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Increased in vivo tyrosine hydroxylase activity in rat telencephalon produced by self-stimulation of the ventral tegmental area
Brain Research, 402 (1987) 109-116 Elsevier
109
BRE 12299
Increased in vivo tyrosine hydroxylase activity in rat telencephalon produced by self-stimulation of the ventral tegmental area Anthony G. Phillips 1, Alexander Jakubovic 2 and Hans C. Fibiger 2 IDepartment of Psychology, University of British Columbia, Vancouver (Canada) and 2Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver (Canada) (Accepted 17 June 1986) Key words: Dopamine; 3,4-Dihydroxyphenylalanine (DOPA); NSD-1015; Tyrosine hydroxylase; Ventral tegmental area; Nucleus accumbens; Striatum; Olfactory tubercle; Brain-stimulation reward; Food reward
Changes in the activity of dopaminergic neurons associated with intracranial self-stimulation of the ventral tegmentum were assessed by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) after inhibition of aromatic amino acid decarboxylase by NSD-1015. When compared to implanted unstimulated controls, DOPA concentrations were elevated significantly in the nucleus accumbens, striatum and olfactory tubercle in the hemisphere ipsilateral to the electrode, after a 30 min session of self-stimulation. The concentration of DOPA in the contralateral nucleus accumbens and striatum did not differ from control levels, although relative to control values it was significantly increased in the contralateral olfactory tubercle. A similar analysis of in vivo tyrosine hydroxylase activity in these brain regions following a 30 min session of lever pressing for food reward on a fixed-ratio (FR-8) schedule failed to reveal any significant changes relative to control subjects. These results are consistent with a role for dopamine in brain-stimulation reward obtained from electrical stimulation of the ventral tegmental area but do not provide evidence for dopaminergic mediation of the rewarding properties of food. INTRODUCTION Despite a long-standing interest in the n e u r o c h e m istry of brain-stimulation r e w a r d (cf. refs. 6, 11, 20, 27), an unequivocal case has yet to be m a d e for the role of a specific n e u r o t r a n s m i t t e r in this i m p o r t a n t neurobiological process. In part, the difficulty stems from the fact that there is no single ' r e w a r d - n e u r o transmitter'. R a t h e r , multiple systems a p p e a r to be concerned with different aspects of r e w a r d processes 21. Biochemical analyses of brain n e u r o t r a n s m i t t e r s have p r o v i d e d the most direct assessment of the neural substrates of brain-stimulation r e w a r d and to date such studies suggest a role for d o p a m i n e ( D A ) in selfstimulation b e h a v i o r elicited by electrical stimulation of the ventral t e g m e n t a l area ( V T A ) . F o r e x a m p l e , self-stimulation in the V T A , but not the posterior hypothalamus, has been associated with increased con-
centrations of the D A m e t a b o l i t e 3,4-dihydroxyphenylacetic acid ( D O P A C ) in the olfactory tubercle is. In that study, no significant changes were observed in either the nucleus accumbens or striatum. H o w e v e r , increased D A t u r n o v e r has b e e n o b s e r v e d in both the nucleus accumbens and striatum of mice after stimulation of D A fibers at the level of the hypothalamus t°. These latter effects were seen only at sites ipsilateral to the stimulating e l e c t r o d e , t h e r e b y ruling out m o t o r activity as a causal factor. W e have o b t a i n e d a similar p a t t e r n of results in rats with elect r o d e placements in the V T A 8. Significant increases in D A m e t a b o l i s m were o b s e r v e d in the nucleus accumbens, olfactory tubercle and to a lesser degree in the striatum. A g a i n these changes were o b s e r v e d only in the h e m i s p h e r e ipsilateral to the site of brainstimulation. In a related e x p e r i m e n t , self-stimulation of the V T A was disrupted by 6 - h y d r o x y d o p a m i n e lesions of the m e s o t e l e n c e p h a l i c D A pathways in the
Correspondence: A.G. Phillips, Department of Psychology, The University of British Columbia, 2136 West Mall, Vancouver, B.C., Canada V6T 1Y7. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
110 ipsilateral, but not in the contralateral hemisphere 8. Collectively, these data suggest that the release of D A is closely associated with brain-stimulation reward obtained from electrodes in the VTA. The present study provided a further assessment of changes in the synthesis of D A associated with VTA self-stimulation by measuring the accumulation of D O P A after aromatic amino acid decarboxylase inhibition by m-hydroxybenzylhydrazine dihydrochloride (NSD-1015). This procedure was used initially to obtain in vivo estimates of the activity of D A neurons in the nigrostriatal and mesolimbic pathways 4,19 and more recently in conjunction with high pressure liquid chromatography with electrochemical detection (HPLC-EC) to study the catabolism of D A in the rat brain 29. In addition to a role in brain-stimulation reward, it has been suggested that D A systems may be involved more generally with the rewarding properties of natural substances such as palatable foods 3°. These two hypotheses were tested in the present study by monitoring tyrosine hydroxylase (TH) activity in the striatum, nucleus accumbens and olfactory tubercle during self-stimulation of the VTA or barpressing for food reward on a fixed-ratio (FR-8) schedule of reinforcement. MATERIALS AND METHODS
Intracranial self-stimulation Male hooded rats of the Long-Evans strain were housed in individual stainless steel cages throughout the experiment. Food and water were available ad libitum. At the time of surgery, body weights ranged from 300 to 350 g. Each rat had a bipolar nichrome electrode (Plastic Products Co., NS-303-0.005 in.) implanted stereotaxically while under pentobarbital anesthesia. The electrode was aimed at the A10 region of the ventral tegmentum using the following coordinates: 2.8 mm anterior, 0.6 mm lateral, and 2.1 mm dorsal to stereotaxic zero (Kopf). The stereotaxic incisor bar was set at 3.0 mm ventral to the interaural line. Testing for self-stimulation was conducted in 6 Plexiglas boxes (46 × 30 × 24 cm) housed within sound attenuating chambers. Depression of a lever (25 × 7.5 cm) delivered an AC sine wave current (60 Hz) of a fixed duration (200 ms) through a flexible lead connected to the chronically implanted elec-
trode assembly. The current intensities varied from 20 to 30ktA and were pre-set by a computer (Nova-3; Manx software) controlling the value of a set of resistors in a 6-channel constant current stimulator. Two weeks after surgery each subject was screened for self-stimulation. After 10 min of shaping, the rat was allowed to self-stimulate for 30 rain. The current intensity for each rat was selected to elicit the highest self-stimulation rate possible. This procedure was continued until each rat had at least 5 days of experience with self-stimulation at maximum current intensities. At this time rats were assigned to one of two groups: VTA self-stimulation (n = 7), or unstimulated controls (n = 7). On the day after the last screening session each rat received an intraperitoneal injection of NSD-1015 (50 mg/kg, Sigma) in a volume of 2 ml/kg, 5 rain prior to behavioral testing. Subjects in the self-stimulation group were killed by cervical fracture immediately after a 30 min self-stimulation session. Control subjects received no brain stimulation and were placed into test boxes in which the levers had been removed and sacrificed after 30 min. The brains were rapidly removed, cut in the coronal plane at the level of the optic chiasm, mounted on a microtome and frozen with CO2. Histological confirmation of the electrode placements was obtained by sectioning the mesencephalon in a cryostat, and examining coronal sections of the brains, stained with Cresyl violet.
Dissection and assay procedures Immediately following decapitation, brains were rapidly removed, a coronal cut was made slightly caudal to the optic chiasm and the anterior section frozen on a microtome. Two consecutive 1-mm coronal slices were taken and placed on ice so that the rostral surface was face up. According to K6nig and Klippe114, the rostral extent of the anterior slice was approximately at the level of A9820/zm while the rostral limit of the second slice was approximately at A8620 /~m. The nucleus accumbens was dissected from the rostral slice. The structure was defined as being ventral to the genu of the corpus callosum and dorsal to the olfactory tubercle (demarcated by small fiber bundles forming the olfactory radiations). The anterior commissure was included in the sample. The olfactory tubercle was taken from both slices. The lateral limit of this structure (both slices) was the ol-
111 factory tract while the septal region served as the medial border. The dorsal border was the ventral limit of the nucleus accumbens. The striatum was dissected from the caudal slice and was defined dorsally and laterally by the corpus callosum. The medial border was formed by the lateral ventricles. The ventral limit was imposed by making a horizontal cut at the level of the anterior commissure. The tissues were weighed, placed in 1.5 ml polypropylene microcentrifuge tubes containing ice-cold 0.2 N HCIO4 with 0.15% of NA2S205 and 0.5% Na2EDTA, disrupted by sonication and centrifuged at 30,000 g for 15 min. The clear supernatant was stored at -80 °C until analyzed. The precipitate was assayed for protein content 15. For the determination of amines and metabolites, 20/~1 aliquots of samples filtered through a membrane of 0.2 ktm pore size (Schleicher and Shuel) were injected directly into the HPLC-EC system. The system consisted of a Beckman model 100A pump, LC-4B amperometric detector with a glassy carbon electrode (BAS), 3390A recorder integrator (Hewlett-Packard) and a ~Bondapak C18 (10 ktm, 3.9 x 150 mm) reverse phase analytical column (Waters), fitted with a precolumn (30 x 3.9 mm). The potential was set at +0.75 V vs an Ag/AgC reference electrode, and the sensitivity at 10-20 nA/V full scale. The eluent consisted of 0.1 M KH2PO 4 buffer containing 2 mM sodium octanesulfonic acid (OSA), 0.15 mM Na2EDTA, 1% methanol, pH 3.4 for D O P A separation (retention time 2.4 min; flow rate 1.7 ml/min). The concentration of D O P A was established by comparing the peak areas with the known standards and is expressed as Bg/g protein. The detection limit under these conditions was approximately 80 pgm/20 ktl tissue sample.
Lever pressing for food reward Eighteen male hooded rats (450-500 g) of the L o n g - E v a n s strain were food deprived to 85-90% of their body weights. Six rats were shaped to lever press for food pellets (Noyes, 45 mg) on a continuous reinforcement schedule (CRF) for 30 min each day. After 5 days on the CRF schedule, the density of reinforcement was gradually decreased until the animals worked on a schedule in which every eighth lever press was reinforced (FR-8). Lever pressing was
maintained on this schedule for 10-12 days. At the same time as the above animals were tested each day, animals from a second group (n = 6) were placed in adjoining chambers in which the levers had been removed from the testing boxes. An animal in this group (yoked controls) automatically received a food pellet each time that its counterpart in the lever pressing group was rewarded with food. Animals in the third group (n = 6), were placed in an adjacent testing box without levers at the same time as the lever pressing group. However, animals in this group never received food pellets in the chamber. Thus, there were 6 squads consisting of 3 animals each: a lever presser, a yoked control, and a no-food control subject. The members of each squad remained constant throughout the experiment. One hour after each daily session each rat received an amount of laboratory chow in its home cage sufficient to maintain its weight at 85% of pre-training body weight. On the test day, all subjects received an intraperitoneal injection of NSD-1015 (50 mg/kg) in a volume of 2 ml/kg, 5 min prior to behavioral testing. Animals from each squad were killed by cervical fracture immediately after the 30 min behavioral session. The brains were rapidly removed, dissected and analyzed for D O P A by HPLC-EC as described above. RESULTS
Intracranial self-stimulation Subjects in both the experimental and control groups responded at comparable rates after the initial training period. On the final day of training, the response rate for the experimental group was .~ -2926 + 182 as compared to ~ = 2820 + 202/30 min for the control subjects. During the 30 min test session immediately preceding sacrifice, animals in the selfstimulation group responded at an average rate of 2998 + 239/30 min. The biochemical analyses of D O P A concentrations revealed significant increases in the striatum, nucleus accumbens and olfactory turbercle in the group that had engaged in self-stimulation behavior for 30 min prior to sacrifice (see Fig. 1). In the striaturn and nucleus accumbens, stimulation-induced increases in the concentrations of D O P A were observed only in the hemisphere ipsilateral to the V T A self-stimulation electrode. Relative to the electrode
112 STRIATUM
ACCUMBENS
OLFACTORY TUBERCLE 1
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ICSS
Fig. 1. DOPA concentrations in the striatum, nucleus accumbens and olfactory tubercle of an implanted unstimulated control group (CTRL) or a group that had engaged in intracranial self-stimulation (ICSS) of the ventral tegmental area for 30 rain prior to sacrifice. Open bars indicate brain regions contralateral to the electrode and shaded bars indicate regions ipsilateral to the electrode placement. Data are expressed as means _+ S.E.M. (~r) Significant difference from ipsilateral hemisphere in control group, P < 0.05. (~r "1") Significant difference from contralateral hemisphere in control group, P < 0.05. containing h e m i s p h e r e in control subjects, D O P A concentrations were increased by 61% in the striaturn and 58% in the nucleus accumbens. No significant differences between self-stimulation and control groups were seen in the striatum and nucleus accumbens contralateral to the electrode placements. A different p a t t e r n of results was o b s e r v e d in the olfactory tubercle. A g a i n , c o m p a r e d to unstimulated controls, D O P A was increased by 63% in the hemisphere ipsilateral to the stimulation electrode. In addition, however, the concentration of D O P A in the contralateral h e m i s p h e r e of the self-stimulation group was increased significantly by 39% c o m p a r e d to the value in the corresponding h e m i s p h e r e of the non-stimulated control group. Nevertheless in the experimental group, the concentration of D O P A was significantly higher in the h e m i s p h e r e ipsilateral to the stimulation electrode when c o m p a r e d to the contralateral hemisphere ( P < 0.05). The statistical significance of these patterns of results was c o n f i r m e d by analyses of variance, as a group (self-stimulation vs control) by h e m i s p h e r e (ipsilateral vs contralateral) interaction. The F values for the interactions were; striatum, Fl,12 = 19.08, P < 0.001; nucleus accumbens, F1,12= 9.65, P < 0.009; olfactory tubercle, F1,12= 7.33, P < 0.01.
A n i m a l s in the lever-pressing group r e s p o n d e d for food at a m e a n rate of 1176 + 170 presses/30 rain on the test day and received an average of 147 _+ 21 pellets. The subjects in the y o k e d group f o o d c o n s u m e d the same n u m b e r of food pellets but did not e n g a g e in o p e r a n t behavior. F o r graphic p r e s e n t a t i o n , the c o n c e n t r a t i o n s of D O P A in each h e m i s p h e r e were a v e r a g e d to give a single value for each brain region from each subject. The m e a n values of D O P A concentration in the striaturn, nucleus accumbens and olfactory t u b e r c l e for each group are shown in Fig. 3. N o significant g r o u p differences in the concentrations of D O P A were ob-
A 2.1
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Fig. 2. Coronal sections of the rat mesencephalon, redrawn from K6nig and KlippelTM, indicating the location of the tips of bipolar electrodes from unstimulated control subjects (0) and subjects in the ICSS group (11).
113 STRIATUM
ACCUMBENS
OLFACTORY TUBERCLE
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Fig. 3. DOPA concentrations in the striatum, nucleus accumbens and olfactory tubercle of an unfed control group (CTRL), a yoked control group that received free pellets at the same time as subjects engaged in lever pressing for food pellets (Lever) on an FR-8 schedule of reinforcement. Data from both hemispheres were combined to give a single value for each brain region for individual subjects. Data are expressed as means + S.E.M. served in any of the 3 brain regions sampled, nor were any significant differences observed as a function of hemisphere. There was a slight trend towards elevated concentrations of D O P A in the striatum of the lever press group, but this was not statistically significant. DISCUSSION Significant increases in D O P A concentrations are observed in the terminal fields of the mesotelencephalic D A system ipsilateral to electrodes in the A10 region of the V T A following self-stimulation at optimal response rates. Accumulation of D O P A following decarboxylase inhibition appears to be a valid index of tyrosine hydroxylase activity in vivo 4A9'29. Therefore, it may be concluded that electrical stimulation of D A neurons at the parameters employed (60 Hz, sine wave) increased the activity of TH, the rate limiting enzyme for the synthesis of D A . While it is recognized that the brain regions sampled here contain both D A and norepinephrine (NE), the levels of D A exceed those of N E by a factor of 50:1 in the striatum and 5:1 in the nucleus accumbens 2s. These differences coupled with the location of the stimulating electrodes which were in close proximity to D A neurons in the V T A suggest that a major portion of the accumulated D O P A occurred in terminals of D A neurons. As such, these data were consistent with a role for D A in brain-stimulation reward.
These results in unanesthetized freely moving animals complement an earlier report of increased D O P A accumulation, following aromatic amino acid decarboxylase inhibition and increased specific activity of D A in the neostriatum after administration of [3H]tyrosine, produced by electrical stimulation of the substantia nigra pars compacta of anesthetized rats 19. In that study 19, electrical stimulation did not affect endogenous tyrosine or D A levels or increase tyrosine specific activity in the neostriatum of the stimulated hemisphere. Therefore it was concluded that the observed increases in D A specific activity could not be attributed to changes in endogenous D A levels, increased transport of tyrosine into the stimulated striatal regions or preferential uptake of labeled tyrosine. By inference, it may be assumed that accumulation of D O P A was not influenced by any of these factors. Other studies have confirmed that in the absence of DOPA-decarboxylation, increased T H activity is reflected by a significant increase in the accumulation of D O P A 4'29. Increased accumulation of D O P A in the nucleus accumbens, striatum and olfactory tubercle during V T A self-stimulation complement findings from this and other laboratories that have used changes in the ratio of both D O P A C and homovanillic acid to D A as indices of dopaminergic activity 8. This pattern of results is consistent with the fact that dopaminergic neurons in the V T A project to both the striatum and nucleus accumbens 5'7. Using bilateral stimulation of
114 the VTA, Simon et al. 25 reported increased D O P A C / D A ratios in both the nucleus accumbens and medial prefrontal cortex, a structure not sampled in the present study. Mitchell et al. 18 noted increased D A activity in the olfactory tubercle following selfstimulation of the VTA. However, these authors failed to observe significant changes in either the nucleus accumbens or striatum, which they attributed to their electrode placements being close to the midline of the brain. Although a yoked stimulation group receiving brain-stimulation in the abscence of lever pressing was not included in the present study, this condition was shown to increase D A turnover in the nucleus accumbens and striatum in a related study s. It remains to be determined whether yoked stimulation is rewarding for subjects with a previous history of self-stimulation behavior. Nevertheless, these biochemical experiments suggest that brain-stimulation reward at sites in the VTA is accompanied by the release of D A in the terminal fields of the mesotelencephalic D A system. The fact that self-stimulation of the VTA is greatly attenuated by lesions of these same pathways suggests that dopaminergic activity is an important component of brain-stimulation reward in this region of the brain 8'22. Although the present findings emphasize dopaminergic substrates of reward there is clear evidence for the involvement of non-dopaminergic pathways in brain-stimulation reward. For example, recent studies with [14C]2-deoxyglucose (2-DG) autoradiography failed to demonstrate metabolic activation of telencephalic D A terminal fields in response to rewarding stimulation of the posterior medial forebrain bundle 9. The 2-DG technique also may be used to confirm activation of D A systems in conjunction with rewarding stimulation of D A cell bodies in the VTA 23 and substantia nigra 9. Pulse duration appears to be a critical variable for selective activation of D A neurons, as 0.2-0.5 ms square wave pulses have been shown to be ineffective for electrically induced release of D A as measured by in vivo voltammetry 17. In contrast, longer square wave pulse durations of 1.0-5.0 ms or 60 Hz sine wave pulses are very effective in causing the release of D A in the striatum 17. Failure to pay careful attention to stimulation parameters may lead to confusion regarding the contribution of dopaminergic and non-dopaminergic pathways to the phenomenon of brain-stimulation reward.
The finding that increased TH activity was restricted to regions ipsilateral to the stimulating electrode is important, as it would appear to rule out lever pressing and other forms of motor behavior as causal factors. A further control for motor performance effects is provided by the absence of increased accumulation of D O P A in animals lever pressing at high rates for food reward. However, it should be noted that mean maximal response rates for food were lower than those obtained in the self-stimulation tests. The failure to obtain evidence of increased D A activity in rats lever pressing on an FR-8 schedule is somewhat inconsistent with previous reports. For example, operant behavior maintained by water reinforcement has been reported to be associated with increased synthesis of D A in the striatum but not in the nucleus accumbens as measured by either the conversion of [3H]tyrosine to [3H]DA 13 or the accumulation of D O P A following inhibition of aromatic amino acid decarboxylase 24, the procedure used in the present study. This discrepancy may be related to the use of water vs food reward, but is unlikely to be due to either food or water deprivation per se, as neither of these manipulations is associated with changes in D A metabolism 12'16. An alternate explanation of these differences may lie in the methods of dissection employed. Our procedure sampled tissue from the anterior region of the striatum (anterior to the decussation of the anterior commissure) and perhaps samples from more posterior areas are required to observe the reported increases in D A activity associated with operant lever pressing behavior. Despite these caveats, it is important that this issue be resolved as the combined data from the self-stimulation and food-reinforcement experiments reported here suggest that D A synthesis is not increased during the performance of a well-established lever pressing response. The present results also speak to the issue of a general role for D A in reward processes. The failure to observe increased activity in D A systems produced by food reward using both the D O P A accumulation measure used here, and the D A metabolite/DA ratio employed in a separate study s, does not support a role for D A in natural reward processes 3°. Nor have we observed increased activity in D A systems following ingestion of a palatable saccharin solution 1, although there is evidence for such increases 1 h after
115 consumption of a large meal of food pellets or liquid diet in the home cage 1'12. A n alternate hypothesis is
primary reinforcement mechanism. Therefore, dopaminergic substrates of brain-stimulation reward
that increased activity in dopaminergic n e u r o n s is associated with processes underlying the anticipation of reward and the initiation of preparatory behaviors that take place prior to consumption of a primary re-
may be related to the activation of systems associated with incentive motivation 3, but not those c o n c e r n e d with analyzing the hedonic properties of sensory signals.
ward. In accordance with this hypothesis, the D A receptor antagonist pimozide has been shown to attenuate conditioned preparatory responses that precede a meal, while failing to block the subsequent ingestion of a palatable liquid diet 2. The a t t e n u a t i o n by ha-
ACKNOWLEDGEMENTS
loperidol of conditioned place preference using food reward 26 may also reflect a disruption of conditioned
and D. Fu is gratefully acknowledged. Supported by Program G r a n t No. 23 from the Medical Research
anticipatory responses rather than i m p a i r m e n t of a
Council of Canada.
REFERENCES
11 Hall, R.D., Bloom, F.E. and Olds, J., Neuronal and neurochemical substrates of reinforcement, Neurosci. Res. Progr. Bull., 15 (1977) 133-314. 12 Heffner, T.G., Hartman, J.A. and Seiden, L.S., Feeding increases dopamine metabolism in the rat brain, Science, 108 (1980) 1168-1170. 13 Heffner, T.G. and Seiden, L.S., Synthesis of 3H-catecholamines from 3H-tyrosine in brain regions of rats during performance of operant behavior, Brain Research, 183 (1980) 403-419. 14 K6nig, J.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas, Williams and Wilkins, Baltimore, 1963. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Luttinger, D.A. and Seiden, L.S., Increased NE metabolism after water deprivation in the rat brain, Brain Research, 208 (1981) 147-165. 17 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the median forebrain bundle, Eur. J. Pharmacol., 109 (1985) 341-348. 18 Mitchell, M.J., Nicoleau, N.M., Arbuthnott, G.W. and Yates, C.M., Increases in dopamine metabolism are not a central feature of intracranial self-stimulation, Life Sci., 30 (1982) 1081-1085. 19 Murrin, L.C. and Roth, R.H., Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis, Mol. Pharmacol., 12 (1976) 463-475. 20 Olds, J., Drives and Reinforcements: Behavioral Studies of Hypothalamic Function, Raven Press, New York, 1977. 21 Phillips, A.G., Brain reward circuitry: a case for separate systems, Brain Res. Bull., 12 (1984) 195-201. 22 Phillips, A.G. and Fibiger, H.C., The role of dopamine in maintaining intracranial self-stimulation in the ventral tegmentum, nucleus accumbens and medial prefrontal cortex, Can. J. Psychol., 32 (1978) 58-66. 23 Porrino, L.J., Esposito, R.U., Seeger, T.F., Crane, A.M., Pert, A. and Sokoloff, L., Metabolic mapping of the brain during rewarding self-stimulation, Science, 224 (1984) 306-309.
1 Blackburn, J.R., Phillips, A.G., Jakubovic, A. and Fibiger, H.C., Increased dopamine metabolism in the nucleus accumbens and striatum following consumption of a nutritive meal but not a palatable non-nutritive saccharin solution, Pharmacol. Biochem. Behav., in press. 2 Blackburn, J.R., Phillips, A.G. and Fibiger, H.C., Dopamine and preparatory behavior. 1: effects of pimozide, Behay. Neurosci., in press. 3 Bolles, R.C., Reinforcement, expectancy and learning, Psych. Rev., 79 (1972) 394-409. 4 Carlsson, A., Kehr, W., Lindquist, M., Magnusson, T. and Atack, C.V., Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev., 24 (1972) 371-384. 5 Carter, D.A. and Fibiger, H.C., Ascending projections of presumed dopamine-containing neurons in the ventral tegmentum of the rat as demonstrated by horseradish peroxidase, Neuroscience, 2 (1977) 569-576. 6 Crow, T.J., Specific monoamine systems as reward pathways: evidence for the hypothesis that activation of the ventral mesencephalic dopaminergic neurons and noradrenergic neurons of the locus coeruleus complex will support selfstimulation responding. In A. Wauquier and E.T. Rolls (Eds.), Brain-Stimulation Reward, North-Holland Publishing Co., Amsterdam, 1976, pp. 211-237. 7 Fallon, J.H. and Moore, R.Y., Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum, J. Comp. Neurol., 180 (1978) 545-580. 8 FiNger, H.C., Jakubovic, A. and Phillips, A.G., The role of dopamine in intracranial self-stimulation of the ventral tegmental area, J. Neurosci., submitted. 9 Gallistel, C.R., Gomita, Y., Yadin, E. and Campbell, K.A., Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward blocking doses of pimozide, J. Neurosci., 5 (1985) 1246-1261. 10 Garrigues, A.M. and Cazala, P., Central catecholamine metabolism and hypothalamic self-stimulation behavior in two inbred strains of mice, Brain Research, 265 (1983) 265-271.
The excellent technical assistance of F . G . L e P i a n e
116 24 Seiden, L.S. and Heffner, T.G., Alteration of brain catecholamine metabolism by environmental and behavioral events: an explanation of drug-behavior interactions. In E. Usdin (Ed.), Catecholamines: Neuropharmacology and Central Nervous System -- Theoretical Aspects, A.R. Liss Inc., New York, 1984, pp. 275-284. 25 Simon, H., Stinus, L., Tassin, J.P., Lavielle, S., Blanc, G., Thierry, A.M., Glowinski, J. and Le Moal, M., Is the dopaminergic mesocorticolimbic system necessary for intracranial self-stimulation? Biochemical and behavioral studies from A10 cell bodies and terminals, Behav. Neural. Biol., 27 (1979) 125-145. 26 Spyraki, C., Fibiger, H.C. and Phillips, A.G., Attenuation by haloperidol of place preference conditioning using food reinforcement, Psychopharmacology, 77 (1982) 379-382. 27 Stein, L., Chemistry of reward and punishment. In D.H. Efron (Ed.), Psychopharmacology, A Review of Progress,
1957-1967, U.S. Government Printing Office, Washington, D.C., 1968, pp. 105-123. 28 Westerink, B.H.C. and Mulder, T.B.A., Determinations of picomole amounts of dopamine, noradrenaline, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylalacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid in nervous tissue after one-step purification on sephadex G-10, using high performance liquid chromatography with a novel type of electrochemical detection, J. Neurochem., 36 (1981) 1449-1462. 29 Westerink, B.H.C. and Wirix, E., On the significance of tyrosine for the synthesis and catabolism of dopamine in rat brain: evaluation by HPLC with electrochemical detection, J. Neurochem., 40 (1983) 758-764. 30 Wise, R.A., Neuroleptics and operant behavior: the anhedonia hypothesis, Behav. Brain Sci., 5 (1982) 39-53.
109
BRE 12299
Increased in vivo tyrosine hydroxylase activity in rat telencephalon produced by self-stimulation of the ventral tegmental area Anthony G. Phillips 1, Alexander Jakubovic 2 and Hans C. Fibiger 2 IDepartment of Psychology, University of British Columbia, Vancouver (Canada) and 2Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver (Canada) (Accepted 17 June 1986) Key words: Dopamine; 3,4-Dihydroxyphenylalanine (DOPA); NSD-1015; Tyrosine hydroxylase; Ventral tegmental area; Nucleus accumbens; Striatum; Olfactory tubercle; Brain-stimulation reward; Food reward
Changes in the activity of dopaminergic neurons associated with intracranial self-stimulation of the ventral tegmentum were assessed by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) after inhibition of aromatic amino acid decarboxylase by NSD-1015. When compared to implanted unstimulated controls, DOPA concentrations were elevated significantly in the nucleus accumbens, striatum and olfactory tubercle in the hemisphere ipsilateral to the electrode, after a 30 min session of self-stimulation. The concentration of DOPA in the contralateral nucleus accumbens and striatum did not differ from control levels, although relative to control values it was significantly increased in the contralateral olfactory tubercle. A similar analysis of in vivo tyrosine hydroxylase activity in these brain regions following a 30 min session of lever pressing for food reward on a fixed-ratio (FR-8) schedule failed to reveal any significant changes relative to control subjects. These results are consistent with a role for dopamine in brain-stimulation reward obtained from electrical stimulation of the ventral tegmental area but do not provide evidence for dopaminergic mediation of the rewarding properties of food. INTRODUCTION Despite a long-standing interest in the n e u r o c h e m istry of brain-stimulation r e w a r d (cf. refs. 6, 11, 20, 27), an unequivocal case has yet to be m a d e for the role of a specific n e u r o t r a n s m i t t e r in this i m p o r t a n t neurobiological process. In part, the difficulty stems from the fact that there is no single ' r e w a r d - n e u r o transmitter'. R a t h e r , multiple systems a p p e a r to be concerned with different aspects of r e w a r d processes 21. Biochemical analyses of brain n e u r o t r a n s m i t t e r s have p r o v i d e d the most direct assessment of the neural substrates of brain-stimulation r e w a r d and to date such studies suggest a role for d o p a m i n e ( D A ) in selfstimulation b e h a v i o r elicited by electrical stimulation of the ventral t e g m e n t a l area ( V T A ) . F o r e x a m p l e , self-stimulation in the V T A , but not the posterior hypothalamus, has been associated with increased con-
centrations of the D A m e t a b o l i t e 3,4-dihydroxyphenylacetic acid ( D O P A C ) in the olfactory tubercle is. In that study, no significant changes were observed in either the nucleus accumbens or striatum. H o w e v e r , increased D A t u r n o v e r has b e e n o b s e r v e d in both the nucleus accumbens and striatum of mice after stimulation of D A fibers at the level of the hypothalamus t°. These latter effects were seen only at sites ipsilateral to the stimulating e l e c t r o d e , t h e r e b y ruling out m o t o r activity as a causal factor. W e have o b t a i n e d a similar p a t t e r n of results in rats with elect r o d e placements in the V T A 8. Significant increases in D A m e t a b o l i s m were o b s e r v e d in the nucleus accumbens, olfactory tubercle and to a lesser degree in the striatum. A g a i n these changes were o b s e r v e d only in the h e m i s p h e r e ipsilateral to the site of brainstimulation. In a related e x p e r i m e n t , self-stimulation of the V T A was disrupted by 6 - h y d r o x y d o p a m i n e lesions of the m e s o t e l e n c e p h a l i c D A pathways in the
Correspondence: A.G. Phillips, Department of Psychology, The University of British Columbia, 2136 West Mall, Vancouver, B.C., Canada V6T 1Y7. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
110 ipsilateral, but not in the contralateral hemisphere 8. Collectively, these data suggest that the release of D A is closely associated with brain-stimulation reward obtained from electrodes in the VTA. The present study provided a further assessment of changes in the synthesis of D A associated with VTA self-stimulation by measuring the accumulation of D O P A after aromatic amino acid decarboxylase inhibition by m-hydroxybenzylhydrazine dihydrochloride (NSD-1015). This procedure was used initially to obtain in vivo estimates of the activity of D A neurons in the nigrostriatal and mesolimbic pathways 4,19 and more recently in conjunction with high pressure liquid chromatography with electrochemical detection (HPLC-EC) to study the catabolism of D A in the rat brain 29. In addition to a role in brain-stimulation reward, it has been suggested that D A systems may be involved more generally with the rewarding properties of natural substances such as palatable foods 3°. These two hypotheses were tested in the present study by monitoring tyrosine hydroxylase (TH) activity in the striatum, nucleus accumbens and olfactory tubercle during self-stimulation of the VTA or barpressing for food reward on a fixed-ratio (FR-8) schedule of reinforcement. MATERIALS AND METHODS
Intracranial self-stimulation Male hooded rats of the Long-Evans strain were housed in individual stainless steel cages throughout the experiment. Food and water were available ad libitum. At the time of surgery, body weights ranged from 300 to 350 g. Each rat had a bipolar nichrome electrode (Plastic Products Co., NS-303-0.005 in.) implanted stereotaxically while under pentobarbital anesthesia. The electrode was aimed at the A10 region of the ventral tegmentum using the following coordinates: 2.8 mm anterior, 0.6 mm lateral, and 2.1 mm dorsal to stereotaxic zero (Kopf). The stereotaxic incisor bar was set at 3.0 mm ventral to the interaural line. Testing for self-stimulation was conducted in 6 Plexiglas boxes (46 × 30 × 24 cm) housed within sound attenuating chambers. Depression of a lever (25 × 7.5 cm) delivered an AC sine wave current (60 Hz) of a fixed duration (200 ms) through a flexible lead connected to the chronically implanted elec-
trode assembly. The current intensities varied from 20 to 30ktA and were pre-set by a computer (Nova-3; Manx software) controlling the value of a set of resistors in a 6-channel constant current stimulator. Two weeks after surgery each subject was screened for self-stimulation. After 10 min of shaping, the rat was allowed to self-stimulate for 30 rain. The current intensity for each rat was selected to elicit the highest self-stimulation rate possible. This procedure was continued until each rat had at least 5 days of experience with self-stimulation at maximum current intensities. At this time rats were assigned to one of two groups: VTA self-stimulation (n = 7), or unstimulated controls (n = 7). On the day after the last screening session each rat received an intraperitoneal injection of NSD-1015 (50 mg/kg, Sigma) in a volume of 2 ml/kg, 5 rain prior to behavioral testing. Subjects in the self-stimulation group were killed by cervical fracture immediately after a 30 min self-stimulation session. Control subjects received no brain stimulation and were placed into test boxes in which the levers had been removed and sacrificed after 30 min. The brains were rapidly removed, cut in the coronal plane at the level of the optic chiasm, mounted on a microtome and frozen with CO2. Histological confirmation of the electrode placements was obtained by sectioning the mesencephalon in a cryostat, and examining coronal sections of the brains, stained with Cresyl violet.
Dissection and assay procedures Immediately following decapitation, brains were rapidly removed, a coronal cut was made slightly caudal to the optic chiasm and the anterior section frozen on a microtome. Two consecutive 1-mm coronal slices were taken and placed on ice so that the rostral surface was face up. According to K6nig and Klippe114, the rostral extent of the anterior slice was approximately at the level of A9820/zm while the rostral limit of the second slice was approximately at A8620 /~m. The nucleus accumbens was dissected from the rostral slice. The structure was defined as being ventral to the genu of the corpus callosum and dorsal to the olfactory tubercle (demarcated by small fiber bundles forming the olfactory radiations). The anterior commissure was included in the sample. The olfactory tubercle was taken from both slices. The lateral limit of this structure (both slices) was the ol-
111 factory tract while the septal region served as the medial border. The dorsal border was the ventral limit of the nucleus accumbens. The striatum was dissected from the caudal slice and was defined dorsally and laterally by the corpus callosum. The medial border was formed by the lateral ventricles. The ventral limit was imposed by making a horizontal cut at the level of the anterior commissure. The tissues were weighed, placed in 1.5 ml polypropylene microcentrifuge tubes containing ice-cold 0.2 N HCIO4 with 0.15% of NA2S205 and 0.5% Na2EDTA, disrupted by sonication and centrifuged at 30,000 g for 15 min. The clear supernatant was stored at -80 °C until analyzed. The precipitate was assayed for protein content 15. For the determination of amines and metabolites, 20/~1 aliquots of samples filtered through a membrane of 0.2 ktm pore size (Schleicher and Shuel) were injected directly into the HPLC-EC system. The system consisted of a Beckman model 100A pump, LC-4B amperometric detector with a glassy carbon electrode (BAS), 3390A recorder integrator (Hewlett-Packard) and a ~Bondapak C18 (10 ktm, 3.9 x 150 mm) reverse phase analytical column (Waters), fitted with a precolumn (30 x 3.9 mm). The potential was set at +0.75 V vs an Ag/AgC reference electrode, and the sensitivity at 10-20 nA/V full scale. The eluent consisted of 0.1 M KH2PO 4 buffer containing 2 mM sodium octanesulfonic acid (OSA), 0.15 mM Na2EDTA, 1% methanol, pH 3.4 for D O P A separation (retention time 2.4 min; flow rate 1.7 ml/min). The concentration of D O P A was established by comparing the peak areas with the known standards and is expressed as Bg/g protein. The detection limit under these conditions was approximately 80 pgm/20 ktl tissue sample.
Lever pressing for food reward Eighteen male hooded rats (450-500 g) of the L o n g - E v a n s strain were food deprived to 85-90% of their body weights. Six rats were shaped to lever press for food pellets (Noyes, 45 mg) on a continuous reinforcement schedule (CRF) for 30 min each day. After 5 days on the CRF schedule, the density of reinforcement was gradually decreased until the animals worked on a schedule in which every eighth lever press was reinforced (FR-8). Lever pressing was
maintained on this schedule for 10-12 days. At the same time as the above animals were tested each day, animals from a second group (n = 6) were placed in adjoining chambers in which the levers had been removed from the testing boxes. An animal in this group (yoked controls) automatically received a food pellet each time that its counterpart in the lever pressing group was rewarded with food. Animals in the third group (n = 6), were placed in an adjacent testing box without levers at the same time as the lever pressing group. However, animals in this group never received food pellets in the chamber. Thus, there were 6 squads consisting of 3 animals each: a lever presser, a yoked control, and a no-food control subject. The members of each squad remained constant throughout the experiment. One hour after each daily session each rat received an amount of laboratory chow in its home cage sufficient to maintain its weight at 85% of pre-training body weight. On the test day, all subjects received an intraperitoneal injection of NSD-1015 (50 mg/kg) in a volume of 2 ml/kg, 5 min prior to behavioral testing. Animals from each squad were killed by cervical fracture immediately after the 30 min behavioral session. The brains were rapidly removed, dissected and analyzed for D O P A by HPLC-EC as described above. RESULTS
Intracranial self-stimulation Subjects in both the experimental and control groups responded at comparable rates after the initial training period. On the final day of training, the response rate for the experimental group was .~ -2926 + 182 as compared to ~ = 2820 + 202/30 min for the control subjects. During the 30 min test session immediately preceding sacrifice, animals in the selfstimulation group responded at an average rate of 2998 + 239/30 min. The biochemical analyses of D O P A concentrations revealed significant increases in the striatum, nucleus accumbens and olfactory turbercle in the group that had engaged in self-stimulation behavior for 30 min prior to sacrifice (see Fig. 1). In the striaturn and nucleus accumbens, stimulation-induced increases in the concentrations of D O P A were observed only in the hemisphere ipsilateral to the V T A self-stimulation electrode. Relative to the electrode
112 STRIATUM
ACCUMBENS
OLFACTORY TUBERCLE 1
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ICSS
Fig. 1. DOPA concentrations in the striatum, nucleus accumbens and olfactory tubercle of an implanted unstimulated control group (CTRL) or a group that had engaged in intracranial self-stimulation (ICSS) of the ventral tegmental area for 30 rain prior to sacrifice. Open bars indicate brain regions contralateral to the electrode and shaded bars indicate regions ipsilateral to the electrode placement. Data are expressed as means _+ S.E.M. (~r) Significant difference from ipsilateral hemisphere in control group, P < 0.05. (~r "1") Significant difference from contralateral hemisphere in control group, P < 0.05. containing h e m i s p h e r e in control subjects, D O P A concentrations were increased by 61% in the striaturn and 58% in the nucleus accumbens. No significant differences between self-stimulation and control groups were seen in the striatum and nucleus accumbens contralateral to the electrode placements. A different p a t t e r n of results was o b s e r v e d in the olfactory tubercle. A g a i n , c o m p a r e d to unstimulated controls, D O P A was increased by 63% in the hemisphere ipsilateral to the stimulation electrode. In addition, however, the concentration of D O P A in the contralateral h e m i s p h e r e of the self-stimulation group was increased significantly by 39% c o m p a r e d to the value in the corresponding h e m i s p h e r e of the non-stimulated control group. Nevertheless in the experimental group, the concentration of D O P A was significantly higher in the h e m i s p h e r e ipsilateral to the stimulation electrode when c o m p a r e d to the contralateral hemisphere ( P < 0.05). The statistical significance of these patterns of results was c o n f i r m e d by analyses of variance, as a group (self-stimulation vs control) by h e m i s p h e r e (ipsilateral vs contralateral) interaction. The F values for the interactions were; striatum, Fl,12 = 19.08, P < 0.001; nucleus accumbens, F1,12= 9.65, P < 0.009; olfactory tubercle, F1,12= 7.33, P < 0.01.
A n i m a l s in the lever-pressing group r e s p o n d e d for food at a m e a n rate of 1176 + 170 presses/30 rain on the test day and received an average of 147 _+ 21 pellets. The subjects in the y o k e d group f o o d c o n s u m e d the same n u m b e r of food pellets but did not e n g a g e in o p e r a n t behavior. F o r graphic p r e s e n t a t i o n , the c o n c e n t r a t i o n s of D O P A in each h e m i s p h e r e were a v e r a g e d to give a single value for each brain region from each subject. The m e a n values of D O P A concentration in the striaturn, nucleus accumbens and olfactory t u b e r c l e for each group are shown in Fig. 3. N o significant g r o u p differences in the concentrations of D O P A were ob-
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Fig. 2. Coronal sections of the rat mesencephalon, redrawn from K6nig and KlippelTM, indicating the location of the tips of bipolar electrodes from unstimulated control subjects (0) and subjects in the ICSS group (11).
113 STRIATUM
ACCUMBENS
OLFACTORY TUBERCLE
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Fig. 3. DOPA concentrations in the striatum, nucleus accumbens and olfactory tubercle of an unfed control group (CTRL), a yoked control group that received free pellets at the same time as subjects engaged in lever pressing for food pellets (Lever) on an FR-8 schedule of reinforcement. Data from both hemispheres were combined to give a single value for each brain region for individual subjects. Data are expressed as means + S.E.M. served in any of the 3 brain regions sampled, nor were any significant differences observed as a function of hemisphere. There was a slight trend towards elevated concentrations of D O P A in the striatum of the lever press group, but this was not statistically significant. DISCUSSION Significant increases in D O P A concentrations are observed in the terminal fields of the mesotelencephalic D A system ipsilateral to electrodes in the A10 region of the V T A following self-stimulation at optimal response rates. Accumulation of D O P A following decarboxylase inhibition appears to be a valid index of tyrosine hydroxylase activity in vivo 4A9'29. Therefore, it may be concluded that electrical stimulation of D A neurons at the parameters employed (60 Hz, sine wave) increased the activity of TH, the rate limiting enzyme for the synthesis of D A . While it is recognized that the brain regions sampled here contain both D A and norepinephrine (NE), the levels of D A exceed those of N E by a factor of 50:1 in the striatum and 5:1 in the nucleus accumbens 2s. These differences coupled with the location of the stimulating electrodes which were in close proximity to D A neurons in the V T A suggest that a major portion of the accumulated D O P A occurred in terminals of D A neurons. As such, these data were consistent with a role for D A in brain-stimulation reward.
These results in unanesthetized freely moving animals complement an earlier report of increased D O P A accumulation, following aromatic amino acid decarboxylase inhibition and increased specific activity of D A in the neostriatum after administration of [3H]tyrosine, produced by electrical stimulation of the substantia nigra pars compacta of anesthetized rats 19. In that study 19, electrical stimulation did not affect endogenous tyrosine or D A levels or increase tyrosine specific activity in the neostriatum of the stimulated hemisphere. Therefore it was concluded that the observed increases in D A specific activity could not be attributed to changes in endogenous D A levels, increased transport of tyrosine into the stimulated striatal regions or preferential uptake of labeled tyrosine. By inference, it may be assumed that accumulation of D O P A was not influenced by any of these factors. Other studies have confirmed that in the absence of DOPA-decarboxylation, increased T H activity is reflected by a significant increase in the accumulation of D O P A 4'29. Increased accumulation of D O P A in the nucleus accumbens, striatum and olfactory tubercle during V T A self-stimulation complement findings from this and other laboratories that have used changes in the ratio of both D O P A C and homovanillic acid to D A as indices of dopaminergic activity 8. This pattern of results is consistent with the fact that dopaminergic neurons in the V T A project to both the striatum and nucleus accumbens 5'7. Using bilateral stimulation of
114 the VTA, Simon et al. 25 reported increased D O P A C / D A ratios in both the nucleus accumbens and medial prefrontal cortex, a structure not sampled in the present study. Mitchell et al. 18 noted increased D A activity in the olfactory tubercle following selfstimulation of the VTA. However, these authors failed to observe significant changes in either the nucleus accumbens or striatum, which they attributed to their electrode placements being close to the midline of the brain. Although a yoked stimulation group receiving brain-stimulation in the abscence of lever pressing was not included in the present study, this condition was shown to increase D A turnover in the nucleus accumbens and striatum in a related study s. It remains to be determined whether yoked stimulation is rewarding for subjects with a previous history of self-stimulation behavior. Nevertheless, these biochemical experiments suggest that brain-stimulation reward at sites in the VTA is accompanied by the release of D A in the terminal fields of the mesotelencephalic D A system. The fact that self-stimulation of the VTA is greatly attenuated by lesions of these same pathways suggests that dopaminergic activity is an important component of brain-stimulation reward in this region of the brain 8'22. Although the present findings emphasize dopaminergic substrates of reward there is clear evidence for the involvement of non-dopaminergic pathways in brain-stimulation reward. For example, recent studies with [14C]2-deoxyglucose (2-DG) autoradiography failed to demonstrate metabolic activation of telencephalic D A terminal fields in response to rewarding stimulation of the posterior medial forebrain bundle 9. The 2-DG technique also may be used to confirm activation of D A systems in conjunction with rewarding stimulation of D A cell bodies in the VTA 23 and substantia nigra 9. Pulse duration appears to be a critical variable for selective activation of D A neurons, as 0.2-0.5 ms square wave pulses have been shown to be ineffective for electrically induced release of D A as measured by in vivo voltammetry 17. In contrast, longer square wave pulse durations of 1.0-5.0 ms or 60 Hz sine wave pulses are very effective in causing the release of D A in the striatum 17. Failure to pay careful attention to stimulation parameters may lead to confusion regarding the contribution of dopaminergic and non-dopaminergic pathways to the phenomenon of brain-stimulation reward.
The finding that increased TH activity was restricted to regions ipsilateral to the stimulating electrode is important, as it would appear to rule out lever pressing and other forms of motor behavior as causal factors. A further control for motor performance effects is provided by the absence of increased accumulation of D O P A in animals lever pressing at high rates for food reward. However, it should be noted that mean maximal response rates for food were lower than those obtained in the self-stimulation tests. The failure to obtain evidence of increased D A activity in rats lever pressing on an FR-8 schedule is somewhat inconsistent with previous reports. For example, operant behavior maintained by water reinforcement has been reported to be associated with increased synthesis of D A in the striatum but not in the nucleus accumbens as measured by either the conversion of [3H]tyrosine to [3H]DA 13 or the accumulation of D O P A following inhibition of aromatic amino acid decarboxylase 24, the procedure used in the present study. This discrepancy may be related to the use of water vs food reward, but is unlikely to be due to either food or water deprivation per se, as neither of these manipulations is associated with changes in D A metabolism 12'16. An alternate explanation of these differences may lie in the methods of dissection employed. Our procedure sampled tissue from the anterior region of the striatum (anterior to the decussation of the anterior commissure) and perhaps samples from more posterior areas are required to observe the reported increases in D A activity associated with operant lever pressing behavior. Despite these caveats, it is important that this issue be resolved as the combined data from the self-stimulation and food-reinforcement experiments reported here suggest that D A synthesis is not increased during the performance of a well-established lever pressing response. The present results also speak to the issue of a general role for D A in reward processes. The failure to observe increased activity in D A systems produced by food reward using both the D O P A accumulation measure used here, and the D A metabolite/DA ratio employed in a separate study s, does not support a role for D A in natural reward processes 3°. Nor have we observed increased activity in D A systems following ingestion of a palatable saccharin solution 1, although there is evidence for such increases 1 h after
115 consumption of a large meal of food pellets or liquid diet in the home cage 1'12. A n alternate hypothesis is
primary reinforcement mechanism. Therefore, dopaminergic substrates of brain-stimulation reward
that increased activity in dopaminergic n e u r o n s is associated with processes underlying the anticipation of reward and the initiation of preparatory behaviors that take place prior to consumption of a primary re-
may be related to the activation of systems associated with incentive motivation 3, but not those c o n c e r n e d with analyzing the hedonic properties of sensory signals.
ward. In accordance with this hypothesis, the D A receptor antagonist pimozide has been shown to attenuate conditioned preparatory responses that precede a meal, while failing to block the subsequent ingestion of a palatable liquid diet 2. The a t t e n u a t i o n by ha-
ACKNOWLEDGEMENTS
loperidol of conditioned place preference using food reward 26 may also reflect a disruption of conditioned
and D. Fu is gratefully acknowledged. Supported by Program G r a n t No. 23 from the Medical Research
anticipatory responses rather than i m p a i r m e n t of a
Council of Canada.
REFERENCES
11 Hall, R.D., Bloom, F.E. and Olds, J., Neuronal and neurochemical substrates of reinforcement, Neurosci. Res. Progr. Bull., 15 (1977) 133-314. 12 Heffner, T.G., Hartman, J.A. and Seiden, L.S., Feeding increases dopamine metabolism in the rat brain, Science, 108 (1980) 1168-1170. 13 Heffner, T.G. and Seiden, L.S., Synthesis of 3H-catecholamines from 3H-tyrosine in brain regions of rats during performance of operant behavior, Brain Research, 183 (1980) 403-419. 14 K6nig, J.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas, Williams and Wilkins, Baltimore, 1963. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Luttinger, D.A. and Seiden, L.S., Increased NE metabolism after water deprivation in the rat brain, Brain Research, 208 (1981) 147-165. 17 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the median forebrain bundle, Eur. J. Pharmacol., 109 (1985) 341-348. 18 Mitchell, M.J., Nicoleau, N.M., Arbuthnott, G.W. and Yates, C.M., Increases in dopamine metabolism are not a central feature of intracranial self-stimulation, Life Sci., 30 (1982) 1081-1085. 19 Murrin, L.C. and Roth, R.H., Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis, Mol. Pharmacol., 12 (1976) 463-475. 20 Olds, J., Drives and Reinforcements: Behavioral Studies of Hypothalamic Function, Raven Press, New York, 1977. 21 Phillips, A.G., Brain reward circuitry: a case for separate systems, Brain Res. Bull., 12 (1984) 195-201. 22 Phillips, A.G. and Fibiger, H.C., The role of dopamine in maintaining intracranial self-stimulation in the ventral tegmentum, nucleus accumbens and medial prefrontal cortex, Can. J. Psychol., 32 (1978) 58-66. 23 Porrino, L.J., Esposito, R.U., Seeger, T.F., Crane, A.M., Pert, A. and Sokoloff, L., Metabolic mapping of the brain during rewarding self-stimulation, Science, 224 (1984) 306-309.
1 Blackburn, J.R., Phillips, A.G., Jakubovic, A. and Fibiger, H.C., Increased dopamine metabolism in the nucleus accumbens and striatum following consumption of a nutritive meal but not a palatable non-nutritive saccharin solution, Pharmacol. Biochem. Behav., in press. 2 Blackburn, J.R., Phillips, A.G. and Fibiger, H.C., Dopamine and preparatory behavior. 1: effects of pimozide, Behay. Neurosci., in press. 3 Bolles, R.C., Reinforcement, expectancy and learning, Psych. Rev., 79 (1972) 394-409. 4 Carlsson, A., Kehr, W., Lindquist, M., Magnusson, T. and Atack, C.V., Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev., 24 (1972) 371-384. 5 Carter, D.A. and Fibiger, H.C., Ascending projections of presumed dopamine-containing neurons in the ventral tegmentum of the rat as demonstrated by horseradish peroxidase, Neuroscience, 2 (1977) 569-576. 6 Crow, T.J., Specific monoamine systems as reward pathways: evidence for the hypothesis that activation of the ventral mesencephalic dopaminergic neurons and noradrenergic neurons of the locus coeruleus complex will support selfstimulation responding. In A. Wauquier and E.T. Rolls (Eds.), Brain-Stimulation Reward, North-Holland Publishing Co., Amsterdam, 1976, pp. 211-237. 7 Fallon, J.H. and Moore, R.Y., Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum, J. Comp. Neurol., 180 (1978) 545-580. 8 FiNger, H.C., Jakubovic, A. and Phillips, A.G., The role of dopamine in intracranial self-stimulation of the ventral tegmental area, J. Neurosci., submitted. 9 Gallistel, C.R., Gomita, Y., Yadin, E. and Campbell, K.A., Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward blocking doses of pimozide, J. Neurosci., 5 (1985) 1246-1261. 10 Garrigues, A.M. and Cazala, P., Central catecholamine metabolism and hypothalamic self-stimulation behavior in two inbred strains of mice, Brain Research, 265 (1983) 265-271.
The excellent technical assistance of F . G . L e P i a n e
116 24 Seiden, L.S. and Heffner, T.G., Alteration of brain catecholamine metabolism by environmental and behavioral events: an explanation of drug-behavior interactions. In E. Usdin (Ed.), Catecholamines: Neuropharmacology and Central Nervous System -- Theoretical Aspects, A.R. Liss Inc., New York, 1984, pp. 275-284. 25 Simon, H., Stinus, L., Tassin, J.P., Lavielle, S., Blanc, G., Thierry, A.M., Glowinski, J. and Le Moal, M., Is the dopaminergic mesocorticolimbic system necessary for intracranial self-stimulation? Biochemical and behavioral studies from A10 cell bodies and terminals, Behav. Neural. Biol., 27 (1979) 125-145. 26 Spyraki, C., Fibiger, H.C. and Phillips, A.G., Attenuation by haloperidol of place preference conditioning using food reinforcement, Psychopharmacology, 77 (1982) 379-382. 27 Stein, L., Chemistry of reward and punishment. In D.H. Efron (Ed.), Psychopharmacology, A Review of Progress,
1957-1967, U.S. Government Printing Office, Washington, D.C., 1968, pp. 105-123. 28 Westerink, B.H.C. and Mulder, T.B.A., Determinations of picomole amounts of dopamine, noradrenaline, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylalacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid in nervous tissue after one-step purification on sephadex G-10, using high performance liquid chromatography with a novel type of electrochemical detection, J. Neurochem., 36 (1981) 1449-1462. 29 Westerink, B.H.C. and Wirix, E., On the significance of tyrosine for the synthesis and catabolism of dopamine in rat brain: evaluation by HPLC with electrochemical detection, J. Neurochem., 40 (1983) 758-764. 30 Wise, R.A., Neuroleptics and operant behavior: the anhedonia hypothesis, Behav. Brain Sci., 5 (1982) 39-53.