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Effects of dopamine-receptor blockade on self-stimulation in the monkey
Phartnacology Biochemistry & Behavior, Vol. 4, pp. 2 1 1 - 2 1 6 . Copyright © 19"/6 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
BRIEF COMMUNICATION Effects of Dopamine-Receptor Blockade on Self-Stimulation in the Monkey I F. M O R A 2 , E. T. R O L L S 3 , M. J. B U R T O N A N D S. G, SHAW
University of Oxford, Department o f Experimental Psychology, Oxford, England ( R e c e i v e d 9 J u n e 1975) MORA, F., E. T. ROLLS, M. J. BURTON AND S. G. SHAW. Effects ofdopamine-receptor blockade on self-stimulation in the monkey. PHARMAC. BIOCHEM. BEHAV. 4(2) 2 1 1 - 2 1 6 , 1976. - In a do~-response experiment it was shown that intraperitoneal injections of 0.062 mg/kg, and 0.1 mg/kg of the dopamine-receptor blocking agent and neuroleptic spiroperidol severely attenuate self-stimulation in the orbitofrontal cortex, hypothalamus, and in the region of the locus coeruleus, in the rhesus monkey and in the squirrel monkey. In the rhesus monkey intracranial injections of 6 ug of spiroperidol bilaterally into the nucleus accumbens or the hypothalamus attenuated self-stimulation of the amygdala, and injections into the orbitofrontal cortex attenuated self-stimulation of the amygdala and lateral hypothalamus. Self-stimulation at other sites tested (including the region of the locus coeruleus) was much less affected by the injections, and injections into the region of the locus coeruleus were ineffective. These results together with other control experiments suggest that spiroperidol can attenuate self-stimulation in the monkey independently of any motor impairment or sedation produced, and that dopamine receptors in particular brain regions are involved in self-stimulation of particular brain sites. Self-stimulation
Dopamine
Spiroperidol
Reward
T H E R E is evidence t h a t d o p a m i n e r e c e p t o r s are involved in s e l f - s t i m u l a t i o n in t h e rat. S e l f - s t i m u l a t i o n can b e o b t a i n e d in the region of d o p a m i n e - c o n t a i n i n g cell b o d i e s [2,41 and is a t t e n u a t e d by the a d m i n i s t r a t i o n of agents w h i c h b l o c k d o p a m i n e r e c e p t o r s such as h a l o p e r i d o l , p i m o z i d e [ 16] and spiroperidol [ 6 , 1 2 ] . R e c e n t l y it has b e e n s h o w n t h a t i n t r a c r a n i a l a n d i n t r a p e r i t o n e a l injections of b l o c k i n g agents for d o p a m i n e r e c e p t o r s a t t e n u a t e s e l f - s t i m u l a t i o n of a n u m b e r o f sites in a d d i t i o n to the lateral h y p o t h a l a m u s 19,111. In this s t u d y we describe an investigation i n t o w h e t h e r d o p a m i n e r e c e p t o r s are involved in s e l f - s t i m u l a t i o n in t h e m o n k e y . First, a d o s e - r e s p o n s e curve o f the effect of i n t r a p e r i t o n e a l injections o f t h e d o p a m i n e - r e c e p t o r blocking a g e n t s p i r o p e r i d o l [ 1 ] o n s e l f - s t i m u l a t i o n o f d i f f e r e n t b r a i n sites is described. T h e n t h e effects o f i n t r a c r a n i a l i n j e c t i o n s o f s p i r o p e r i d o l i n t o d i f f e r e n t b r a i n sites are described. T h e aims o f this s e c o n d e x p e r i m e n t were to d e t e r m i n e w h e t h e r t h e r e are p a r t i c u l a r areas of the b r a i n w h i c h are p a r t i c u l a r l y sensitive to the effects o f spiroperidol o n s e l f - s t i m u l a t i o n , and to d e t e r m i n e w h e t h e r all self-stimulation sites are equally affected b y d o p a m i n e r e c e p t o r b l o c k a d e . T h e plan o f the e x p e r i m e n t was therefore to inject a given dose o f s p i r o p e r i d o l into d i f f e r e n t
Monkey
b r a i n areas, and to measure t h e effects on s e l f - s t i m u l a t i o n at d i f f e r e n t sites. This e x p e r i m e n t a l design also indicates w h e t h e r a side effect such as m o t o r i m p a i r m e n t or s e d a t i o n a c c o u n t s for a n y a t t e n u a t i o n of self-stimulation observed (see below). T h e sites c h o s e n for self-stimulation and for injections of s p i r o p e r i d o l were the o r b i t o f r o n t a l cortex, amygdala, nucleus a c c u m b e n s , lateral h y p o t h a l a m u s and region o f t h e locus coeruleus. T h o s e areas were c h o s e n because t h e r e is e v i d e n c e t h a t t h e y are involved in s e l f - s t i m u l a t i o n in the m o n k e y , in t h a t s e l f - s t i m u l a t i o n occurs in t h o s e areas and in t h a t single u n i t s in each of these areas are activated (usually t r a n s - s y n a p t i c a l l y ) in self-stimulation o f each of the o t h e r areas {3, 9, 11 I. METHOD
Animals T h r e e rhesus m o n k e y s (Macaca mulatta) and t w o squirrel m o n k e y s (Saimiri sciureus) weighing 2 . 8 - 3 . 2 5 kg and 0 . 5 0 - 0 . 7 0 kg were i m p l a n t e d u n d e r N e m b u t a l anesthesia (0.6 m g / k g ) with bilateral stainless-steel c a n n u l a e (0.6 m m o.d.). T h e c a n n u l a e could be used for local i n t r a c r a n i a l i n j e c t i o n b y inserting 0.3 m m o.d. c a n n u l a e c o n n e c t e d to flexible p o l y t h e n e t u b e s i n t o t h e m , or for brain s t i m u l a t i o n
1This work was supported by The Medic',d Research Council. We thank Dr. A. M. Sanguinetti and Mr. I. Ilughes for assistance. ~ Supported by The European Training Program in Brain and Behaviour Research. ~Send reprint requests to Dr. E. T. Rolls, University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, Great Britain. 211
212 by passing stainless-steel wires insulated e x c e p t for 0.2 mm at the tip through the cannulae. Using the atlas o f Snider and Lee [ 1 4 ] , the cannulae were aimed in the rhesus monkeys at the orbitofrontal cbrtex, nucleus accumbens, lateral hypothalamus, amygdala, and locus coeruleus. In the squirrel monkeys comparable sites were used [5]. One week after surgery the m o n k e y s were tested for selfstimulation. The monkeys sat in a primate chair. A 0.3 sec train of 0.5 msec capacitatively-coupled negative pulses at a frequency of 100 Hz was applied to the electrode under test when the m o n k e y s contacted a bar in front of them. When self-stimulation was found at a particular site the threshold for self-stimulation was determined by repeatedly decreasing and increasing the current, and the stability of the self-stimulation threshold and rate were Confirmed over several days. The current was set to be just above the threshold current (i.e. the lowest current which would maintain continuous self-stimulation). Under these conditions the baseline self-stimulation rates for each structure in every m o n k e y were determined.
Pro cedure A dose-response curve of the effect of the dopaminereceptor blocking agent spiroperidol [ 1 ] on self-stimulation was made as follows. The spiroperidol (generously supplied by Janssen Pharmaceutica, Beerse, Belgium) was injected intraperitoneally in doses of 0.016, 0.062, and 0.1 mg/kg. Control injections were of the solvent only, 0.01 M tartaric acid. The monkeys were tested every 3 days. On a test day first the baseline rate of self-stimulation at every site was tested. T h e n the IP injections were given, and one hour later the self-stimulation rate was measured for each electrode for 10 one min periods, with 5 min periods allowed for changing the stimulation site and stabilization of the rate. The sites were tested in the following order: orbitofrontal cortex, amygdala, lateral h y p o t h a l a m u s and locus coeruleus. The self-stimulation rate over a period of I0 min for each electrode was calculated as a percentage of the corresponding mean baseline rate for that electrode to facilitate comparison between the electrodes. The plan of the experiments with intracranial injections was to compare the effects of injections of spiroperidol into different brain areas on self-stimulation of many different brain sites. In this particular e x p e r i m e n t we used two rhesus monkeys and one of the squirrel monkeys. For the rhesus m o n k e y s 2 ul of 3 ug/ul spiroperidol were injected bilaterally into a given site. Control injections of the solvent (0.01 M tartaric acid) were also given. The procedure was as for the 1P injection tests. The self-stimulation rate over a period of 10 rain for each electrode was calculated as a percentage of the corresponding mean control rate. In the squirrel m o n k e y the procedure was as for the rhesus m o n k e y s except that the spiroperidol (1 ug/gl in 1 ul volumes) was injected bilaterally until an effect was obtained. Haloperidol in a concentration of 2.5 ug/ul was also tested. Additional experiments with different doses were also performed (see Table 1).
Histology At the conclusion of the experiments every site was verified histologically. The animals were anesthetized with Nembutal and a lesion (0.1 mA, 60 sec) was made to assist
MORA, ROLLS, B U R T O N AND SHAW identification of the region in which stimulation and injections were given. The m o n k e y s were perfused with isotonic saline followed by 10 percent formal-saline. The brains were frozen and sections were cut at 50 u and stained with thionin. As shown in the sections from one of the rhesus monkeys illustrated in Fig. 3, the cannulae aimed at orbitofrontal cortex, lateral hypothalamus and amygdala were accurately placed, and the cannulae aimed at the nucleus accumbens ended in this particular m o n k e y just below the nucleus accumbens close to the dura, so that the injections probably reached the nucleus accumbens, but self-stimulation did not occur probably because the stimulation electrode in protruding from the cannula touched the dura. The cannulae aimed at the locus coeruleus appeared to end within 2 mm of the locus coeruleus. RESULTS
Intraperitoneal Injection Dose-response curves for the effect of spiroperidol on self-stimulation in the rhesus m o n k e y s and the squirrel m o n k e y s are shown in Fig. 1. In this figure we represent the mean and standard error of the rate per rain measured over a period of 10 min for each electrode 1 hr after injection in both groups of rhesus and squirrel monkeys. The results for each electrode were expressed as a percentage of the mean control (i.e. placebo) rate for that electrode. The mean rates for each electrode under the control condition, and on each testing day before injection, remained constant throughout the experiments. The experiments were repeated twice in rhesus m o n k e y 1 at each drug dose and the same results were obtained in the two separate experimental sessions (see Fig. 2). It is shown in Fig. 1 that in b o t h the rhesus monkeys and the squirrel m o n k e y s self-stimulation at all the sites tested was severely attenuated by a dose of spiroperidol of 0.062 or 0. I mg/kg. At the lower dose of 0.016 mg/kg self-stimulation at most sites was unaffected, but selfstimulation o f the orbitofrontal cortex was a t t e n u a t e d in two o f the rhesus monkeys and one o t the squirrel monkeys. The typical time course of the attenuation of self-stimulation by spiroperidol, and the repeatability of the results are illustrated in the two separate runs of the experiment in rhesus m o n k e y 1 shown in Fig. 2. The results shown in Figs. 1 and 2 suggest that with a dose of 0.016 mg/kg of spiroperidol the effect on self-stimulation depends on which sell-stimulation site is tested, and suggest that orbitofrontal cortex self-stimulation is particularly sensitive to d o p a m i n e - r e c e p t o r blockade.
Intracranial Injection The self-stimulation rate (expressed as a percentage of the preinjection control rate) for different sites one hr after injection of solutions of spiroperidol bilaterally into different brain areas of the monkeys is shown in Table I. The error figures represent an estimate of the standard errors calculated from l0 one-minute observations of self-stimulation rate; and the significance of the difference of these observations from the preinjection control rates using a t-test and confirmed with a U test is shown by *p<0.02 or +p<0.001. The largest effect found with a 6 #g dose was attenuation of the rate of self-stimulation of the amygdala by 70 percent by injection into the nucleus accumbens (see Table
SPIROPERIDOL AND SELF-STIMULATION
Rhesus o J~ (~
213
Squirrel
monkey
monkey
120 eHypothalamus • L. coeruleus • Orbito=frontal
• L. coeruleus • Hypothalamus • Orbito-frontal
~ 100 .~
t-
0
80
60 O
E
40
I
20
0
I
0.016
I
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i
0"062 0-1
0.062 0.1 0.016 Spiroperidol (rag/kg)
FIG. 1. Dose-response curves for the effect of IP injections of spiroperidol on self-stimulation rate at different sites in 3
rhesus monkeys and 2 squirrel monkeys. The self-stimulation rate for a particular electrode is shown as a percentage of the self-stimulation rate after a placebo injection, and for the rhesus monkey is the mean of two separate experimental determinations of every point. The vertical lines indicate an estimate of the standard error of the ten one-minute observations of self-stimulation rate. The self-stimulation electrodes were in the hypothalamus, the orbitofrontal cortex, and near the locus coeruleus. 1). Similar results, a n d dose-related effects, were seen w i t h 9 gg o f s p i r o p e r i d o l in w h i c h t h e a t t e n u a t i o n of selfs t i m u l a t i o n o f t h e a m y g d a l a was 99 p e r c e n t . This e f f e c t was c o n f i r m e d w i t h t h e squirrel m o n k e y in w h i c h 5 ~g o f h a l o p e r i d o l i n j e c t e d i n t o the same s t r u c t u r e ( n u c l e u s a c c u m b e n s ) totally a t t e n u a t e d s e l f - s t i m u l a t i o n o f t h e a m y g dala. O b s e r v a t i o n s in o t h e r s t r u c t u r e s were t h a t i n j e c t i o n s into t h e o r b i t o f r o n t a l c o r t e x (O. F. C o r t e x ) partially a t t e n u a t e d s e l f - s t i m u l a t i o n of t h e h y p o t h a l a m u s ( b y 46 p e r c e n t ) a n d o f t h e a m y g d a l a ( b y 32 p e r c e n t ) , and injections i n t o t h e h y p o t h a l a m u s a t t e n u a t e d self-stimulation of the a m y g d a l a ( b y 35 p e r c e n t ) . T h e r e was never a n y large effect of i n j e c t i o n s o f s p i r o p e r i d o l i n t o t h e region o f t h e locus c o e r u l e u s or o f i n j e c t i o n s i n t o o t h e r areas o n s e l f - s t i m u l a t i o n o f t h e region o f t h e locus coeruleus. T h e s e results were c o n f i r m e d in a d d i t i o n a l e x p e r i m e n t s (see T a b l e 1). T h e l a t e n c y for a m e a s u r a b l e effect was 1 0 - 2 0 min, a n d the effect r e a c h e d its m a x i m u m at 2 0 - 3 0 m i n and r e m a i n e d at this level for m o r e t h a n 4 hr.
DISCUSSION
[ntraperitoneal Injections T h e e x p e r i m e n t w i t h i n t r a p e r i t o n e a l injections s h o w s t h a t in t h e m o n k e y s p i r o p e r i d o l a t t e n u a t e s intracranial s e l f - s t i m u l a t i o n at t h e d i f f e r e n t sites tested at doses of 0 . 0 6 2 and 0.1 mg/kg. A t t h e lower dose of 0 . 0 1 6 m g / k g some e v i d e n c e was o b t a i n e d t h a t o r b i t o f r o n t a l c o r t e x s e l f - s t i m u l a t i o n is particularly sensitive to d o p a m i n e r e c e p t o r b l o c k a d e . A l t h o u g h the a n i m a l s could still work for glucose at a dose of 0 . 0 6 2 m g / k g o f spiroperidol, a general effect o f t h e s p i r o p e r i d o l o n b e h a v i o r ( s u c h as a m o t o r i m p a i r m e n t or s e d a t i o n ) was suspected, since at t h e higher dose o f 0.1 m g / k g t h e m o n k e y s looked drowsy. To determine whether spiroperidol attenuated self-stimulation b y this t y p e o f general effect on b e h a v i o r or b y a specific effect o n s e l f - s t i m u l a t i o n , in E x p e r i m e n t 2 s p i r o p e r i d o l was i n j e c t e d directly i n t o d i f f e r e n t b r a i n sites. A n a t t e n u a t i o n o f s e l f - s t i m u l a t i o n at o n l y some s e l f - s t i m u l a t i o n sites w o u l d
214
MORA, ROLLS, B U R T O N AND SHAW eo H y p o t h Q l a m u s I O L. coeruleus &&Orbito-frontal
show t h a t t h e effect on b e h a v i o r was specific, and would provide evidence on the neural system w h i c h was involved in self-stimulation of these particular self-stimulation sites.
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5 65 95 125 155 Time ( i n j e c t i o n - t e s t ) (rain)
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FIG. 2. The effect of 0.016 mg/kg of spiroperidol IP on self-stimulation with electrodes in the hypothalamus, orbitofrontal cortex and in the region of locus cocruleus, in rhesus monkey 1. The solid and open symbols represenl the two separate experimental determinations of every point. The self-stimulation rate for a particular electrode is shown as a percentage of the self-stimulation rate after a placebo injection. C represents the control rate.
Or bitofrontal Cortex
Arnygdala
T h e finding w i t h intracranial injections of spiroperidol t h a t self-stimulation in the region o f the locus c o e r u l e u s was almost u n a f f e c t e d even w h e n self-stimulation at some o t h e r sites was severely a t t e n u a t e d suggests that the a t t e n u a t i o n of self-stimulation f o u n d at the affected site was not due to a general effect o n b e h a v i o r such as m o t o r i m p a i r m e n t or sedation. In a d d i t i o n , a f t e r the tests for self-stimulation the m o n k e y s were able to p e r f o r m the m o t o r response task to o b t a i n 20 p e r c e n t glucose s o l u t i o n (see T a b l e 1). T h u s it appears t h a t the a t t e n u a t i o n o f self-stimulation f o u n d at s o m e sites can n o t be e x p l a i n e d by a single effect such as m o t o r i m p a i r m e n t [81. T h e finding that injections of spiroperidol i n t o the o r b i t o f r o n t a l cortex a t t e n u a t e d self-stimulation of the a m y g d a l a and lateral h y p o t h a l a m u s suggests t h a t the o r b i t o f r o n t a l cortex is involved in self-stimulation of some limbic structures. This suggestion is c o n s i s t e n t with first, the electrophysiological findings that single u n i t s in the o r b i t o f r o n t a l cortex are activated in self-stimulation of the lateral h y p o t h a l a m u s and a m y g d a l a and vice versa [ 3 ] , second, that anesthetization o f the sulcal p r e f r o n t a l cortex in the rat ( w h i c h m a y
Nucleus
Lateral
accumbens
Hypot halamus
FIG. 3. Sites of cannulae used for injection and for stimulation in the rhesus monkey. No self-stimulation occurred at the
nucleus accumbens site in this monkey, because the stimulation site was close to the base of the brain.
*p
SW
t~
N. Accumbens
Hypothalamus
R2
O.P. Cortex
Rl
Amygdala
N. Accumbens
RI
R2
Amygdala
RI
N. Accumbens
L. Coeruleus
Rl and R2
R2
Hypothalamus
Rl and R2
O.F. Cortex
67
Amygdala
Rl and R2
R2
32
N. Accumbens
Rl and R2 4.3
98
84
25
12
-
103 t
90 f 4.3
4.3
87 f 10.0
74 f
-
1
_
0
33
l12?
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-
6
3*
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68 f 10*
-
O.F. Cortex
Rl and R2
INJECTIONS
_ _
82 f 1.3
81 f 3.3
92 _+4.6
-
_
II7
_
0
36
4
18
-
-
-
-
-
-
N. Accumbens
_
100 ? 5.0
92 * 0.6
I60 + 3.0
87 f 4.6
Rate for Glucose as Percent of Control Rate
ON SELF-STIMULATION
_
-
83
50
85
Additional Experiments
4.0
2.6
6.3
82 f 9.2
L. Coeruleus
47
71
64
120 t
82 f
96 c
54 f l3.2*
Hypothalamus
1
OF SPIROPERIDOL
Rate (Percent of Control Rate)
Amygdala
Monkeys
Self-Stimulation
OF INTRACRANIAL
O.F. Cortex
Injection Site Spiroperidol
EFFECTS
TABLE
2rl
(Haloperidol) 2.5 f.dPl
3 rslrl
3 f&d
2 PI 2 PI
4 rglpl
3 /%/Id
3 /%/~I
3 &rl
4 rslrl
Concentration
3 ctl
2 rl
2pl
3 I%
3 Ml
2 ctl
2 nl
2 IJl
2 ccl
2 rl
Volume Injected Bilaterally
E ol
5 ?I c!e
5
216
MORA, ROLLS, B U R T O N A N D SHAW
c o r r e s p o n d to the o r b i t o f r o n t a l cortex o f the m o n k e y ) [7] a t t e n u a t e d self-stimulation of the lateral h y p o t h a l a m u s [ 1 0 ] , third, t h a t there are d o p a m i n e r g i c t e r m i n a l s in the region of the p r e f r o n t a l c o r t e x in the rat [ ! 5 ], and f o u r t h , that single cells in this p r e f r o n t a l region w h i c h are activated during self-stimulation of the lateral h y p o t h a l a m u s of t h e rat alter their firing rate w h e n a d o p a m i n e r g i c agonist { a p o m o r p h i n e ) or a n t a g o n i s t ( s p i r o p e r i d o l ) is a d m i n i s t e r e d systemically in low doses (personal observations). Thus,
d o p a m i n e r g i c c o n n e c t i o n s to the o r b i t o f r o n t a l cortex m a y be involved in self-stimulation of a n u m b e r o f d i f f e r e n t sites. A l t h o u g h it is difficult to i n t e r p r e t at this stage, it is of interest t h a t injections into the nucleus a c c u m b e n s and lateral h y p o t h a l a m u s a t t e n u a t e d self-stimulation o f the amygdala. It is k n o w n that single u n i t s in these t w o areas are a c t i v a t e d during s e l f - s t i m u l a t i o n [ 3 ].
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9. 10. 1 I. 12. 13.
14. 15.
16.
RolLs, E. T. The neural basis of brain-stimulation reward. Progress in Neurobiology 3:71-160, 1974. Rolls, E. T. and S. J. Cooper. Anesthetization and stimulation of the sulcal prefrontal cortex and brain-stimulation reward. Physiol. Behav. 12: 563-571, 1974. Rolls, E. T. 7"he Brain and Reward. London: Pergamon Press, 1975. Rolls, E. T., P. H. Kelly and S. G. Shaw. Noradrenaline, dopamine, and brain-stimulation reward. Pharmac. Biochem. Behav. 2: 735-740, 1974. Rolls, E. T., B. J. Rolls, P. H. Kelly, S. G. Shaw, R. J. Wood and R. Dale. The relative attenuation of self-stimulation, eating and drinking produced by dopamine-receptor blockade. Psychopharmacologia 3 8 : 2 1 9 - 2 3 0 , 1974. Snider, R. S. and J. C. Lee. A Stereotaxicatlas o]'the monkey brain (Macaca mulatta). Chicago: University of Chicago Press, 1961. Thierry, A. M., J. P. Tassin, G. Blanc and J. Glowinski. Topographic and pharmacological study of the dopaminergic mesocortical system. In: Brain-stimulation Reward, edited by A. Wauquier and E. T. Rolls. Amsterdam: North-Holland, 1976. Wauquier, A. and C. J. E. Niemegeers. lntracranial self-stimulation in rats as a function of various stimulus parameters. II. lnlluence of haloperidol, pimozide and pipamperone on medial forebrain stimulation with monopolar electrodes. Psychopharmacologia 27: t 91 --202, 1972.
BRIEF COMMUNICATION Effects of Dopamine-Receptor Blockade on Self-Stimulation in the Monkey I F. M O R A 2 , E. T. R O L L S 3 , M. J. B U R T O N A N D S. G, SHAW
University of Oxford, Department o f Experimental Psychology, Oxford, England ( R e c e i v e d 9 J u n e 1975) MORA, F., E. T. ROLLS, M. J. BURTON AND S. G. SHAW. Effects ofdopamine-receptor blockade on self-stimulation in the monkey. PHARMAC. BIOCHEM. BEHAV. 4(2) 2 1 1 - 2 1 6 , 1976. - In a do~-response experiment it was shown that intraperitoneal injections of 0.062 mg/kg, and 0.1 mg/kg of the dopamine-receptor blocking agent and neuroleptic spiroperidol severely attenuate self-stimulation in the orbitofrontal cortex, hypothalamus, and in the region of the locus coeruleus, in the rhesus monkey and in the squirrel monkey. In the rhesus monkey intracranial injections of 6 ug of spiroperidol bilaterally into the nucleus accumbens or the hypothalamus attenuated self-stimulation of the amygdala, and injections into the orbitofrontal cortex attenuated self-stimulation of the amygdala and lateral hypothalamus. Self-stimulation at other sites tested (including the region of the locus coeruleus) was much less affected by the injections, and injections into the region of the locus coeruleus were ineffective. These results together with other control experiments suggest that spiroperidol can attenuate self-stimulation in the monkey independently of any motor impairment or sedation produced, and that dopamine receptors in particular brain regions are involved in self-stimulation of particular brain sites. Self-stimulation
Dopamine
Spiroperidol
Reward
T H E R E is evidence t h a t d o p a m i n e r e c e p t o r s are involved in s e l f - s t i m u l a t i o n in t h e rat. S e l f - s t i m u l a t i o n can b e o b t a i n e d in the region of d o p a m i n e - c o n t a i n i n g cell b o d i e s [2,41 and is a t t e n u a t e d by the a d m i n i s t r a t i o n of agents w h i c h b l o c k d o p a m i n e r e c e p t o r s such as h a l o p e r i d o l , p i m o z i d e [ 16] and spiroperidol [ 6 , 1 2 ] . R e c e n t l y it has b e e n s h o w n t h a t i n t r a c r a n i a l a n d i n t r a p e r i t o n e a l injections of b l o c k i n g agents for d o p a m i n e r e c e p t o r s a t t e n u a t e s e l f - s t i m u l a t i o n of a n u m b e r o f sites in a d d i t i o n to the lateral h y p o t h a l a m u s 19,111. In this s t u d y we describe an investigation i n t o w h e t h e r d o p a m i n e r e c e p t o r s are involved in s e l f - s t i m u l a t i o n in t h e m o n k e y . First, a d o s e - r e s p o n s e curve o f the effect of i n t r a p e r i t o n e a l injections o f t h e d o p a m i n e - r e c e p t o r blocking a g e n t s p i r o p e r i d o l [ 1 ] o n s e l f - s t i m u l a t i o n o f d i f f e r e n t b r a i n sites is described. T h e n t h e effects o f i n t r a c r a n i a l i n j e c t i o n s o f s p i r o p e r i d o l i n t o d i f f e r e n t b r a i n sites are described. T h e aims o f this s e c o n d e x p e r i m e n t were to d e t e r m i n e w h e t h e r t h e r e are p a r t i c u l a r areas of the b r a i n w h i c h are p a r t i c u l a r l y sensitive to the effects o f spiroperidol o n s e l f - s t i m u l a t i o n , and to d e t e r m i n e w h e t h e r all self-stimulation sites are equally affected b y d o p a m i n e r e c e p t o r b l o c k a d e . T h e plan o f the e x p e r i m e n t was therefore to inject a given dose o f s p i r o p e r i d o l into d i f f e r e n t
Monkey
b r a i n areas, and to measure t h e effects on s e l f - s t i m u l a t i o n at d i f f e r e n t sites. This e x p e r i m e n t a l design also indicates w h e t h e r a side effect such as m o t o r i m p a i r m e n t or s e d a t i o n a c c o u n t s for a n y a t t e n u a t i o n of self-stimulation observed (see below). T h e sites c h o s e n for self-stimulation and for injections of s p i r o p e r i d o l were the o r b i t o f r o n t a l cortex, amygdala, nucleus a c c u m b e n s , lateral h y p o t h a l a m u s and region o f t h e locus coeruleus. T h o s e areas were c h o s e n because t h e r e is e v i d e n c e t h a t t h e y are involved in s e l f - s t i m u l a t i o n in the m o n k e y , in t h a t s e l f - s t i m u l a t i o n occurs in t h o s e areas and in t h a t single u n i t s in each of these areas are activated (usually t r a n s - s y n a p t i c a l l y ) in self-stimulation o f each of the o t h e r areas {3, 9, 11 I. METHOD
Animals T h r e e rhesus m o n k e y s (Macaca mulatta) and t w o squirrel m o n k e y s (Saimiri sciureus) weighing 2 . 8 - 3 . 2 5 kg and 0 . 5 0 - 0 . 7 0 kg were i m p l a n t e d u n d e r N e m b u t a l anesthesia (0.6 m g / k g ) with bilateral stainless-steel c a n n u l a e (0.6 m m o.d.). T h e c a n n u l a e could be used for local i n t r a c r a n i a l i n j e c t i o n b y inserting 0.3 m m o.d. c a n n u l a e c o n n e c t e d to flexible p o l y t h e n e t u b e s i n t o t h e m , or for brain s t i m u l a t i o n
1This work was supported by The Medic',d Research Council. We thank Dr. A. M. Sanguinetti and Mr. I. Ilughes for assistance. ~ Supported by The European Training Program in Brain and Behaviour Research. ~Send reprint requests to Dr. E. T. Rolls, University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, Great Britain. 211
212 by passing stainless-steel wires insulated e x c e p t for 0.2 mm at the tip through the cannulae. Using the atlas o f Snider and Lee [ 1 4 ] , the cannulae were aimed in the rhesus monkeys at the orbitofrontal cbrtex, nucleus accumbens, lateral hypothalamus, amygdala, and locus coeruleus. In the squirrel monkeys comparable sites were used [5]. One week after surgery the m o n k e y s were tested for selfstimulation. The monkeys sat in a primate chair. A 0.3 sec train of 0.5 msec capacitatively-coupled negative pulses at a frequency of 100 Hz was applied to the electrode under test when the m o n k e y s contacted a bar in front of them. When self-stimulation was found at a particular site the threshold for self-stimulation was determined by repeatedly decreasing and increasing the current, and the stability of the self-stimulation threshold and rate were Confirmed over several days. The current was set to be just above the threshold current (i.e. the lowest current which would maintain continuous self-stimulation). Under these conditions the baseline self-stimulation rates for each structure in every m o n k e y were determined.
Pro cedure A dose-response curve of the effect of the dopaminereceptor blocking agent spiroperidol [ 1 ] on self-stimulation was made as follows. The spiroperidol (generously supplied by Janssen Pharmaceutica, Beerse, Belgium) was injected intraperitoneally in doses of 0.016, 0.062, and 0.1 mg/kg. Control injections were of the solvent only, 0.01 M tartaric acid. The monkeys were tested every 3 days. On a test day first the baseline rate of self-stimulation at every site was tested. T h e n the IP injections were given, and one hour later the self-stimulation rate was measured for each electrode for 10 one min periods, with 5 min periods allowed for changing the stimulation site and stabilization of the rate. The sites were tested in the following order: orbitofrontal cortex, amygdala, lateral h y p o t h a l a m u s and locus coeruleus. The self-stimulation rate over a period of I0 min for each electrode was calculated as a percentage of the corresponding mean baseline rate for that electrode to facilitate comparison between the electrodes. The plan of the experiments with intracranial injections was to compare the effects of injections of spiroperidol into different brain areas on self-stimulation of many different brain sites. In this particular e x p e r i m e n t we used two rhesus monkeys and one of the squirrel monkeys. For the rhesus m o n k e y s 2 ul of 3 ug/ul spiroperidol were injected bilaterally into a given site. Control injections of the solvent (0.01 M tartaric acid) were also given. The procedure was as for the 1P injection tests. The self-stimulation rate over a period of 10 rain for each electrode was calculated as a percentage of the corresponding mean control rate. In the squirrel m o n k e y the procedure was as for the rhesus m o n k e y s except that the spiroperidol (1 ug/gl in 1 ul volumes) was injected bilaterally until an effect was obtained. Haloperidol in a concentration of 2.5 ug/ul was also tested. Additional experiments with different doses were also performed (see Table 1).
Histology At the conclusion of the experiments every site was verified histologically. The animals were anesthetized with Nembutal and a lesion (0.1 mA, 60 sec) was made to assist
MORA, ROLLS, B U R T O N AND SHAW identification of the region in which stimulation and injections were given. The m o n k e y s were perfused with isotonic saline followed by 10 percent formal-saline. The brains were frozen and sections were cut at 50 u and stained with thionin. As shown in the sections from one of the rhesus monkeys illustrated in Fig. 3, the cannulae aimed at orbitofrontal cortex, lateral hypothalamus and amygdala were accurately placed, and the cannulae aimed at the nucleus accumbens ended in this particular m o n k e y just below the nucleus accumbens close to the dura, so that the injections probably reached the nucleus accumbens, but self-stimulation did not occur probably because the stimulation electrode in protruding from the cannula touched the dura. The cannulae aimed at the locus coeruleus appeared to end within 2 mm of the locus coeruleus. RESULTS
Intraperitoneal Injection Dose-response curves for the effect of spiroperidol on self-stimulation in the rhesus m o n k e y s and the squirrel m o n k e y s are shown in Fig. 1. In this figure we represent the mean and standard error of the rate per rain measured over a period of 10 min for each electrode 1 hr after injection in both groups of rhesus and squirrel monkeys. The results for each electrode were expressed as a percentage of the mean control (i.e. placebo) rate for that electrode. The mean rates for each electrode under the control condition, and on each testing day before injection, remained constant throughout the experiments. The experiments were repeated twice in rhesus m o n k e y 1 at each drug dose and the same results were obtained in the two separate experimental sessions (see Fig. 2). It is shown in Fig. 1 that in b o t h the rhesus monkeys and the squirrel m o n k e y s self-stimulation at all the sites tested was severely attenuated by a dose of spiroperidol of 0.062 or 0. I mg/kg. At the lower dose of 0.016 mg/kg self-stimulation at most sites was unaffected, but selfstimulation o f the orbitofrontal cortex was a t t e n u a t e d in two o f the rhesus monkeys and one o t the squirrel monkeys. The typical time course of the attenuation of self-stimulation by spiroperidol, and the repeatability of the results are illustrated in the two separate runs of the experiment in rhesus m o n k e y 1 shown in Fig. 2. The results shown in Figs. 1 and 2 suggest that with a dose of 0.016 mg/kg of spiroperidol the effect on self-stimulation depends on which sell-stimulation site is tested, and suggest that orbitofrontal cortex self-stimulation is particularly sensitive to d o p a m i n e - r e c e p t o r blockade.
Intracranial Injection The self-stimulation rate (expressed as a percentage of the preinjection control rate) for different sites one hr after injection of solutions of spiroperidol bilaterally into different brain areas of the monkeys is shown in Table I. The error figures represent an estimate of the standard errors calculated from l0 one-minute observations of self-stimulation rate; and the significance of the difference of these observations from the preinjection control rates using a t-test and confirmed with a U test is shown by *p<0.02 or +p<0.001. The largest effect found with a 6 #g dose was attenuation of the rate of self-stimulation of the amygdala by 70 percent by injection into the nucleus accumbens (see Table
SPIROPERIDOL AND SELF-STIMULATION
Rhesus o J~ (~
213
Squirrel
monkey
monkey
120 eHypothalamus • L. coeruleus • Orbito=frontal
• L. coeruleus • Hypothalamus • Orbito-frontal
~ 100 .~
t-
0
80
60 O
E
40
I
20
0
I
0.016
I
"I"
i
0"062 0-1
0.062 0.1 0.016 Spiroperidol (rag/kg)
FIG. 1. Dose-response curves for the effect of IP injections of spiroperidol on self-stimulation rate at different sites in 3
rhesus monkeys and 2 squirrel monkeys. The self-stimulation rate for a particular electrode is shown as a percentage of the self-stimulation rate after a placebo injection, and for the rhesus monkey is the mean of two separate experimental determinations of every point. The vertical lines indicate an estimate of the standard error of the ten one-minute observations of self-stimulation rate. The self-stimulation electrodes were in the hypothalamus, the orbitofrontal cortex, and near the locus coeruleus. 1). Similar results, a n d dose-related effects, were seen w i t h 9 gg o f s p i r o p e r i d o l in w h i c h t h e a t t e n u a t i o n of selfs t i m u l a t i o n o f t h e a m y g d a l a was 99 p e r c e n t . This e f f e c t was c o n f i r m e d w i t h t h e squirrel m o n k e y in w h i c h 5 ~g o f h a l o p e r i d o l i n j e c t e d i n t o the same s t r u c t u r e ( n u c l e u s a c c u m b e n s ) totally a t t e n u a t e d s e l f - s t i m u l a t i o n o f t h e a m y g dala. O b s e r v a t i o n s in o t h e r s t r u c t u r e s were t h a t i n j e c t i o n s into t h e o r b i t o f r o n t a l c o r t e x (O. F. C o r t e x ) partially a t t e n u a t e d s e l f - s t i m u l a t i o n of t h e h y p o t h a l a m u s ( b y 46 p e r c e n t ) a n d o f t h e a m y g d a l a ( b y 32 p e r c e n t ) , and injections i n t o t h e h y p o t h a l a m u s a t t e n u a t e d self-stimulation of the a m y g d a l a ( b y 35 p e r c e n t ) . T h e r e was never a n y large effect of i n j e c t i o n s o f s p i r o p e r i d o l i n t o t h e region o f t h e locus c o e r u l e u s or o f i n j e c t i o n s i n t o o t h e r areas o n s e l f - s t i m u l a t i o n o f t h e region o f t h e locus coeruleus. T h e s e results were c o n f i r m e d in a d d i t i o n a l e x p e r i m e n t s (see T a b l e 1). T h e l a t e n c y for a m e a s u r a b l e effect was 1 0 - 2 0 min, a n d the effect r e a c h e d its m a x i m u m at 2 0 - 3 0 m i n and r e m a i n e d at this level for m o r e t h a n 4 hr.
DISCUSSION
[ntraperitoneal Injections T h e e x p e r i m e n t w i t h i n t r a p e r i t o n e a l injections s h o w s t h a t in t h e m o n k e y s p i r o p e r i d o l a t t e n u a t e s intracranial s e l f - s t i m u l a t i o n at t h e d i f f e r e n t sites tested at doses of 0 . 0 6 2 and 0.1 mg/kg. A t t h e lower dose of 0 . 0 1 6 m g / k g some e v i d e n c e was o b t a i n e d t h a t o r b i t o f r o n t a l c o r t e x s e l f - s t i m u l a t i o n is particularly sensitive to d o p a m i n e r e c e p t o r b l o c k a d e . A l t h o u g h the a n i m a l s could still work for glucose at a dose of 0 . 0 6 2 m g / k g o f spiroperidol, a general effect o f t h e s p i r o p e r i d o l o n b e h a v i o r ( s u c h as a m o t o r i m p a i r m e n t or s e d a t i o n ) was suspected, since at t h e higher dose o f 0.1 m g / k g t h e m o n k e y s looked drowsy. To determine whether spiroperidol attenuated self-stimulation b y this t y p e o f general effect on b e h a v i o r or b y a specific effect o n s e l f - s t i m u l a t i o n , in E x p e r i m e n t 2 s p i r o p e r i d o l was i n j e c t e d directly i n t o d i f f e r e n t b r a i n sites. A n a t t e n u a t i o n o f s e l f - s t i m u l a t i o n at o n l y some s e l f - s t i m u l a t i o n sites w o u l d
214
MORA, ROLLS, B U R T O N AND SHAW eo H y p o t h Q l a m u s I O L. coeruleus &&Orbito-frontal
show t h a t t h e effect on b e h a v i o r was specific, and would provide evidence on the neural system w h i c h was involved in self-stimulation of these particular self-stimulation sites.
~z5 u
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t_
o
o
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~.,
•
•
o
o
~
•
•
Intracranial Injections
o
o I
•
~i.
,- 75 0 ._
~ so
•
~.
~
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•
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0
C
5
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I
I
[
I
5 65 95 125 155 Time ( i n j e c t i o n - t e s t ) (rain)
!
185
FIG. 2. The effect of 0.016 mg/kg of spiroperidol IP on self-stimulation with electrodes in the hypothalamus, orbitofrontal cortex and in the region of locus cocruleus, in rhesus monkey 1. The solid and open symbols represenl the two separate experimental determinations of every point. The self-stimulation rate for a particular electrode is shown as a percentage of the self-stimulation rate after a placebo injection. C represents the control rate.
Or bitofrontal Cortex
Arnygdala
T h e finding w i t h intracranial injections of spiroperidol t h a t self-stimulation in the region o f the locus c o e r u l e u s was almost u n a f f e c t e d even w h e n self-stimulation at some o t h e r sites was severely a t t e n u a t e d suggests that the a t t e n u a t i o n of self-stimulation f o u n d at the affected site was not due to a general effect o n b e h a v i o r such as m o t o r i m p a i r m e n t or sedation. In a d d i t i o n , a f t e r the tests for self-stimulation the m o n k e y s were able to p e r f o r m the m o t o r response task to o b t a i n 20 p e r c e n t glucose s o l u t i o n (see T a b l e 1). T h u s it appears t h a t the a t t e n u a t i o n o f self-stimulation f o u n d at s o m e sites can n o t be e x p l a i n e d by a single effect such as m o t o r i m p a i r m e n t [81. T h e finding that injections of spiroperidol i n t o the o r b i t o f r o n t a l cortex a t t e n u a t e d self-stimulation of the a m y g d a l a and lateral h y p o t h a l a m u s suggests t h a t the o r b i t o f r o n t a l cortex is involved in self-stimulation of some limbic structures. This suggestion is c o n s i s t e n t with first, the electrophysiological findings that single u n i t s in the o r b i t o f r o n t a l cortex are activated in self-stimulation of the lateral h y p o t h a l a m u s and a m y g d a l a and vice versa [ 3 ] , second, that anesthetization o f the sulcal p r e f r o n t a l cortex in the rat ( w h i c h m a y
Nucleus
Lateral
accumbens
Hypot halamus
FIG. 3. Sites of cannulae used for injection and for stimulation in the rhesus monkey. No self-stimulation occurred at the
nucleus accumbens site in this monkey, because the stimulation site was close to the base of the brain.
*p
SW
t~
N. Accumbens
Hypothalamus
R2
O.P. Cortex
Rl
Amygdala
N. Accumbens
RI
R2
Amygdala
RI
N. Accumbens
L. Coeruleus
Rl and R2
R2
Hypothalamus
Rl and R2
O.F. Cortex
67
Amygdala
Rl and R2
R2
32
N. Accumbens
Rl and R2 4.3
98
84
25
12
-
103 t
90 f 4.3
4.3
87 f 10.0
74 f
-
1
_
0
33
l12?
65?
-
6
3*
30 f lot
68 f 10*
-
O.F. Cortex
Rl and R2
INJECTIONS
_ _
82 f 1.3
81 f 3.3
92 _+4.6
-
_
II7
_
0
36
4
18
-
-
-
-
-
-
N. Accumbens
_
100 ? 5.0
92 * 0.6
I60 + 3.0
87 f 4.6
Rate for Glucose as Percent of Control Rate
ON SELF-STIMULATION
_
-
83
50
85
Additional Experiments
4.0
2.6
6.3
82 f 9.2
L. Coeruleus
47
71
64
120 t
82 f
96 c
54 f l3.2*
Hypothalamus
1
OF SPIROPERIDOL
Rate (Percent of Control Rate)
Amygdala
Monkeys
Self-Stimulation
OF INTRACRANIAL
O.F. Cortex
Injection Site Spiroperidol
EFFECTS
TABLE
2rl
(Haloperidol) 2.5 f.dPl
3 rslrl
3 f&d
2 PI 2 PI
4 rglpl
3 /%/Id
3 /%/~I
3 &rl
4 rslrl
Concentration
3 ctl
2 rl
2pl
3 I%
3 Ml
2 ctl
2 nl
2 IJl
2 ccl
2 rl
Volume Injected Bilaterally
E ol
5 ?I c!e
5
216
MORA, ROLLS, B U R T O N A N D SHAW
c o r r e s p o n d to the o r b i t o f r o n t a l cortex o f the m o n k e y ) [7] a t t e n u a t e d self-stimulation of the lateral h y p o t h a l a m u s [ 1 0 ] , third, t h a t there are d o p a m i n e r g i c t e r m i n a l s in the region of the p r e f r o n t a l c o r t e x in the rat [ ! 5 ], and f o u r t h , that single cells in this p r e f r o n t a l region w h i c h are activated during self-stimulation of the lateral h y p o t h a l a m u s of t h e rat alter their firing rate w h e n a d o p a m i n e r g i c agonist { a p o m o r p h i n e ) or a n t a g o n i s t ( s p i r o p e r i d o l ) is a d m i n i s t e r e d systemically in low doses (personal observations). Thus,
d o p a m i n e r g i c c o n n e c t i o n s to the o r b i t o f r o n t a l cortex m a y be involved in self-stimulation of a n u m b e r o f d i f f e r e n t sites. A l t h o u g h it is difficult to i n t e r p r e t at this stage, it is of interest t h a t injections into the nucleus a c c u m b e n s and lateral h y p o t h a l a m u s a t t e n u a t e d self-stimulation o f the amygdala. It is k n o w n that single u n i t s in these t w o areas are a c t i v a t e d during s e l f - s t i m u l a t i o n [ 3 ].
REFERENCES 1. And~n, N. E., S. G. Butcher, H. Corrodi, K. Fuxe and U. Ungerstedt. Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. Eur. J. Pharmac. I1: 303-314, 1970. 2. Anlezark, G. M., G. W. Arbuthnott, J. E. Christie and T. J. (;row. Electrical self-stimulation with electrodes in the region of the interpeduncular nucleus. J. PhysioL, Lond. 234:103 P, 1973. 3. Burton, M. J., E. T. Rolls, t:. Mora and S. J. Cooper. Ncurophysiological mapping of self-stimulation pathways in the monkey. ¢in preparation). 4. Crow, T. J. A map of the rat mesencephalon for electrical sclfistimulation. Brain Res. 36: 265-273, 1972. 5. Emmers, A. and K. Akert. A Stereotaxic Atlas o]the Bra• o f the Squirrel Monkey. Madison: The University of Wisconsin Press, 1963. 6. Kelly, P. H., IL T. Rolls and S. G. Shaw. Functions of catccbolamines in brain-stimulation reward. Brain Res. 36: 363 364, 1974. 7. leonard, C. M. The prefrontal cortex in the rat. i. Cortical projections of the mediodorsal nucleus. I1. Efferent connections. Brain Res. 1 2 : 3 2 1 - 3 4 3 , 1969. 8. Mora, F., A. M. Sanguinetti, E. T. Rolls and S. (;. Shaw. Differential effects on self-stimulation and motor behaviour produced by microintracranial injections of a dopamine-receptor blocking agent. Neuroscience Letters I : 179- 184, 1975.
9. 10. 1 I. 12. 13.
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