Denervation of the locus coeruleus projections by treatment with
the selective neurotoxin DSP-4[N (2-chloroethyl)-N -ethyl-2-bromobenzylamine]reduces dopamine release potential in the
nucleus accumbens shell in conscious rats
Riina Ha ¨idkind a ,Toomas Kivastik b ,Marika Eller a ,Ivo Kolts c ,
Lars Oreland d ,Jaanus Harro a,*
a
Department of Psychology,Centre of Behavioural and Health Sciences,University of Tartu,EE-50410T artu,Estonia
b
Department of Pharmacology,University of T artu,EE-50410Tartu,Estonia c
Department of Anatomy,University of T artu,EE-50410Tartu,Estonia
d
Department of Neuroscience,Pharmacology,University of Uppsala,Biomedical Centre,Uppsala,Sweden
Received 14June 2002;received in revised form 5July 2002;accepted 18July 2002
Abstract
Pretreatment with DSP-4,a neurotoxin highly selective for the locus coeruleus (LC)noradrenergic projections,2weeks before in vivo microdialysis in conscious rats had no effect on baseline extracellular dopamine (DA)levels in the nucleus accumbens shell,but reduced dose-dependently the dopamine response to depolarisation induced by 50mM KCl.DA metabolism in the frontal cortex,as measured ex vivo,was increased in animals treated with a low (10mg/kg)but not with a high dose (50mg/kg)of DSP-4,possibly indicating an increased sensitivity to stress in these animals and thus suggesting differential regulation of DA in the forebrain by the LC lesions.The reduced DA release potential in the nucleus accumbens after DSP-4treatment suggests that weakening of the LC input to DA nerve cells contributes to motivational deficits.q 2002Elsevier Science Ireland Ltd.All rights reserved.
Keywords :Nucleus accumbens;Frontal cortex;Dopamine;DSP-4[N (2-chloroethyl)-N -ethyl-2-bromobenzylamine];Locus coeruleus;In vivo microdialysis
Dopaminergic (DA-ergic)neurotransmission,particu-larly DA release in the nucleus accumbens,has been impli-cated in the mechanism of action of addictive drugs [1,4],and reduction in DA-ergic neurotransmission in the accum-bens may serve as the basis for anhedonia and loss of energy in depression [6].It is well established that there is a nora-drenergic (NA-ergic)stimulatory input from the locus coer-uleus (LC)to the mesencephalic DA-ergic cell groups (see ref.[6]for references),which provide DA-ergic innervation to forebrain regions.There is some evidence that lesions of the LC projections can be a reason for a reduced DA release in the accumbens.Thus,in anaesthetised rats,a 28%reduc-tion in basal DA overflow was observed using in vivo microdialysis in the nucleus accumbens after pretreatment with DSP-4,a selective NA-ergic neurotoxin [13].This
study also observed a reduction in extracellular DA in the striatum after DSP-4treatment.On the other hand,we have previously been unable to see any change in either basal or KCl-induced DA release in the frontal cortex after DSP-4pretreatment [12].In a recent study in conscious rats no decrease in the extracellular DA in the nucleus accumbens core was found after DSP-4treatment [10].The aim of the present investigation was to study whether partial or nearly complete denervation of the LC by DSP-4tr
eatment influ-ences DA overflow in the nucleus accumbens shell in conscious animals.
Male Wistar rats (300–400g,Kuopio,Finland)were housed individually under 12h light/dark cycle (lights on at 07:00h)with food and water available ad libitum.DSP-4[N (2-chloroethyl)-N -ethyl-2-bromobenzylamine](Astra,Sweden)was administered as a single dose of 10or 50mg/kg (expressed as for hydrochloride)intraperitoneally (IP)at the start of individual housing.Each dose was weighed separately,dissolved in distilled water and imme-
Neuroscience Letters 332(2002)
79–82
0304-3940/02/$-see front matter q 2002Elsevier Science Ireland Ltd.All rights reserved.PII:S0304-3940(02)00817-0
www.elsevier/locate/neulet
*Corresponding author.Tel.:1372-7-375-911;fax:1372-7-375-900.
E-mail address:jharro@ (J.Harro).
diately injected.Control animals received an injection of distilled water.After the administration of DSP-4the animals ’body weight was monitored daily.Surgery was carried out 1week after the toxin treatment.The animals were anaesthetised with chloral hydrate (350mg/kg IP)and mounted in a Kopf stereotactic frame.Guide cannulas for the microdialysis probes (MAB 6;AgnThos AB,Sweden)were implanted above the left nucleus accumbens shell according to the following coordinates relative to bregma:AP:11.7,ML:11.2,and DV:22.8(the final DV coordi-nate after probe insertion 28.2;according to Paxinos and Watson [15]).Three stainless steel screws and dental acrylic was used to fix the cannula to the scull.After the surgery the animals were given 1week to recover.During this period the rats were handled and weighed daily.
Microdialysis was conducted in awake freely moving rats.In the morning of the experiment day (at about 08:00
h)the animals were transported to a separate experiment room.The animals remained in their individual home cages throughout the experiment,though the metal cage covers were replaced with plastic ones having an open ceil-ing.Microdialysis probes with 2mm active polyethersul-phone membrane,cut-off 15kD (MAB 6,AgnTho ’s AB,Sweden)were connected to a syringe pump (World Preci-sion Instruments,USA)and a CMA/142microsampler (CMA,Sweden)via a two-channel swivel.Probes were inserted in the morning of the day of experiment and perfused with arti ficial cerebrospinal fluid (147mM NaCl,3.0mM KCl,1.3mM CaCl 2,1.0mM MgCl 2,1mM Na 2H PO 4,and 0.2mM NaH 2PO 4;pH 7.3–7.4)at a constant rate 1m l/min.After the probe insertion the perfusate was discarded during the first 120min.This was followed by collection of 15dialysate fractions (each for 20min)into the vials pre filled with 10m l of 0.3M perchloric acid.After the tenth sample the system was switched to the perfusion solu-tion containing 50mM KCl and left so for 40min.Upon completion of the experiment the animals were deeply anaesthetised with chloral hydrate (350mg/kg IP)and decapitated;the brains were removed,immediately frozen in ice cold acetone,and kept at 2808C.The brains were sectioned in a cryostatic microtome (Microm GmbH,Germany)after the whole frontal cortex had been taken for separate analysis;the probe placements were determined according to the atlas by Paxinos and Watson [15]and data of animals with probe placements outside the nucleus accumbens shell were excluded from the analysis.
The quantity of DA in the samples was determined by high-performance liquid chromatography with electroche-mical detection.The chromatography system consisted of Hewlett Packard series 1100pump and autosampler,a LiChrospher 100RP-18column (250£3mm,5m m),an ESA 5021conditioning cell (1300mV),an ESA 5011analytical cell (first electrode 2300mV,second electrode 1400mV)and an ESA Coulochem II controller unit.The mobile phase composition was:0.05M sodium citrate buffer,pH 5.5,0.02mM EDTA,2mM sodium octylsupho-nate,0.3mM triethylamine,12%methanol.The content of monoamines and their metabolites in brain tissue was deter-mined as previously described [9].After exclusion of animals with incorrect placement of microdialysis probes or incomplete biochemical data due to sampler failures,the number of animals in analysis was 18(control n ¼5,DSP-410mg/kg n ¼8,DSP-450mg/kg n ¼5).Repeated measures analysis of variance (ANOVA)was used,followed by a post-hoc Fisher ’s analysis per time point.As shown in Fig.1,no signi ficant effect of LC denerva-tion by DSP-4pretreatment was observed on basal extra-cellular DA in the nucleus accumbens shell in conscious rats.Nevertheless,local depolarisation elicited by perfusion with 50mM KCl was affected by this treatment,as revealed by signi ficant two-way ANOVA sample £treatment inter-action (F ð2;28Þ¼3:51,P ,0:0001).Post-hoc Fisher ’s protected least signi ficant difference tests revealed that the
R.Ha ¨idkind et al./Neuroscience Letters 332(2002)79–82
80Fig.1.Effect of DSP-4on DA levels in the nucleus accumbens
shell in conscious rats.The values (mean ^SEM)are expressed as fmol/25m l of sample.Solid bar indicates infusion of 50mM KCl.For statistical evaluation of the data,see text.
Table 1
Effect of DSP-4on monoamine levels in the rat frontal cortex a
Vehicle
DSP-4(10mg/kg)DSP-4(50mg/kg)NA    2.75^0.54  2.38^0.240.72^0.18b,c DA 0.53^0.120.59^0.110.52^0.09HVA 0.20^0.020.28^0.03d,e 0.20^0.02DOPAC 0.12^0.020.19^0.020.14^0.025-HT    2.45^0.55  2.47^0.22  2.57^0.265-HIAA
2.91^0.37
2.98^0.43
3.02^0.26
a
The values (mean ^SEM)are expressed as pmol/mg wet weight tissue.b
Signi ficant difference from the Vehicle group,P ,0.001;post-hoc Fischer ’s PLSD test after signi ficant ANOVA.c
Signi ficant difference from the DSP-410mg/kg group,P ,0.005;post-hoc Fischer ’s PLSD test after signi ficant ANOVA.d
Signi ficant difference from the Vehicle group,P ,0.05;post-hoc Fischer ’s PLSD test after signi ficant ANOVA.e
Signi ficant difference from the DSP-450mg/kg group,P ,0.05;post-hoc Fischer ’s PLSD test after signi ficant ANOVA.
peak extracellular DA was significantly(P¼0:011) reduced after the higher(50mg/kg)dose of DSP-4,whereas the effect of the lower dose(10mg/kg)missed the conven-tional level of significance(P¼0:076).DSP-4treatment reduced NA levels in the frontal cortex(Fð2;13Þ¼10:8, P,0:005).However,only the higher dose reduced NA levels significantly(Table1).DSP-4pretreatment had
no effect on cortical levels of5-hydroxytryptamine(5-HT)and DA,but there was a significant effect on homovanillic acid levels(Fð2;13Þ¼4:71,P,0:05).Thus,the lower but not the higher dose of DSP-4increased HVA levels in the fron-tal cortex.
DSP-4is a noradrenergic neurotoxin highly specific to the nerve terminals originating from the LC[3,16],while it has no direct effect on DA-or5-HT-ergic neurons[11].DSP-4does not affect tissue DA levels but both DA utilisation and3,4-dihydroxyphenylacetic acid production rates have been found to be lower after DSP-4treatment[2,5].There is also striatal DA D2receptor upregulation as well as behavioural supersen-sitivity to the effects of amphetamine after DSP-4treatment [8].In contrast to thefindings of Lategan et al.[13]in anaes-thetised rats,a recent study in conscious animals did notfind any reduction of extracellular DA in the nucleus accumbens core after DSP-4treatment[10].Similarly,we did notfind any significant reduction of extracellular DA in the nucleus accumbens shell in conscious rats.As determined by DA over-flow,induced by local depolarisation,there is,however,a significantly lower DA release potential in LC-denervated animals,which may translate into lower DA release in the nucleus accumbens in response to stimuli activating the LC. Chemical denervation of the LC projections by DSP-4treat-ment reduced the effectiveness of positive reinforcers[14]. Thus it is conceivable that weakening of the LC input to DA nerve cells,which results in diminished DA output in t
he accumbens,contributes to motivational deficits,occurring, e.g.in depression[6].On the other hand,as low basal extra-cellular DA in the nucleus accumbens shell has been also associated with peak drug-seeking behaviour[4],the possibi-lity that functional weakness of the LC contributes to drug addiction(which is frequently associated with depression) should be examined.
Unexpectedly,there was evidence for an increased DA metabolism in the frontal cortex after the lower dose of the neurotoxin.Increases in frontal cortical HVA have been suggested to be strongly indicative as a reaction of stress [17,18],which has previously also been observed in this laboratory[9].We have previously found that DSP-4treat-ment has a biphasic effect in rats submitted to forced swim-ming:10mg/kg of DSP-4increased immobility but50mg/ kg rather reduced this measure[7].It is thus tempting to speculate that partial LC denervation(10mg/kg DSP-4) renders animals more susceptible to stress.Nevertheless, such an interpretation as well as the idea that the effect of LC lesions on DA-ergic neurotransmission is different for the mesolimbic and the mesocortical pathways would require specifically designed experiments which control for stress and compare both DA release and metabolism in these respective projection areas in parallel.
This work was supported by grants from the Estonian Science Foundation(No4531),the Estonian Ministry of Education(No0814),and the Swedish Academy of Sciences.Prof.Svante Ross(Astra)kindly p
rovided DSP-4.We are grateful to Prof.Tomas Ho¨kfelt for support in setting up the microdialysis lab and Ms Margit Kirk for skilful technical assistance.
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