134

Brain Research, 523 (1990) 134-138

Elsevier BRES 24173

Effects of locus coeruious lesions on the release dopa,-nine in the rat nucleus accumbens and determined by intracerebral microdtetysis

as

Andries J. Lategan, Marc R. Marien and Francis C. Colpaert Neurobiology Division, FONDAX-Groupe de Recherche SERVIER, Puteaux (France)

(Accepted 27 March 1990) Key words: Locus ceruleus; 6-Hydroxydopamine;Intracerebral microdialysis;Dopamine release; Caudate nucleus; Nucleus accumbens;

(+)-Amphetamine

Bilateral 6-hydroxydopaminelesions of the rat locus coeruleus (a) depleted forebrain norepinephrine levels by 67%, (b) reduced the basal release of dopamine in the nucleus accumbens and caudate nucleus by 26% and 19%, respectively,and (c) reduced (+)-amphetamine-induced release in the nucleus accumbens and caudate nucleus. The locus coeruleus appears to exert a tonic excitatory influence on striatal and limbic dopamine release in vivo.

While the expression of the cardinal motor symptoms of Parkinson's disease (i.e. hypokinesia, tremor and rigidity) appear to be a result of decreased dopaminergic neurotransmission in the nigrostriatal pathway 15'33, it has been hypothesized7 that the progression of the disease process may be governed by noradrenergic mechanisms originating in the locus coeruleus (LC). Although anatomical evidence in support of this theory is minimal, considerable neuropharmacological data 3 indicate that noradrenergic mechanisms significantly modify locomotor activity and stereotypy, behaviors which are mediated 18 by dopaminergic neurotransmission in the nucleus accumbens (NACC) and the caudate nucleus (CN), respectively16'23"28'32"35. For example, in rats, the locomotor hyperactivity induced by (+)-amphetamine is antagonized by pretreatment with the noradrenergic neurotoxin DSP-427 and the a-antagonist prazosin 11. Similarly, the locomotor response of rats to (+)-amphetamine is inhibited by the a-adrenergic agonist clonidine 25~ 34, at doses which reduce the firing rate of LC neurons 37, regularize cell firing in the ventral tegmental area (VTA) 13, and decrease dopamine (DA) turnover in the striatum 12. Bilateral LC lesions, produced either electrolytically5'21 or chemically with 6-hydroxydopamine (6O H D A ) 5,12'21'38 or DSP-41°, decrease spontaneous locomotor activity5'1°'z°'21'38 and striatal DA turnover ~2 in rats. LC lesions also modify the behavioral responses to DA receptor agonists and antagonists, resulting in in-

creased susceptibilities to the cataleptogenic effects of neuroleptics 19'2°, to stereotypy induced by apomorphine 19'2°, and to locomotor activation produced by putative D2 agonists such as LY 17155521. In addition, in rats with unilateral LC lesions, both apomorphine and amphetamine elicit contralateral circling24'29. Taken together, these studies have suggested that the disruption of neurotransmission originating in the LC reduces a facilitative noradrenergic input to mesolimbic and nigrostriatal dopaminergic pathways. To directly investigate the biochemical basis of this proposed interaction, the present study has examined the influence of the LC on the dynamics of DA release in vivo. The effect of a bilateral LC lesion on the basal and (+)-amphetamine-induced release of endogenous DA was monitored simultaneously in the NACC and CN of the rat using brain microdialysis techniques. A preliminary report of these data has been presented elsewhere 22. Bilateral 6-OHDA lesions of the LC were produced in male Wistar rats (Ico:WI [I.O.P.S. AF/Han], 230-240 g) using stereotaxic procedures described previously21. In sham-treated animals, the injection cannula was lowered to 1 mm dorsal to the LC and no injection was made. At 7 or 8 days after surgery, sham and lesioned rats were run contemporaneously in dialysis experiments. The animals were anesthetized with urethane (1.2 g/kg i.p.) and stereotaxically implanted with microdialysis probes (CMA/10, Carnegie-Medicin, Stockholm) terminating in

Correspondence: A.J. Lategan, Neurobiology Division - P10, FONDAX-Groupe de Recherche SERVIER, 7 rue Ampere, 92800 Puteaux,

France. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V, (Biomedical Division)

135 the left nucleus accumbens (NACC) and right caudate nucleus (CN) at the following coordinates: NACC (2 mm probe), 2.3 mm anterior to bregma, 1.7 mm lateral to midline suture, 7.4 mm ventral from dura; CN (3 mm probe), 1.0 mm anterior, 2.8 mm lateral, 6.0 mm ventral (incisor bar position -2.3 mm). The probes were peffused at 2 #l/min with Ringer's solution (in mM: NaCI 147, KCI 4.0, CaCI 3.4) commencing at between 09.20 and 09.50 h. After a 3.5 h equilibration period, dialysates were collected into ice-cooled microtubes containing 10/~1 of a mixture of internal standard (isoproterenol) and 0.15 mM disodium E D T A in 0.1 M HC1. Three 30 min dialysate samples were collected for basal release measurements. Animals were then injected i.p. with (+)-amphetamine (1.25 mg/kg) and nine 20 min dialysate samples were collected over the subsequent 3 h period (animals' body temperature was maintained at 38 °C). At the end of the experiment, animals were decapitated and portions of the frontal cortex and cerebellum were quickly dissected, weighed and frozen on dry ice prior to monoamine determinations. The remainder of the brain was retained for sectioning, Cresyl violet staining and microscopic examination of both the LC lesion and the dialysis probe placement sites. Monoamine and metabolite levels in the dialysate and tissue samples were determined by HPLC with electrochemical detection using a Waters Catecholamine Analysis System 21. Dialysate samples were injected directly (35 /A) into the HPLC system. Tissue samples were homogenized in 10 vols. of 0.4 M perchloric acid containing 0.15 mM disodium EDTA and internal standard (isoproterenol) and centrifuged at 20,000 g for 10 min. The supernatants were filtered through 0.22 #m pore microfiltration tubes prior to injection. Catecholamine levels were calculated by the peak area ratios of the endogenous substance to the internal standard, using a regression curve derived from authentic standards which had been carried through the sample preparation procedures. Dialysate DA levels in each experiment were corrected for individual probe recoveries. The recovery of DA by the probes, determined in vitro 39 after each experiment was 7-13% and 15-31% for the 2 and 3 mm probes, respectively. Out of a total of 22 rats, data from 9 animals were discarded on the basis of unilateral lesions (n = 2) or improper probe placements (n = 7). Thus, 5 pairs of animals remained (5 LC lesioned and 5 sham treated animals) from which data were compiled. Bilateral 6-OHDA lesions of the LC resulted in a selective depletion of norepinephrine (NE) in the frontal cortex and cerebellum; regional levels were reduced by 67 and 75%, respectively. In comparison, frontal cortical levels of DA and serotonin (5-HT) and their major acid

TABLE I Basal release of dopamine (DA) in the nucleus accumbens (NACC) and caudate nucleus (CN) at 7-8 days following bilateral 6-OHDA lesioning of the LC

Basal release is defined as the average amount of DA measured in 3 consecutive 30 min dialysate samples collected after 3.5 h following the implantationof the microdialysisprobes. Values are corrected for the recovery of DA by the probes in vitro and are presented as the means -+ S.E.M. Five pairs of animals were used. Basal D A release (pg/30 min sample)

Sham control LC lesioned % change

Nucleus accumbens

Caudate nucleus

128.8 + 10 94.8 + 8* -26%

151.4 + 15 122.2 + 14" -19%

*P < 0.05 compared to sham-operated controls, Student's t-test for paired data.

metabolites DOPAC and 5-HIAA, respectively, were not significantly changed (Table II). At 7-8 days following bilateral LC lesions, the basal release of endogenous D A was significantly reduced in the NACC and CN, by 26% and 19%, respectively (Table I). The basal values of D O P A C and HVA were also lower in the LC lesioned animals (data not shown). The time course of (+)-amphetamine-induced DA release in both the NACC and CN is shown in Fig. 1. Following the injection of (+)-amphetamine (1.25 mg/kg i.p.), the levels of DA in the dialysates from both regions increased rapidly. The maximal D A response was evident during the second sample collection period (20-40 min) postdrug. Similar to a previous report 32, the peak effects

TABLE II Monoamine and metabolite levels in the frontal cortex and cerebellum at 7-8 days following bilateral 6-OHDA lesioning of the LC. Values are means + S. E.M. Numbers in parentheses indicate the number of animals per group

DOPAC, dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid. Amine or metabolite level (ng/g wet weight) Sham control (5)

L C lesioned (5)

382 _+19 72 + 8 1113 _+81 18 + 2 665 + 58

124 _+15" 84 _+13 1048 _+93 17 + 1 613 _+65

334 + 15

84 + 10"

% Change

Frontal cortex

Norepinephrine Dopamine Serotonin DOPAC 5-HIAA

-67 + 17 -6 -6 -8

Cerebellum

Norepinephrine

-75

*P < 0.001 compared to sham-operated controls, Student's t-test.

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TIME AFTER D-AMPHETAMINE INJECTION (MINUTES) Fig. 1. Effect of (+)-amphetamine (1.25 mg/kg i.p.) on the in vivo release of endogenous DA in the nucleus accumbens (NACC) and caudate nucleus (CN) of LC lesioned (O O) and sham-operated (11 m) rats. Release values (pg DA/sample) are corrected for the DA recoveries by the microdialysis probes in vitro and are presented as the means + S.E.M. Dialysate samples prior to amphetamine administration are 30 min collections: samples postdrug are 20 rain collections. *P < 0.05 compared to sham controls, Student's t-test for paired data.

elicited by this dose of (+)-amphetamine represented a 21-25 fold increase over the basal DA release rates. After this time, DA release gradually returned towards basal levels but remained significantly elevated for up to 3 h following the drug treatment. (+)-Amphetamine-induced DA release was consistently lower in both the NACC and CN of the LClesioned animals compared to sham-operated controls (Fig. 1), though the difference was statistically significant only during the interval of maximal DA release in the NACC (i.e. a 37% reduction at 20-40 min postdrug, P < 0.05) and between 40-60 min in the CN (i.e. a 21% reduction, P < 0.05). Significance was maintained after the subtraction of basal from postdrug values of release. The present study provides direct in vivo biochemical evidence that the LC exerts a tonic excitatory influence on limbic and striatal DA release and may mediate in part the action of (+)-amphetamine in these systems. Using intracerebral microdialysis to monitor changes in extracellular DA levels within the CN and NACC of the same animals, we observed that bilateral LC lesions significantly reduced the basal release of DA in both regions. This reduction in DA release is consistent with previous pharmacological evidence suggesting that forebrain DA synthesis and turnover in vivo is regulated through changes in noradrenergic neurotransmission 2' 12.3o, and may account for the decrease in spontaneous motor activities observed after LC lesioning s'l°'2°m'38. The decrease in DA release following the LC lesion was not likely due to an indirect effect of the 6-OHDA injections upon midbrain DAergic neurons. Unlike the extensive depletion of cortical NE, the levels of frontal cortical DA, as an index of the integrity of DA neurons 14 originating predominantly in the VTA, were not signifi-

cantly affected by the lesioning procedures. Previous reports that lesioning of the LC augments the behavioral responses of rats to various DA agonist and antagonist drugs have suggested an involvement of supersensitive postsynaptic D2 receptors 19-21'24'29 in mediating these drug effects. A direct examination of this hypothesis with DA ligand binding experiments has not been reported. However, an upregulation of D 2 receptors 9 and an increase in the number of D 2 binding sites in the striatum 26 is known to occur in response to extensive nigrostriatal denervations in which the synaptic concentration of DA is reduced by at least 16-40% (see ref. 1). Thus, an upregulation of postsynaptic D 2 receptors following LC lesioning can be hypothesized on the basis of the 26-19% reductions in basal DA release observed in the present study. Although LC lesions reduced basal DA release in both the NACC and CN, (+)-amphetamine-induced release was also reduced significantly in the NACC and CN, although at different times. However, the release was consistently lower in the LC lesioned animals in both structures. Interestingly, pretreatments with the NE neurotoxin DSP-4 significantly reduced the locomotor hyperactivity but not the stereotypies induced by (+)amphetamine 27. Because of the mediation of these drug-induced behaviors by DA release in the NACC and CN, respectively33, the present results provide furthm evidence to indicate a selective facilitation of limbic and striatal DA neurotransmission by central NE neurons in mediating the locomotor stimulant effects of (+)-amphetamine. Projections from the LC to the substantia nigra (SN), VTA, NACC, but not to the CN, have been described 3' ~7.3~ However, functional studies to determine the

137 n e u r o a n a t o m i c a l sites through which noradrenergic mechanisms might influence D A pathway activity are not unequivocal. F o r e x a m p l e , electrical stimulation of the L C exerts an excitatory effect on cells in the SN 6, and the injection of N E into this region elicits signs of l o c o m o t o r activation 24, yet the systemic injection of the a - a d r e n ergic agonist cionidine does not affect nigral cell firing 37. N E or n o r a d r e n e r g i c drug infusions into the CN or N A C C have been r e p o r t e d to either increase 4"~6, decrease 36, o r have no effect 8"24"28 on l o c o m o t o r behav-

iors. F u r t h e r m o r e , the influence of the LC on D A transmission m a y be m e d i a t e d indirectly via synaptic relays with o t h e r transmitter systems in the cortex and raphe nucleus 3"12"31. Microdialysis e x p e r i m e n t s in combination with regional drug injections m a y be a useful a p p r o a c h to further investigate the organization o f the functional circuitry underlying the role of the LC in modulating D A transmission (e.g. release in the mesostriatal and mesolimbic systems).

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