- Email: [email protected]
Lesion of dopaminergic terminals in the amygdala produces enhanced locomotor response to d -amphetamine and opposite changes in dopaminergic activity in prefrontal cortex and nucleus accumbens
Brain Research, 447 (1988) 335-340 Elsevier
335
BRE 13488
Lesion of dopaminergic terminals in the amygdala produces enhanced locomotor response to D-amphetamine and opposite changes in dopaminergic activity in prefrontal cortex and nucleus accumbens* H. Simon 1, K. Taghzouti I, H. Gozlan 2, J.M. Studler 2, A. Louilot ~, D. Herve 2, J. Glowinski 2, J.P. Tassin 2 and M. Le Moal 1 ILaboratoire de Psychobiologie des Comportements Adaptatifs, I.N.S.E.R.M.U.259 -- UniversitE de Bordeaux !I, Domaine de Carreire, Bordeaux (France) and 2Chaire de Nevropharmacologie, L N. S. E. R . M . U . 114 -- CollEge de France, Paris (France)
(Accepted 20 Oclober 1987) Key words: Dopaminergic mesencephalic neuron; Amygda!a; Nucleus accumbens; Prefrontal cortex; Dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio; D-Amphetamine; Locomotor activity
In a previous study using differential pulse voltammetry we demonstrated an interaction between dopaminergic actiwty in the amygdala and the nucleus accumbens. In the present study, by post-mortem biochemical measurements, we showed that bilateral 6OHDA lesions of DA innervation of the amygdala leads to an increase in DA activity in the nucleus accumbens (DOPAC/DA ratio + 24%) and a reduction (DOPAC/DA ratio-40%) in the prefrontal cortex. In addition, after these lesions in the amygdala, there was an increas,:d behavioral sensitivity to D-amphetamine, demonstrated by enhanced locomotor activity. Increased ~nderstanding of the interregulations between dopaminergic activity in forebrain structures may help explain forebrain functions and/or dysfunctions.
INTRODUCTION
There is increasing evidence to suggest that dopaminergic (DA) neurons do not have a specific role in the control of behavior 13'25'27. It appears rather that DA neurons facilitate the functions of various forebrain structures. From the results of recent studies 26"28-33, we suggested that DA neurons act on the projection areas, not only by ensuring an optimal functional level of the neuronal integrative projection systems, but also by fine tuning the respective activity of several areas to facilitate transfer to information from one region to another. This implies that the various DA pathways act in a coordinated manner, and consequently that there are inter-regula-
tions between them. In vivo voltammetric studies 2° have shown that DA activity in the nucleus accumbens is influenced by DA activity in the amygdala. Acute DA blockade in the amygdala is followed by an increase in the height of the dihydroxyphenylacetic acid (DOPAC) peak in the nucleus accumbens. Conversely, facilitation of DA transmission in the amygdala induces a reduction in the DOPAC peak in the nucleus accumbens. In order to extend these findings, experiments were designed to investigate the following. (1) To confirm these results by post-mortem determinations of dopamine and its main metabolite, DOPAC, in the relevant brain regions. This would also establish whether there are long-term effects after loss of DA
* These results have been published, in part, as a poster form at the 8th European Neurosciences Congress, The Hague, 1984 Correspondence: H. Simon, Laboratoire de Psychobiologie des Comportements Adaptatifs, I.N.S.E.R.M.U.259 - - Universitt~ de Bordeaux II, Domaine de Carreire m rue Camille Saint-Sa~ns, 33077 Bordeaux Cedcx, France. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
336 activity in the amygdala. (2) To find if such inter-regulations can be observed in other DA structures in the forebrain. (3) To correlate these biochemical interactions with changes in behavior. We analyzed the effects of bilateral 6-OHDA lesions of DA terminals in the amygdala on the locomotor response to D-amphetamine, and also on DA and DOPAC levels, measured post-mortem in the nucleus accumbens and prefrontal cortex. MATERIALS AND METHODS
Adult male Sprague-Dawley rats were housed singly with free access to food and water in a temperature-controlled environment (22 + 1 °C). They weighed between 280 and 300 g at the time of surgery. Light cycle was maintained between 06.00 and 20.00 h, and all behavioral testings were carried out between 09.00 and 14.00 h. Thirty to 45 min prior to surgery, rats receiv~:~i an intraperitoneal injection of desmethylimipra~Tine (Pertrofan, Geigy, 25 mg/kg) in order to preve~ the destruction of noradrenergic terminals by ~-,-hydroxydopamine. Sixteen rats were operated under chloral hydrate anesthesia (150 mg/kg, i.p.). The animals were placed in a Kopf stereotaxic apparatus with tooth bar 5 mm above the interaural line. The skull was exposed and burr holes drilled above the appropriate coordinate targets. The experirr=:ntal rats (n -- 10) received a bilateral 6-hydroxydopamine (6-OHDA) infusion at the level of basolateral nucleus of the amygdala according to the foll~wing coordinates: 0.8 mm posterior to the bregma, 4.~ mm lateral to the midline and 8.8 mm below the skul~ surface. Each site was infused with 6 pg/2 pl of '= U - K _'2 ~ .~ r~ l . J / ' l k,, (free base, Sigma). The neurotoxin was dissolve at a concentration of 3 pg (free base) per 1/zl in a ve:ficle solution containing 0.2 mg/ml of ascorbic acid and injected over 7 rain through a 0.3 mm diameter stainless steel cannula connected via a polyethylene: tubing to a 10 kd microsyringe driven by a pump at constant speed. The cannulae were left in place ~ an additional 5 min after the end of the infusion: c~ntrol rats (n = 6) underwent the same procedure ~~:cept that they only received the vehicle solution. Locomotor activity was measured in a circular corridor, 12 cm wide and 170 cm long, equipped with 4 photocell beams situated 3 cm above the floor. Inter-
ruption of the beams was recorded automatically on an event recorder situated outside the testing room. Behavioral testing began 3 weeks after surgery. Spontaneous locomotor activity was first measured during a 90 min habituation period (from 09.00 to 10.30 h). Each rat then received a saline injection, and was replaced in the circular corridor for an additional 90 min period. After the injection of 1.5 mg/kg of (+)-amphetamine, locomotor activity was recorded over 10 min intervals for a total duration of 90 min.
Neurochemical assays One week after the end of the behavioral testing, the rats were killed by decapitation. The brain was rapidly removed and chilled on an ice-cooled plate. Brain regions (n. accumbens, amygdala and prefrontal cortex) were dissected according to the method
~m
e~e CON O. . . . O AMY (-) 0 - - - - 0 AI~Y (+)
0----"(~ F -/i
40C
~
/ 300
; I
\
200
;~.._ ,s-/.,
X~_. . . .
100
o
0
I
20
I
ao
I
40
I
so
I
60
I
ro
I
so
J
90
TIME (MIN) Fig. 1. Time course of amphetamine-induced locomotor response following bilateral 6 - O H D A injection into the amygdala. A M Y ( + ) = rats with lesions (n = 6), hyperresponsive to amphetamine; A M Y ( - ) = rats with lesions, hyporesponsive to amphetamine 01 = 4) with respect to A M Y ( + ) rats or responding in a similar way to that of controls (CON, n = 6).
337 described by Tassin et al. 34. D A , noradrenaline ( N A ) and D O P A C concentrations were measured either by using high-pressure liquid chromatography with electrochemical detection (n. accumbens, amygdala) 36 or by a radioenzymatic method (prefrontal cortex) 11 RESULTS Injection of 1,5 mg/kg of D-amphetamine led to two kinds of locomotor response in rats with 6O H D A lesions in the amygdala (Fig. 1). One group of rats with lesion (n = 4) responded similarly to controls ( A M Y - ) , and the other group (n = 6) increased responding (AMY+). The A M Y + rats were 2 - 3 times more active than the A M Y - and control animals (C). Analysis of variance between the 3 groups showed a significant group-effect (F2.13 = 14.23; P < 0.001). A post-hoc comparison of the group mean by the Newman-Keuls' test indicated a statistical significance between A M Y + vs C (N = 5.35; P < 0.001);
3.000
I
ICON
AMY(-) ~] AMY(+)
"
~if~-2'0001-
A M Y + vs A M Y - (N = 6.72; P < 0.001), but no statistical significance between A M Y - vs C (N = 1.36, NS). The higher score in controls and A M Y - rats was approximately of ! 60 counts per 10 miD, while in the A M Y + rats it reached 420 counts per 10 miD. The period of peak locomotor response to D-amphetamine was the same for the 3 groups. It took place between the 30th and 50th minute. No time-effect was observed between the 3 groups (F2.13 = 0.35, NS). The total locomotor activity of rats in the 3 phases of testing is shown in Fig. 2A. It is interesting to note that the 3 groups of rats (C, A M Y - , A M Y +) behaved similarly in the preinjection period (F2,13 = 0.636, NS) and after the saline injection (F2,13 = 0.202, NS). The two groups of experimental rats ( A M Y - and A M Y + ) discriminated on the basis of their behavioral data also differed in neurochemical status (Fig. 2B). The A M Y - rats had a D O P A C / D A ratio in the prefrontal cortex similar to that of controls ( A M Y - = 0.44 + 0 . 0 3 - C = 0.41 + 0.03; t8 = 0.6, NS). The ra-
15o
• i 100I
110oo till O'-
injPre ection inSal iectiineon1.5 id-amph nmg/kg jection
PFC
ACC
Fig. 2. Correlation between locomotor response to amphetamine and brain neurochemical status of rats with 6-OHDA lesion in the amygdala. A: total locomotor activity; 90 rain habituation phase (pre-injection), saline injection phase and o-amphetamine injection phase. B: DOPAC/DA ratio in the prefrontal cortex (PFC) and in the nucleus accumbens (ACC) expressed as the percentage of control values (prefrontal cortex: DA = 69.6 + 7 pg/mg tissue; DOPAC = 28.3 + 2 pg/mg tissue; and nucleus accumbens: DA = 8.37 + 0.14 ng/mg tissue; DOPAC = 0.86 + 0.05 ng/mg tissue. Statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05.
338 tio was somewhat reduced in the nucleus accumbens (AMY- = 0.147 + 0.09-C = 0.172 + 0.016; t8 = 2.2, P < 0.05). The endogenous concentrations of DA and DOPAC in each structure were not significantly different between the two groups. For the AMY + rats the DOPAC/DA ratio was decreased by 40% in the frontal cortex (t~0 = 3.7; P < 0.01) and was increased by 24% in the nucleus accumbens (tl0 = 3.2; P < 0.05) with respect to controls (Fig. 2). These changes in DOPAC/DA ratio were due to alteration in levels of DOPAC. DA concentrations were unaltered. DOPAC was decreased by 40% in the frontal cortex and increased by 35% in the nucleus accumbens. In the amygdala, the 6-OHDA injection produced a fall of 84% in endogenous concentrations of DA (control values: 533.49 + 25.50 pg/mg tissue) and 40% in NA (control values: 671.02 + 56.81 pg/mg tissue), for both the AMY- and AMY + groups. DISCUSSION
The present results confirm and extend our previous findings from in vivo differential pulse voltammetry. Firstly, 6-OHDA lesions of DA afferents to the amygdala led to an increase in DA activity in the nucleus accumbens, measured in vitro. This increase was in the same range as that found by measurement of DOPAC levels in the nucleus accumbens in vivo, following blockade of DA transmission in the amygdala by local infusion of DA receptor antagonists such as a-flupentixoi or sulpiride 2°. Secondly, the inter-regulation between the a'nygda!a and the nucleus accumbens, demonstrated in the acute studies, was also observed 5 weeks after interruption of DA transmission in the amygdala. In another experiment (unpublished results), increase in DA activity in the nucleus accumbens was observed as long as 13 weeks after lesions of dopaminergic terminals in the amygdala. Thirdly, the biochemical results showed that this inter-regulation phenomenon extended to the forebrain, since lesion of DA terminals in the amygdala redut:ed DA activity in the prefrontal cortex. The anatomical substrate of these inter-regulations remains to be determined. Two main pathways can be involved: one at the level of DA terminals in the fore-
brain, and another terminating in the dopaminergic cell bodies in the ventral mesencephalon. Direct anatomical connections from amygdala to nucleus accumbens 15as'37, to prefrontal cortex ~7and to the ventral tegmental area 21"22 have been demonstrated. However, the involvement of polysynaptic connections cannot be excluded. For example, alteration of DA activity in the amygdala could affect the functioning of DA activity in the nucleus accumbens by interfering with DA pathways in the prefrontal cortex. It has been shown that there is an increase in levels of DOPAC in the nucleus accumbens after local injection of DA antagonists into the cortex 19. This may mimic the qffect of DA lesions in the amygdala. The second main result of this study concerns the behavioral effect of 6-OHDA lesions of the amygdala. Such lesions do not affect the spontaneous locomotor activity of rats, but they do increase the locomotor response to peripheral injection of D-amphetamine. Interestingly, this hypersensitivity to D-amphetamine was only obtained in the group of rats with reciprocal alterations in DA activity in prefrontal cortex and in nucleus accumbens. The reasons why some animals were hyperresponsive to D-amphetamine (AMY +) while others were not (AMY-) cannot readily be explained from the results of the biochemical assays after lesions in the amygdala. Indeed, both groups had the same deficits in DA and NA in the amygdala. It is possible that other neuromediators were affected in different ways by these !e:ions. Since there is a laterality of DA function in the amygdala 2, hemispheric dominance might also account for these differences. The enhanced locomotor response to D-amphetamine in rats with increased DA activity in the n. accumbens is consistent with literature data. Many studies have shown that spontaneous locomotor activity and locomotor response to D-amphetamine are controlled by DA neurons in the n. acccumbens 3'14'16. Further, we have shown in previous studies that manipulations which have opposite effects on DA activity in the n. accumbens (increase) and prefrontal cortex (redaction) - - in similar way that we obtained in the present experiment - - produced locomotor hyperactivity ~12,35 It must also be emphasized that the behavioral effects of disruption of DA in the amygdala are not restricted to alterations in locomotor activity. For ex-
339 ample, we have shown that specific D A lesions in the amgydala increase the acquisition of self-administration of D-amphetamine 6.
Kindling in the amygdala of the rat alters D A transmission in the nucleus accumbens 5"9a°, and also affects l o c o m o t o r activity7"8.z3. It has also been re-
A relationship b e t w e e n D A functions in a m y g d a l a and nucleus accumbens m a y also account for some of the direct effects of d o p a m i n e d e m o n s t r a t e d in animal studies, as well as in some h u m a n autopsy findings. For example, injection of d o p a m i n e in the amygdala of the rat leads to changes in l o c o m o t o r activity 2, which have b e e n t h o u g h t to be m e d i a t e d via an effect on D A t u r n o v e r in the nucleus accumbens.
ported that there are alterations of D A in the amygdala 24 and nucleus accumbens 4 in the brains of schizophrenic patients.
REFERENCES
12 Herv6, D., Simon, H., Blanc, G., Le Moal, M., Giowinski, J. and Tassin, J.P., Opposite changes in dopamine utilization in the nucleus accumbens and the frontal cortex after electrolytic lesion of the median raphe in the rat, Brain Research, 216 (1982) 422-428. 13 Iversen, S.D., Cortical monoamines and behavior. In L. Descarries, T.R. Reader and H.H. Jasper (Eds.), Neurology and Neurobiology, Vol. 10, Monoamine Innervation of Cerebral Cortex, Liss, New York, 1984, pp. 321-349. 14 Iversen, S.D. and Koob, G.F., Behavioral implications of dopaminergic neurons in the mesolimbic system. In E. Costa and G.L. Gessa (Eds.), Nonstriatai Dopamine Neurons. Adv. Biochem. P~ychopharmacol., Vol. 16, Raven, New York, i977, pp. 589-595. 15 Kelley, A.E., Domesick, V.B. and Nauta, W.J.H., The amygdalostriatal projection in the rat. An anatomical study by anterograde and retrogf:'de tracing method, Neuroscience, 7 (1982) 615-630. 16 Kelly, P.H., Seviour, P.W. and Iversen, S.D., Amphetamine and apomorphine response in the rat following 6OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Reseats-h, 94 (1975) 507-522. 17 Krettek, J.E. and Price, J.L., A direct input from the amygdala to the thalamus and the cerebral cortex, Brah~ Research, 67 (1974) 169-174. 18 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 19 Louilot, A., Le Moal, M. and Simon, H., Functional interdependence between the dopaminergic innervation of the frontal cortex and the nucleus accumbens demonstrated by in vivo voltammetry, Neurosci. Lett., Suppl. 26 (1986) $27. 20 Louilot, A., Simon, H., Taghzouti, K. and Le Moal, M., Modulation of dopaminergic activity in the nucleus accumbens following facilitation or blockade of the depaminergic transmission in the amygdala: a study by in vivo differential pulse voltammetry, Brain Research, 346 (1985) 141-145. 21 Maeda, H. and Mogenson, G.J., Electrophysiological responses of neurons of the ventral tegmental area to electrical stimulation of amygdala and lateral septum, Neurosciettce, 6 (1981) 367-376. 22 Phillipson, O.T., Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat, J. Comp Neurol., 187 (1979) 117-144.
1 Blanc, G., HervC D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P., Response to stress of mesocortico-frontal dopaminergic neurons in rats after longterm isolation, Nature (Lond.), 284 (1980) 265-267. 2 Bradbury, A.J., Costall, B., Domeney, A.M. and Naylor, R.J., Laterality of dopamine function and neuroleptic action in the amygdala in the rat, Neuropharmacoiogy, 24 (1985) 1163-1170. 3 Costall, B. and Naylor, R.J., The behavioral effects of dopamine applied intracerebrally to areas of the mesolimbic system, Eur. J. Pharmacol., 32 (1975) 87-92. 4 Crow, T.J., Cross, A.J., Johnson, J.A., Johnstone, E.C., Joseph, M.H., Owen, F., Owens, D.G.C. and Poulter, M., Catecholamines and schizophrenia: an assessment of the evidence. In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engel (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 259-269. 5 Csernansky, J.G., Csernansky, C.A., Bonnet, K.A. and Hollister, L.E., Dopaminergic supersensitivity follow ferric chloride-induced limbic seizures, Biol. Psychiat., 20 (1985) 723-733. 6 Demini~re, J.M., Taghzouti, K., Tassin, J.P., Le Moal, M. and Simon, H., Increased sensitivity to amphetamine and facilitation of amphetamine self-administration after 6-hydroxydopamine lesions of the amygdala, Psychopharmacology, in press. 7 Ehlers, C.L., Koob, G.F. and Bloom, F.E., Post-ictal locomotor activity in three different rat models of epilepsy, Brain Research, 250 (1982) 178-182. 8 Ehlers, C.L. and Koob, G.F., Locomotor behavior following kindling in three different brain sites, Brain Research. 326 (1985) 71-79. 9 Engel, J. and Sharpless, N.S., Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 10 Gee, K.W., Hollinger, M.A., Bowyer, J.F. and Kiilam, E.K., Modification of dopaminergic receptor sensitivity in rat brain after amygdaloid kindling, Exp. Neurol., 66 (',979) 771-777. 11 Gauchy, C., Tassin, J.P., Glowinski, J. and Cheramy, A., Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological samples, J. Neurochem., 26 (1976) 471-480.
In conclusion, the inter-regulation between D A activity in several forebrain structures is an important feature of the functioning of these neurons which could have some importance in the study of biological mechanisms involved in mental disorders.
340 23 Post, R.M., Squillace, K.M., Pert, A. and Sass, W., The effect of amygdala kindling on spontaneous and cocaine-induced motor activity and lidocaine seizures, Psychopharmacology, 72 (1981) 189-196. 24 Reynolds, G.P., Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia, Nature (Lond.), 305 (1983) 527-529. 25 Simon, H. and Le Moal, M., Mesencephalie dopaminergic neurons: functional role. In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engel (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 293-307. 26 Simon, H., Taghzouti, K. and Le Moal, M., Deficits in spatial-memory tasks following lesions of septal dopaminergic terminals in the rat, Behav. Brain Res., 19 (1986) 7-16. 27 Stricker, E.M. and Zigmond, M.J., Brain catecholamines and motivated behavior: specific or nonspecific contributions? In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engei (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 259-269. 28 Taghzouti, K., Garrigues. A.M., Labouesse, J., Le Moal, M. and Simon, H., Bovine serum albumin-haloperidol as a tool for the study of dopaminergic transmission: behavioral and neurochemical effect following single injection in the nucleus accumbens, Life Sci., 40 (1987) 127-137. 29 Taghzouti, K., Le Moal, M. and Simon, H., Enhanced frustrative nonreward effect following 6-hydroxydopamine lesions of the lateral septum in the rat, Behav. Neurosci., 99 (1985) 1066-1073. 30 Taghzouti, K., Louilot, A., Herman, J.P., Le Moal, M. and Simon, H., Alternation behavior, spatial discrimination and reversal disturbances following 6-hydroxydopamine lesions in the nucleus accumbens of the rat, Behav. Neur. Biol., 44 (1985) 354-363.
31 Taghzouti, K., Simon, H. and Le Moal, M., Disturbances in exploratory behavior and functional recovery in the Y and radial mazes following dopamine depletion of the lateral septum, Behav. Neut. Biol., 45 (1986) 48-56. 32 Taghzouti, K., Simon, H., Louilot, A., Herman, J.P. and Le Moal, M., Behavioral study after local injection of 6-hydroxydopamine into the nucleus accumbens in the rat, Brain Research, 344 (1985) 9-20. 33 Taghzouti, K., Simon, H., Tazi, A., Dantzer, R. and Le Moal, M., The effect of 6-OHDA lesions of the lateral septum on schedule-induced polydipsia, Behav. Brain Res., 15 (1985) 1-8. 34 Tassin, J.P., Lavielle, S., Herr6, D., Blanc, G., Thierry, A.M., Alvarez, C., Berger, B. and Glowinski, J., Collateral sprouting and reduced activity of the rat mesocortical dopaminergic neurons after selective destruction of the ascending noradrenergic bundles, Neurosci., 4 (1979) 1569-1582. 35 Tassin, J.P., Simon. H., Glowinski, J. and Bockaert, J., Modulations of the sensitivity of dopaminergic receptors in the prefrontal cortex and the nucleus aceumbens: relationship with locomotor activity. In R. Collu, J.R. Ducharme, A. Barbeau, and M.D. Tolis (Eds.), Brain Peptides and Hormones, Raven, New York, 1982, pp. 17-30. 36 Wagner, J., Vitali, P., Palfreyman, M.G.,, Zraika, M. and Huot, S., Simultaneous determination of 3,4 dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3methoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by high performance liquid chromatography with electrochemical detection, I. Neurochem., 38 (1982) 1241-1254. 37 Yim, C.H. and Mogenson, G.J., Response of nucleus accumbens neurons to amygdala stimulation and its modification by dopamine, Brain Research, 239 (1982) 401-415.
335
BRE 13488
Lesion of dopaminergic terminals in the amygdala produces enhanced locomotor response to D-amphetamine and opposite changes in dopaminergic activity in prefrontal cortex and nucleus accumbens* H. Simon 1, K. Taghzouti I, H. Gozlan 2, J.M. Studler 2, A. Louilot ~, D. Herve 2, J. Glowinski 2, J.P. Tassin 2 and M. Le Moal 1 ILaboratoire de Psychobiologie des Comportements Adaptatifs, I.N.S.E.R.M.U.259 -- UniversitE de Bordeaux !I, Domaine de Carreire, Bordeaux (France) and 2Chaire de Nevropharmacologie, L N. S. E. R . M . U . 114 -- CollEge de France, Paris (France)
(Accepted 20 Oclober 1987) Key words: Dopaminergic mesencephalic neuron; Amygda!a; Nucleus accumbens; Prefrontal cortex; Dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio; D-Amphetamine; Locomotor activity
In a previous study using differential pulse voltammetry we demonstrated an interaction between dopaminergic actiwty in the amygdala and the nucleus accumbens. In the present study, by post-mortem biochemical measurements, we showed that bilateral 6OHDA lesions of DA innervation of the amygdala leads to an increase in DA activity in the nucleus accumbens (DOPAC/DA ratio + 24%) and a reduction (DOPAC/DA ratio-40%) in the prefrontal cortex. In addition, after these lesions in the amygdala, there was an increas,:d behavioral sensitivity to D-amphetamine, demonstrated by enhanced locomotor activity. Increased ~nderstanding of the interregulations between dopaminergic activity in forebrain structures may help explain forebrain functions and/or dysfunctions.
INTRODUCTION
There is increasing evidence to suggest that dopaminergic (DA) neurons do not have a specific role in the control of behavior 13'25'27. It appears rather that DA neurons facilitate the functions of various forebrain structures. From the results of recent studies 26"28-33, we suggested that DA neurons act on the projection areas, not only by ensuring an optimal functional level of the neuronal integrative projection systems, but also by fine tuning the respective activity of several areas to facilitate transfer to information from one region to another. This implies that the various DA pathways act in a coordinated manner, and consequently that there are inter-regula-
tions between them. In vivo voltammetric studies 2° have shown that DA activity in the nucleus accumbens is influenced by DA activity in the amygdala. Acute DA blockade in the amygdala is followed by an increase in the height of the dihydroxyphenylacetic acid (DOPAC) peak in the nucleus accumbens. Conversely, facilitation of DA transmission in the amygdala induces a reduction in the DOPAC peak in the nucleus accumbens. In order to extend these findings, experiments were designed to investigate the following. (1) To confirm these results by post-mortem determinations of dopamine and its main metabolite, DOPAC, in the relevant brain regions. This would also establish whether there are long-term effects after loss of DA
* These results have been published, in part, as a poster form at the 8th European Neurosciences Congress, The Hague, 1984 Correspondence: H. Simon, Laboratoire de Psychobiologie des Comportements Adaptatifs, I.N.S.E.R.M.U.259 - - Universitt~ de Bordeaux II, Domaine de Carreire m rue Camille Saint-Sa~ns, 33077 Bordeaux Cedcx, France. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
336 activity in the amygdala. (2) To find if such inter-regulations can be observed in other DA structures in the forebrain. (3) To correlate these biochemical interactions with changes in behavior. We analyzed the effects of bilateral 6-OHDA lesions of DA terminals in the amygdala on the locomotor response to D-amphetamine, and also on DA and DOPAC levels, measured post-mortem in the nucleus accumbens and prefrontal cortex. MATERIALS AND METHODS
Adult male Sprague-Dawley rats were housed singly with free access to food and water in a temperature-controlled environment (22 + 1 °C). They weighed between 280 and 300 g at the time of surgery. Light cycle was maintained between 06.00 and 20.00 h, and all behavioral testings were carried out between 09.00 and 14.00 h. Thirty to 45 min prior to surgery, rats receiv~:~i an intraperitoneal injection of desmethylimipra~Tine (Pertrofan, Geigy, 25 mg/kg) in order to preve~ the destruction of noradrenergic terminals by ~-,-hydroxydopamine. Sixteen rats were operated under chloral hydrate anesthesia (150 mg/kg, i.p.). The animals were placed in a Kopf stereotaxic apparatus with tooth bar 5 mm above the interaural line. The skull was exposed and burr holes drilled above the appropriate coordinate targets. The experirr=:ntal rats (n -- 10) received a bilateral 6-hydroxydopamine (6-OHDA) infusion at the level of basolateral nucleus of the amygdala according to the foll~wing coordinates: 0.8 mm posterior to the bregma, 4.~ mm lateral to the midline and 8.8 mm below the skul~ surface. Each site was infused with 6 pg/2 pl of '= U - K _'2 ~ .~ r~ l . J / ' l k,, (free base, Sigma). The neurotoxin was dissolve at a concentration of 3 pg (free base) per 1/zl in a ve:ficle solution containing 0.2 mg/ml of ascorbic acid and injected over 7 rain through a 0.3 mm diameter stainless steel cannula connected via a polyethylene: tubing to a 10 kd microsyringe driven by a pump at constant speed. The cannulae were left in place ~ an additional 5 min after the end of the infusion: c~ntrol rats (n = 6) underwent the same procedure ~~:cept that they only received the vehicle solution. Locomotor activity was measured in a circular corridor, 12 cm wide and 170 cm long, equipped with 4 photocell beams situated 3 cm above the floor. Inter-
ruption of the beams was recorded automatically on an event recorder situated outside the testing room. Behavioral testing began 3 weeks after surgery. Spontaneous locomotor activity was first measured during a 90 min habituation period (from 09.00 to 10.30 h). Each rat then received a saline injection, and was replaced in the circular corridor for an additional 90 min period. After the injection of 1.5 mg/kg of (+)-amphetamine, locomotor activity was recorded over 10 min intervals for a total duration of 90 min.
Neurochemical assays One week after the end of the behavioral testing, the rats were killed by decapitation. The brain was rapidly removed and chilled on an ice-cooled plate. Brain regions (n. accumbens, amygdala and prefrontal cortex) were dissected according to the method
~m
e~e CON O. . . . O AMY (-) 0 - - - - 0 AI~Y (+)
0----"(~ F -/i
40C
~
/ 300
; I
\
200
;~.._ ,s-/.,
X~_. . . .
100
o
0
I
20
I
ao
I
40
I
so
I
60
I
ro
I
so
J
90
TIME (MIN) Fig. 1. Time course of amphetamine-induced locomotor response following bilateral 6 - O H D A injection into the amygdala. A M Y ( + ) = rats with lesions (n = 6), hyperresponsive to amphetamine; A M Y ( - ) = rats with lesions, hyporesponsive to amphetamine 01 = 4) with respect to A M Y ( + ) rats or responding in a similar way to that of controls (CON, n = 6).
337 described by Tassin et al. 34. D A , noradrenaline ( N A ) and D O P A C concentrations were measured either by using high-pressure liquid chromatography with electrochemical detection (n. accumbens, amygdala) 36 or by a radioenzymatic method (prefrontal cortex) 11 RESULTS Injection of 1,5 mg/kg of D-amphetamine led to two kinds of locomotor response in rats with 6O H D A lesions in the amygdala (Fig. 1). One group of rats with lesion (n = 4) responded similarly to controls ( A M Y - ) , and the other group (n = 6) increased responding (AMY+). The A M Y + rats were 2 - 3 times more active than the A M Y - and control animals (C). Analysis of variance between the 3 groups showed a significant group-effect (F2.13 = 14.23; P < 0.001). A post-hoc comparison of the group mean by the Newman-Keuls' test indicated a statistical significance between A M Y + vs C (N = 5.35; P < 0.001);
3.000
I
ICON
AMY(-) ~] AMY(+)
"
~if~-2'0001-
A M Y + vs A M Y - (N = 6.72; P < 0.001), but no statistical significance between A M Y - vs C (N = 1.36, NS). The higher score in controls and A M Y - rats was approximately of ! 60 counts per 10 miD, while in the A M Y + rats it reached 420 counts per 10 miD. The period of peak locomotor response to D-amphetamine was the same for the 3 groups. It took place between the 30th and 50th minute. No time-effect was observed between the 3 groups (F2.13 = 0.35, NS). The total locomotor activity of rats in the 3 phases of testing is shown in Fig. 2A. It is interesting to note that the 3 groups of rats (C, A M Y - , A M Y +) behaved similarly in the preinjection period (F2,13 = 0.636, NS) and after the saline injection (F2,13 = 0.202, NS). The two groups of experimental rats ( A M Y - and A M Y + ) discriminated on the basis of their behavioral data also differed in neurochemical status (Fig. 2B). The A M Y - rats had a D O P A C / D A ratio in the prefrontal cortex similar to that of controls ( A M Y - = 0.44 + 0 . 0 3 - C = 0.41 + 0.03; t8 = 0.6, NS). The ra-
15o
• i 100I
110oo till O'-
injPre ection inSal iectiineon1.5 id-amph nmg/kg jection
PFC
ACC
Fig. 2. Correlation between locomotor response to amphetamine and brain neurochemical status of rats with 6-OHDA lesion in the amygdala. A: total locomotor activity; 90 rain habituation phase (pre-injection), saline injection phase and o-amphetamine injection phase. B: DOPAC/DA ratio in the prefrontal cortex (PFC) and in the nucleus accumbens (ACC) expressed as the percentage of control values (prefrontal cortex: DA = 69.6 + 7 pg/mg tissue; DOPAC = 28.3 + 2 pg/mg tissue; and nucleus accumbens: DA = 8.37 + 0.14 ng/mg tissue; DOPAC = 0.86 + 0.05 ng/mg tissue. Statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05.
338 tio was somewhat reduced in the nucleus accumbens (AMY- = 0.147 + 0.09-C = 0.172 + 0.016; t8 = 2.2, P < 0.05). The endogenous concentrations of DA and DOPAC in each structure were not significantly different between the two groups. For the AMY + rats the DOPAC/DA ratio was decreased by 40% in the frontal cortex (t~0 = 3.7; P < 0.01) and was increased by 24% in the nucleus accumbens (tl0 = 3.2; P < 0.05) with respect to controls (Fig. 2). These changes in DOPAC/DA ratio were due to alteration in levels of DOPAC. DA concentrations were unaltered. DOPAC was decreased by 40% in the frontal cortex and increased by 35% in the nucleus accumbens. In the amygdala, the 6-OHDA injection produced a fall of 84% in endogenous concentrations of DA (control values: 533.49 + 25.50 pg/mg tissue) and 40% in NA (control values: 671.02 + 56.81 pg/mg tissue), for both the AMY- and AMY + groups. DISCUSSION
The present results confirm and extend our previous findings from in vivo differential pulse voltammetry. Firstly, 6-OHDA lesions of DA afferents to the amygdala led to an increase in DA activity in the nucleus accumbens, measured in vitro. This increase was in the same range as that found by measurement of DOPAC levels in the nucleus accumbens in vivo, following blockade of DA transmission in the amygdala by local infusion of DA receptor antagonists such as a-flupentixoi or sulpiride 2°. Secondly, the inter-regulation between the a'nygda!a and the nucleus accumbens, demonstrated in the acute studies, was also observed 5 weeks after interruption of DA transmission in the amygdala. In another experiment (unpublished results), increase in DA activity in the nucleus accumbens was observed as long as 13 weeks after lesions of dopaminergic terminals in the amygdala. Thirdly, the biochemical results showed that this inter-regulation phenomenon extended to the forebrain, since lesion of DA terminals in the amygdala redut:ed DA activity in the prefrontal cortex. The anatomical substrate of these inter-regulations remains to be determined. Two main pathways can be involved: one at the level of DA terminals in the fore-
brain, and another terminating in the dopaminergic cell bodies in the ventral mesencephalon. Direct anatomical connections from amygdala to nucleus accumbens 15as'37, to prefrontal cortex ~7and to the ventral tegmental area 21"22 have been demonstrated. However, the involvement of polysynaptic connections cannot be excluded. For example, alteration of DA activity in the amygdala could affect the functioning of DA activity in the nucleus accumbens by interfering with DA pathways in the prefrontal cortex. It has been shown that there is an increase in levels of DOPAC in the nucleus accumbens after local injection of DA antagonists into the cortex 19. This may mimic the qffect of DA lesions in the amygdala. The second main result of this study concerns the behavioral effect of 6-OHDA lesions of the amygdala. Such lesions do not affect the spontaneous locomotor activity of rats, but they do increase the locomotor response to peripheral injection of D-amphetamine. Interestingly, this hypersensitivity to D-amphetamine was only obtained in the group of rats with reciprocal alterations in DA activity in prefrontal cortex and in nucleus accumbens. The reasons why some animals were hyperresponsive to D-amphetamine (AMY +) while others were not (AMY-) cannot readily be explained from the results of the biochemical assays after lesions in the amygdala. Indeed, both groups had the same deficits in DA and NA in the amygdala. It is possible that other neuromediators were affected in different ways by these !e:ions. Since there is a laterality of DA function in the amygdala 2, hemispheric dominance might also account for these differences. The enhanced locomotor response to D-amphetamine in rats with increased DA activity in the n. accumbens is consistent with literature data. Many studies have shown that spontaneous locomotor activity and locomotor response to D-amphetamine are controlled by DA neurons in the n. acccumbens 3'14'16. Further, we have shown in previous studies that manipulations which have opposite effects on DA activity in the n. accumbens (increase) and prefrontal cortex (redaction) - - in similar way that we obtained in the present experiment - - produced locomotor hyperactivity ~12,35 It must also be emphasized that the behavioral effects of disruption of DA in the amygdala are not restricted to alterations in locomotor activity. For ex-
339 ample, we have shown that specific D A lesions in the amgydala increase the acquisition of self-administration of D-amphetamine 6.
Kindling in the amygdala of the rat alters D A transmission in the nucleus accumbens 5"9a°, and also affects l o c o m o t o r activity7"8.z3. It has also been re-
A relationship b e t w e e n D A functions in a m y g d a l a and nucleus accumbens m a y also account for some of the direct effects of d o p a m i n e d e m o n s t r a t e d in animal studies, as well as in some h u m a n autopsy findings. For example, injection of d o p a m i n e in the amygdala of the rat leads to changes in l o c o m o t o r activity 2, which have b e e n t h o u g h t to be m e d i a t e d via an effect on D A t u r n o v e r in the nucleus accumbens.
ported that there are alterations of D A in the amygdala 24 and nucleus accumbens 4 in the brains of schizophrenic patients.
REFERENCES
12 Herv6, D., Simon, H., Blanc, G., Le Moal, M., Giowinski, J. and Tassin, J.P., Opposite changes in dopamine utilization in the nucleus accumbens and the frontal cortex after electrolytic lesion of the median raphe in the rat, Brain Research, 216 (1982) 422-428. 13 Iversen, S.D., Cortical monoamines and behavior. In L. Descarries, T.R. Reader and H.H. Jasper (Eds.), Neurology and Neurobiology, Vol. 10, Monoamine Innervation of Cerebral Cortex, Liss, New York, 1984, pp. 321-349. 14 Iversen, S.D. and Koob, G.F., Behavioral implications of dopaminergic neurons in the mesolimbic system. In E. Costa and G.L. Gessa (Eds.), Nonstriatai Dopamine Neurons. Adv. Biochem. P~ychopharmacol., Vol. 16, Raven, New York, i977, pp. 589-595. 15 Kelley, A.E., Domesick, V.B. and Nauta, W.J.H., The amygdalostriatal projection in the rat. An anatomical study by anterograde and retrogf:'de tracing method, Neuroscience, 7 (1982) 615-630. 16 Kelly, P.H., Seviour, P.W. and Iversen, S.D., Amphetamine and apomorphine response in the rat following 6OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Reseats-h, 94 (1975) 507-522. 17 Krettek, J.E. and Price, J.L., A direct input from the amygdala to the thalamus and the cerebral cortex, Brah~ Research, 67 (1974) 169-174. 18 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 19 Louilot, A., Le Moal, M. and Simon, H., Functional interdependence between the dopaminergic innervation of the frontal cortex and the nucleus accumbens demonstrated by in vivo voltammetry, Neurosci. Lett., Suppl. 26 (1986) $27. 20 Louilot, A., Simon, H., Taghzouti, K. and Le Moal, M., Modulation of dopaminergic activity in the nucleus accumbens following facilitation or blockade of the depaminergic transmission in the amygdala: a study by in vivo differential pulse voltammetry, Brain Research, 346 (1985) 141-145. 21 Maeda, H. and Mogenson, G.J., Electrophysiological responses of neurons of the ventral tegmental area to electrical stimulation of amygdala and lateral septum, Neurosciettce, 6 (1981) 367-376. 22 Phillipson, O.T., Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat, J. Comp Neurol., 187 (1979) 117-144.
1 Blanc, G., HervC D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P., Response to stress of mesocortico-frontal dopaminergic neurons in rats after longterm isolation, Nature (Lond.), 284 (1980) 265-267. 2 Bradbury, A.J., Costall, B., Domeney, A.M. and Naylor, R.J., Laterality of dopamine function and neuroleptic action in the amygdala in the rat, Neuropharmacoiogy, 24 (1985) 1163-1170. 3 Costall, B. and Naylor, R.J., The behavioral effects of dopamine applied intracerebrally to areas of the mesolimbic system, Eur. J. Pharmacol., 32 (1975) 87-92. 4 Crow, T.J., Cross, A.J., Johnson, J.A., Johnstone, E.C., Joseph, M.H., Owen, F., Owens, D.G.C. and Poulter, M., Catecholamines and schizophrenia: an assessment of the evidence. In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engel (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 259-269. 5 Csernansky, J.G., Csernansky, C.A., Bonnet, K.A. and Hollister, L.E., Dopaminergic supersensitivity follow ferric chloride-induced limbic seizures, Biol. Psychiat., 20 (1985) 723-733. 6 Demini~re, J.M., Taghzouti, K., Tassin, J.P., Le Moal, M. and Simon, H., Increased sensitivity to amphetamine and facilitation of amphetamine self-administration after 6-hydroxydopamine lesions of the amygdala, Psychopharmacology, in press. 7 Ehlers, C.L., Koob, G.F. and Bloom, F.E., Post-ictal locomotor activity in three different rat models of epilepsy, Brain Research, 250 (1982) 178-182. 8 Ehlers, C.L. and Koob, G.F., Locomotor behavior following kindling in three different brain sites, Brain Research. 326 (1985) 71-79. 9 Engel, J. and Sharpless, N.S., Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 10 Gee, K.W., Hollinger, M.A., Bowyer, J.F. and Kiilam, E.K., Modification of dopaminergic receptor sensitivity in rat brain after amygdaloid kindling, Exp. Neurol., 66 (',979) 771-777. 11 Gauchy, C., Tassin, J.P., Glowinski, J. and Cheramy, A., Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological samples, J. Neurochem., 26 (1976) 471-480.
In conclusion, the inter-regulation between D A activity in several forebrain structures is an important feature of the functioning of these neurons which could have some importance in the study of biological mechanisms involved in mental disorders.
340 23 Post, R.M., Squillace, K.M., Pert, A. and Sass, W., The effect of amygdala kindling on spontaneous and cocaine-induced motor activity and lidocaine seizures, Psychopharmacology, 72 (1981) 189-196. 24 Reynolds, G.P., Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia, Nature (Lond.), 305 (1983) 527-529. 25 Simon, H. and Le Moal, M., Mesencephalie dopaminergic neurons: functional role. In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engel (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 293-307. 26 Simon, H., Taghzouti, K. and Le Moal, M., Deficits in spatial-memory tasks following lesions of septal dopaminergic terminals in the rat, Behav. Brain Res., 19 (1986) 7-16. 27 Stricker, E.M. and Zigmond, M.J., Brain catecholamines and motivated behavior: specific or nonspecific contributions? In E. Usdin, A. Carlsson, A. Dahlstr6m and J. Engei (Eds.), Catecholamines: Neuropharmacology and Central Nervous System. Theoretical Aspects, Liss, New York, 1984, pp. 259-269. 28 Taghzouti, K., Garrigues. A.M., Labouesse, J., Le Moal, M. and Simon, H., Bovine serum albumin-haloperidol as a tool for the study of dopaminergic transmission: behavioral and neurochemical effect following single injection in the nucleus accumbens, Life Sci., 40 (1987) 127-137. 29 Taghzouti, K., Le Moal, M. and Simon, H., Enhanced frustrative nonreward effect following 6-hydroxydopamine lesions of the lateral septum in the rat, Behav. Neurosci., 99 (1985) 1066-1073. 30 Taghzouti, K., Louilot, A., Herman, J.P., Le Moal, M. and Simon, H., Alternation behavior, spatial discrimination and reversal disturbances following 6-hydroxydopamine lesions in the nucleus accumbens of the rat, Behav. Neur. Biol., 44 (1985) 354-363.
31 Taghzouti, K., Simon, H. and Le Moal, M., Disturbances in exploratory behavior and functional recovery in the Y and radial mazes following dopamine depletion of the lateral septum, Behav. Neut. Biol., 45 (1986) 48-56. 32 Taghzouti, K., Simon, H., Louilot, A., Herman, J.P. and Le Moal, M., Behavioral study after local injection of 6-hydroxydopamine into the nucleus accumbens in the rat, Brain Research, 344 (1985) 9-20. 33 Taghzouti, K., Simon, H., Tazi, A., Dantzer, R. and Le Moal, M., The effect of 6-OHDA lesions of the lateral septum on schedule-induced polydipsia, Behav. Brain Res., 15 (1985) 1-8. 34 Tassin, J.P., Lavielle, S., Herr6, D., Blanc, G., Thierry, A.M., Alvarez, C., Berger, B. and Glowinski, J., Collateral sprouting and reduced activity of the rat mesocortical dopaminergic neurons after selective destruction of the ascending noradrenergic bundles, Neurosci., 4 (1979) 1569-1582. 35 Tassin, J.P., Simon. H., Glowinski, J. and Bockaert, J., Modulations of the sensitivity of dopaminergic receptors in the prefrontal cortex and the nucleus aceumbens: relationship with locomotor activity. In R. Collu, J.R. Ducharme, A. Barbeau, and M.D. Tolis (Eds.), Brain Peptides and Hormones, Raven, New York, 1982, pp. 17-30. 36 Wagner, J., Vitali, P., Palfreyman, M.G.,, Zraika, M. and Huot, S., Simultaneous determination of 3,4 dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3methoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by high performance liquid chromatography with electrochemical detection, I. Neurochem., 38 (1982) 1241-1254. 37 Yim, C.H. and Mogenson, G.J., Response of nucleus accumbens neurons to amygdala stimulation and its modification by dopamine, Brain Research, 239 (1982) 401-415.