed to provide evidence indicating that this was due to the restoration of the 'reward' properties of nigral stimulation rather than the reversal of some other effect of DDC such as the sedation and illness that this drug has been shown to produce 27,29. Stinus and Thierry 3~ have also provided data that suggest that NA systems mediate ICSS obtained from electrodes in the region of the A10 cell bodies. These workers observed that AMPTmediated inhibition of ICSS in the VTA could be reversed by DL-dihydroxyphenylserine (DOPS), a compound which can be decarboxylated to form NA. However, while this finding tends to implicate NA it should be pointed out that because the striatum contains high levels of DOPA decarboxylase activity, this treatment would also result in NA being synthesized in the striatum. Inasmuch as NA can stimulate DA receptors in the striatum a4, the DOPS-induced reinstatement of ICSS in the VTA does not necessarily implicate a central NA pathway in this behavior. We conclude that non-catecholaminergic systems in the vicinity of the substantia nigra are important neural substrates of ICSS and that the dopaminergic NSB serves a non-essential perhaps modulatory function. These findings stand in contrast to recent results from this laboratory which suggested that the NSB serves a critical function in the rewarding properties of neostriatal ICSS z3. In view of these earlier results, the present observations were not anticipated. When taken together, however, these findings indicate that the NSB can serve an essential function in the rewarding properties of ICSS obtained from some sites (neostriatum), but in other regions (lateral hypothalamus 21 and substantia nigra) other neuronal pathways can maintain ICSS in the absence of this system. Clearly then, the role of the NSB in ICSS is not a simple one and additional experiments are required to define more precisely the role of this and other DA projections in central reinforcement mechanisms. In any event, the present observations argue for multiple and perhaps redundant pathways in the vicinity of the substantia nigra which can support nigral ICSS. In this regard, Clavier and Corcoran 4 have recently observed that lesions of the prefrontal sulcal cortex produced substantial reductions in nigral but not dorsal tegmental ICSS. Confirming Leonard 15, these lesions were also found to produce extensive orthograde degeneration in the substantia nigra and it was concluded that this decending projection may be a primary neural substrate for the reinforcing effects of nigral ICSS. Finally, other systems which cannot at present be excluded include the nigrothalamic pathway and the numerous other non-catecholaminergic projections which ascend and descend through the substantia nigra and ventral tegmental area 8,6. ACKNOWLEDGEMENTS Supported by the Non-Medical Use of Drugs Directorate and the Medical Research Council. H.C.F. is a M.R.C. Scholar. We gratefully acknowledge the excellent technical assistance of S. Atmadja, B. Richter and G. Murray.

285 REFERENCES 1 Agid, Y., Javoy, F., Glowinski, J., Bouvet, D. and Sotelo, C., Injection of 6-hydroxydopamine into the substantia nigra of the rat. II. Diffusion and specificity, Brain Research, 58 (1973) 291-301. 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 Clavier, R.M., Atmadja, S. and Fibiger, H. C., Nigrothalamic projection in the rat as demonstrated by orthograde and retrograde tracing techniques, Brain Res. Bull., 1 (1976) 379-384. 4 Clavier, R. M. and Corcoran, M. E., Attenuation of self-stimulation from substantia nigra but not dorsal tegmental noradrenergic bundle by lesions of sulcal prefrontal cortex, Brain Reserach, 113 (1976) 59-69. 5 Clavier, R. M. and Routtenberg, A., Ascending monoamine-containing fiber pathways related to intracranial self-stimulation : histochemical fluorescence study, Brain Research, 72 (1974) 25-40. 6 Clavier, R. M. and Routtenberg, A., Fibers associated with brain stem self-stimulation: FinkHeimer study, Brain Research, 105 (1976) 325-332. 7 Cooper, B. R. and Breese, G. R., A role for dopamine in the psychopharmacology of electrical selfstimulation of the lateral hypothalamus, substantia nigra, and locus coeruleus. In The Functional Significance of Brain Monoaminergic Systems. Pharmacological and Biochemical Approaches, 1976, In press. 8 Cooper, B. R., Cott, J. M. and Breese, G. R., Effects ofcatecholamine-depleting drugs and amphetamine on self-stimulation of brain following various 6-hydroxydopamine treatments, Psychopharmacologia (BerL), 37 (1974) 235-248. 9 Crow, T. J., A map of the rat mesencephalon for electrical self-stimulation, Brain Research, 36 (1972) 265-273. 10 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta PhysioL scand., 62 Suppl. 232 (1964) 1-55. 11 Fibiger, H. C., Carter, D. A. and Phillips, A. G., Decreased intracranial self-stimulation after neuroleptics or 6-hydroxydopamine: Evidence for mediation by motor deficits rather than by reduced reward, Psychopharmacologia (BerL), 47 (1976) 21-27. 12 Fibiger, H. C., Phillips, A. G. and Zis, A. P., Deficits in instrumental responding after 6-hydroxydopamine lesions of the nigro-neostriatal dopaminergic projection, Pharmacol. Biochem. Behav., 2 (1974) 87-96. 13 Fouriezos, G. and Wise, R. A., Pimozide-induced extinction of intracranial self-stimulation: response patterns rule out motor or performance deficits, Brain Research, 103 (1976) 377-380. 14 lversen, L. L., Dopamine receptors in the brain, Science, 188 (1975) 1084-1089. 15 Leonard, C. M., The prefrontal cortex of the rat. I. Cortical projection of the medio-dorsal nucleus. II. Efferent connections, Brain Research, 12 (1969) 321-343. 16 Liebman, J. M. and Butcher, L. L., Effects on self-stimulation behaviour of drugs influencing dopaminergic neurotransmission mechanisms, Acta pharmacol. (Kbh.), 277 (1973) 305-318. 17 Lindvall, O. and Bj6rklund, A., The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method, Acta physiol, scand., Suppl. 412 (1974) 4-48. 18 Lippa, A. S., Antelman, S. M., Fisher, A. E. and Canfield, D. R., Neurochemical mediation of reward: a significant role for dopamine?, PharmacoL Biochem. Behav., 1 (1972) 23-28. 19 McGeer, E. G. and McGeer, P. L., Catecholamine content of the spinal cord, Canad. J. Biochem. PhysioL, 40 (1962) 1141-1151. 20 Mora, F., Rolls, E. T., Burton, M. J. and Shaw, S. G., Effects of dopamine-receptor blockade on self-stimulation in the monkey, Pharmacol. Biochem. Behav., 4 (1976) 211-216. 21 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. 22 Phillips, A. G., Brooke, S. M. and Fibiger, H. C., Effects of amphetamine isomers and neuroleptics on self-stimulation from the nucleus accumbens and dorsal noradrenergic bundle, Brain Research, 85 (1975) 13-22. 23 Phillips, A. G., Carter, D. A. and Fibiger, H. C., Dopaminergic substrates of intracranial selfstimulation in the caudate-putamen, Brain Research, 104 (1976) 221-232. 24 Phillips, A. G. and Fibiger, H. C., Dopaminergic and noradrenergic substrates of positive reinforcement: differential effect of D- and L-amphetamine, Science, 179 (1973) 575-577.

286 25 Price, M. T. C. and Fibiger, H. C., Apomorphine and amphetamine stereotypy after 6-hydroxydopamine lesions of the substantia nigra, Europ. J. PharmacoL, 29 (1974) 249-252. 26 Price, M. T. C. and Fibiger, H. C., Discriminated escape learning and response to electric shock after 6-hydroxydopamine lesions of the nigro-neostriatal dopaminergic projection, Pharmacol. Biochern. Behav., 3 (1975) 285-290. 27 Roberts, D. C. S. and Fibiger, H. C., Conditioned taste aversion induced by diethyldithiocarbamate (DDC), Neurosci. Lett., 2 (1976) 339-342. 28 Roberts, D. C. S., Zis, A. P. and Fibiger, H. C., Ascendingcatecholamine pathways and amphetamine -induced locomotor activity : importance of dopamine and apparent non-involvement of norepinephrine, Brain Research, 93 (1975) 441454. 29 Roll, S. K., lntracranial self-stimulation and wakefulness: effect of manipulating brain catecholamines, Science, 168 (1970) 1370 1372. 30 Routtenberg, A. and Malsbury, C., Brainstem pathways of reward, J. comp, physiol. Psychol., 68 (1969) 22-30. 31 Stein, L., Self-stimulation of the brain and the central stimulant action of amphetamine, Fed. Proc., 23 (1964) 836-850. 32 Stinus, L. and Thierry, A. M., Self-stimulation and catecholamines. II. Blockade of self-stimulation by treatment with alpha-oaethyl-para-tyrosine and the reinstatement by catecholamine precursor administration, Brain Research, 64 (1973) 189-198. 33 Stinus, L., Thierry, A. M. and Cardo, B., Self-stimulation and local injections of 6-hydroxydopamine into the rat brain: enhanced behavioural depressive effects of ¢t-methylparatyrosine, Pharmacol. Bioehem. Behav., 3 (1975) 19-23. 34 Thornburg, J. F. and Moore, K. E., Dopamine and norepinephrine uptake by rat brain synaptosomes: relative inhibitory potencies of L- and D-amphetamine and amantadine, Res. Commun. Chem. Path. PharmacoL, 5 (1973) 81-89. 35 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand., Suppl. 367 (1971) 1~-8. 36 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, A cta physiol, scand., Suppl. 367 (197t) 69-93. 37 Von Voigtlander, P. F. and Moore, K. E., Involvement of nigro-striatal neurons in the in vivo release of dopamine by amphetamine, amantadine and tyramine, J. PharmacoL exp. Ther., 184 (1973) 542-552.