Reprinted from

Brain Research 8i4 (1998) 34-40

Research report

Lewis and Fischer rats: a comparison of dopamine transporter and receptorslevels

Gonzalo Flores, Graham K. Wood, David Barbeau, Rémi Quirion, Lalit K. Srivastava *

Douglas Hospital Research Centre. Departments 01 Psychiatry and Neurology and Neurosurgery. McGi// Univer.\'ity. Montreal. Qc. Canada. H4H J R3

ELSEVIER

BRAINRESEARCH

ELSEVIER Brain Research 814 (1998) 34-40

Research report

Lewis and Fischer rats: a comparison of dopamine transporter and receptorslevels

Gonzalo Flores, Graham K. Wood, David Barbeau, Rémi Quirion, Lalit K. Srivastava .

Abstract

Previous reports have shown that fue inbred strains of rat, Lewis (LEW) and Fischer 344 (F344), differ in several behavioural andbiochernical indices of mesolimbic doparnine (DA) function. Specifically, fuese two strains differ in their behavioural and neurochernicalresponse to novel environments, and acure amphetarnine or cocaine challenge as well as in their susceptibility to addiction. To investigateif differences in DA Dl-like, D2-like, D3 receptors and DA transporter could be correlated with fuese behavioural differences betweenstrains, a comparative autoradiographic study of DA receptors and transporter within fue striatal and accumbal regions was undertaken.We observed strain and region specific differences in binding levels for DA D2-like and D3 receptors and for fue DA transporter.Namely, DA transporter levels in fue striatum, nucleus accumbens and olfactory tubercle of LEW rats were significantly lower than inF344 rats. DA D3 densities in fue shell of fue nucleus accumbens and olfactory tubercle of LEW rats were lower than fue levels found infue F344 rats. Finally, LEW rats have a lower levels of D2-like receptors in fue striatum and fue core of fue nucleus accumbens comparedto F344 rats. These data suggest that differences in DA transporter and DA receptors may in part contribute to differences in DA relatedbehaviour seen between fuese two strains. @ 1998 Elsevier Science B. V. All rights reserved.

Keywords; Lewis rat; Fisher 344 rat; Nucleus accumbens; Doparnine receptor; Doparnine transporter

1. Introduction

The inbred strains of rat, Lewis (LEW) and Fisher 344(F344), differ in behavioural, pharmacological and bio-chemical indices of mesolimbic and nigrostriatal dopaminetransmission. Compared to F344 rats, LEW rats expresshigher novelty-induced locomotor activity and have higherlocomotor and stereotypic responses to the indirectdopamine (DA) agonists, cocaine and methamphetamine[3,8]. Accordingly, it has algO been shown that fue extracel-lular concentrations of DA in fue nucleus accumbens,following cocaine or methamphetamine injection, arehigher in LEW than F344 rats [3]. Moreover, LEW ratsdisplay higher levels of tyrosine hydroxylase in fue ventraltegmental afea, fue origin of nucleus accumbens DA affer-ents [2]. LEW rats algO express lower amounts of neurofil-ament proteins in fue ventral tegmental afea [12], and

higher levels of adenylate cyclase and cAMP-dependentprotein kinases in both fue nucleus accumbens and ventraltegmental area. Accordingly, the concentrations of Giaand G~ in fuese structures are apparently lower [13]. It hasbeen suggested that fuese differences in biochemical pa-rameters may reflect differences in fue functional state offue mesolimbic doparnine system between fuese two strains[13].

Interestingly, LEW and F344 rats not only differ in theaforementioned acute conditions, but also show be-havioural differences in putative animal models of addic-tion or schizophrenia - two neuropsychological disordersin which dysfunction of fue mesolimbic doparnine systemhas been proposed [22,24]. LEW rats have a greaterpropensity to orally self administer cocaine, opioids andalcohol [8,26,28] and demonstrate a greater conditionedplace preference to cocaine and morphine [18] when com-pared to F344 rats. Furthermore, responses to neonatallesions of the ventral hippocampus, which has recentlybeen developed as an animal model of schizophrenia,differ between fuese two strains [20]. Neonatally lesioned

. Corresponding author. Douglas Hospital Research Centre, Neuro-

science Division, 6875 Lasalle Blvd., Verdun, Qc, Callada, H4H lR3.

Fax: + 1-514-762-3034; E-mail: mdls@musica.mcgill.ca

0006-8993/98/$ - see front matter @ 1998 EIsevier Science B.V. AII rights reserved.

PII: SOO06-8993(98)01011-7

Douglas Hospital Research Centre, Departments of Psychiatry and Neurology and Neurosurgery, McGill University. Montreal. Qc, Canada, H4H lR3

Accepted 22 September 1998

G. Flores et al. / Brain Research 814 (1998) 34-40

2.2. Brain processing

F344 (n = 4) and LEW (n = 4) rats were sacrificed bydecapitation and their brains rapidly removed, frozen inisopentane maintained at - 40°C and stored at - 80°C

until use. 15 ¡Lm frozen sections was cut on the corona!plane, using a Leitz cryostat. Sections were collected oncleaned, gelatin-coated microscope slides (foursectionsjslides), thaw-mounted, desiccated under vacuumat 4°C overnight and then stored at - 80°C until fue day of

fue experimento

2.3. Receptor autoradiography

F344 rats postpubertally develop hyper-responsiveness tostress and dopamine agonists, while LEW rats are unaf-fected by similar lesions [20].

By analysing differences in mesolimbic dopamine re-ceptors and transporter in fuese two strains, it mar bepossible to determine what causes fue increased extracellu-lar DA and concomitant increase in locomotor response inLEW rats. Additionally, such results mar help explain theseemingly incongruous result of George et al. [9], whichdemonstrated that amphetamine produces a diminishedeffect in LEW rats compared to F344 rats. These datacould also offer some insights into fue predisposition ofLEW rats to develop addiction or their predisposition notto show hyper-responsiveness following neonatal ventralhippocampallesions [8,20].

Previous studies failed to observe differences in eitherfue maximal density or affinity of DA DI or D2 receptors[9] or D2 receptor rnRNA [21] in fue striatal regions ofLEW and F344 rats. However fuese studies were per-formed using membrane binding homogenates, a techniquethat does not allow examination of mesolimbic subregions.Moreover, fuese studies did not study fue DA D3 receptorsubtype, which is enriched in fue mesolimbic regions, orfue DA transporter, a vital component of the DA systemand fue direct site of action of both methamphetamine andcocaine [4,10,17,25]. Furthermore, a possible reduced levelof the DA transporter in LEW rats is suggested by fueresults of Camp et al. [3], who found that both metham-phetamine and cocaine produced an increased duration offue drug response concomitant with a prolonged increasein DA levels. These findings mar suggest that there arefewer DA transporters in LEW rats, leading to prolongedhalf-life of synaptic DA, similar to fue situation seen indopamine transporter knockout rice [11]. In the presentstudy, we investigated the regional distribution and levelsof DA receptor subtypes (Dl-like, D2-1ike and D3) andDA transporters in fue basal ganglia of LEW and F344 ratsusing quantitative autoradiography. Subregion specific dif-ferences were noted between fuese two strains for the D2and D3 receptors as well as for the DA transporter. Thepossible significance of fuese findings for DA relatedbehaviours are discussed.

2. Materials and methods

2.1. Animals and housing

Nine- to ten-week old male F344 and LEW rats wereused (Charles Rivers, St.-Constant, Quebec). Animals werehoused two per cage in a temperature and humidity con-trolled environment on a 12-12 h light-dark cycle withfree access to food and water. McGill University AnimalCare Committee, in accordance with the guidelines of fueCanadian Council for Animal Care, has previously ap-proved all procedures described in this study.

Coronal brain sections taken at fue level of fue nucleusaccumbens and striatum ( + 1.70 to 2.20 mm from Bregmaaccording to Paxinos and Watson's Atlas [23]) were usedin fue following protocols. According to the protocol ofFlores et al. [5] for Dl-like receptors binding, sectionswere flfst preincubated for 10 min at room temperature inbuffer containing 50 mM Tris-HCl (Sigma, Sto Louis,MO) pH 7.4, 154 mM NaCl, 1 mm EDTA (Sigma) and0.1 % bovine serum albumin (Sigma). Sections were thenincubated for 90 min at room temperature in same bufferwith fue addition of 2 nM [3H]SCH-23390 (74 Ci/mmol;NEN, Boston, MA) and 30 nM ketanserin (RBI, Natick,MA) to eliminate possible binding of fue ligand to seroton-ergic 5-HT2 sites. Non-specific binding was determined onadjacent brain sections by adding 1 11M (+ )-butaclamol(RBI) to the binding buffer. Incubations were terminatedby dipping the slides in ice-cold buffer followed by twoconsecutive 10-min washes in buffer. After a final dippingin ice-cold distilled water, slides were dried at roomtemperature and apposed to [3H]Hyperfilm (Amersham,Oakville, ON) for 5 days, along side calibrated tritiumstandards (Amersham).

For DA D2-like receptor binding, again using the proto-col of Flores et al. [5], slides were first preincubated for 10min at room temperature in buffer containing 50 mMTris-HCl, pH 7.4, 120 mM NaCl, 1 mM EDTA, 5 mMKCl, 1.5 mM CaC12 and 4 mM MgC12. Sections were thenincubated for 2 h at room temperature in fue same buffercontaining 1 nM [3H]YM-09152-2 (86 Ci/mmol; NEN).8-0H-DPAT (50 nM; RBI) was added in each case tomask possible binding of fue ligands to serotonergic 5-HT1a sites. Non-specific binding was determined on adja-cent brain sections by adding 1 mM ( + )-butaclamol in thebinding buffer. Incubations were terminated by dipping fueslides in ice-cold buffer followed by two consecutive 10min washes in the same buffer. After a final dipping inice-cold distilled water, slides were dried at room tempera-ture and apposed to [3H]Hyperfilm for 12 days, along sidecalibrated tritium standards.

[3H]-7-0H-DPAT (Amersham) binding to DA D3 re-ceptors was assessed following the procedure of Levesque

G. Flores et al. / Brain Research 814 (1998) 34-4036

twice for 1 min each in ice-cold 50 rnM Tris-HCl, pH 7.4.After a brief dipping in ice-cold distilled water, brainsections were rapidly dried and apposed to [3H]Hyperfilmfor 6 weeks, along sirle calibrated tritium standards.

2.4. Data analysii

\ Films were developed and then analysed using a com-puterised image analysis system (MCID-4, Imaging Re-search, St.-Catherine, ON). Binding data were analysed inbasal ganglia subregions according to Paxinos and Wat-son' s Atlas [23] namely fue dorsolateral, dorsomedial andventral striatum, fue shell and core of fue nucleus accum-bens and fue olfactory tubercle (Fig. 1), and expressed asfmoljmg wet tissue weight. Data were then compared by aTwo-way ANOV A: F344 and LEW strains of rats byanatomical regions, followed by a simple effects post-hoctest with p < 0.05 being considered significant.

3. Results

Dopamine receptors and transporter binding levels werecompared in subregions of fue basal ganglia of F344 andLEW rats. The distribution of DI receptors was relativelyhomogeneous throughout fue basal ganglia with no signifi-cant differences in DA DI receptor level observed betweenfue two strains under study (Two-way ANOV A, betweenstrains: F¡.36 = 1.875, p = 0.1781; interaction strains xregions: FS.36 = 1.000, p = 0.4379) (Table 1). The striataldistribution of DA D2-like receptors showed a lateral-me-dial and dorsal-ventral gradient, with levels in fue nucleusaccumbens being about half of those seen in fue striatum.F344 and LEW rats exhibited similar levels of DA D2receptors in fue shell of fue nucleus accumbens and olfac-tory tubercle. A Two-way ANOV A revealed that fueTewere significant effects of strain (F¡,36 = 58.40, p <0.0001) as well as an interaction of strain by brain region(FS,36 = 8.654, P < 0.0001). Furthermore, in all three sub-regions of fue striatum (dorsolateral (F¡,36 = 34.58, p <

Olltlc!!)r)' Tuhercle

Fig. 1. Regions of interest used to analyse fue doparnine indices ofFischer 344 and Lewis rats. LSI, interrnediate lateral septal nucleus; Icj,islands of Calleja and Pir, piriforrn cortex. Reproduced from Paxinos and

Watson's Atlas [23].

Table 1Quantitative evaluation oí Dl-like DA receptors in subregions oí fuestriaturn

Dorsolateral striatumDorsomedial striaturnVentral striatumAccumbens coreAccumbens shellOlfactory tubercle

et al. [19] with rninor modifications [5]. Tissue sectionswere first preincubated for 30 rnin in buffer containing 50mM Tris-HCI, pH 7.4, 100 mM NaCI and 300 mM GTP(RBI). Sections were then incubated for 2 h at roomtemperature with 2 nM [3H]-7-0H-DPAT, 50 mM Tris-HCI, pH 7.4, 100 mM NaCI, 300 mM GTP and 5 mMDTG (to block binding to (J" sites). A total of 1 mM DAwas used in adjacent sections to determine non-specificlabelling. Incubations were terminated by washing sectionstwice for 10 rnin each in ice-cold 50 mM Tris-HCI, pH7.4. After a brief dipping in ice-cold distilled water, brillosections were rapidly dried and apposed to [3H]Hyperfilmfor 6 weeks, along sirle calibrated tritium standards.

DA transporter binding was assayed essentially accord-ing to fue method of Kaufman et al. [16]. The sectionswere fIrst preincubated for 20 rnin at 4°C in 50 mMTris-HCI, pH 7.4, containing 100 mM NaCI. Sectionswere then incubated for 2 h at 4°C in fue sarne buffercontaining 10 nM [3H]WIN-35428 (84 Cijmmol; NEN).Non-specific binding was determined on adjacent brillosections by adding 1 mM GBR-12909 (RBI) in fue buffer.Incubations were terminated by washing fue brillo sections

Receptor levels are expressed in fmoljmg of wet tissue and represent tilemean:t S.E.M. from four animals per group of pooled values obtainedfrom four sections on one slide for each animal. Striatal subregionsdefined according to Paxinos and Watson's Atlas [23]. No significantdifferences observed between tile two strains.

G. Flores et al. / Brain Research 814 (1998) 34-40 37

Fischer 344Table 2Quantitative evaJuation of D2-1ike DA receptors in subregions of fuestriatum

Dorsolateral striatumDorsomedial StriatumVentral-striatumAccumbens coreAccumbens shellOlfactory tubercle

Receptor levels are expressed in fmol/mg of wet tissue and represent fuemean:t S.E.M. from tour animals per group of pooled values obtainedfrom tour sections on one slide for each animal. Striatal subregions aredefined according to Paxinos and Watson's Atlas [23]. * p < 0.001;* * p < 0.0001 compared with Pischer 344 rats.

Lewis0.0001), dorsomedial (F1.36 = 17.92, P < 0.001) and ven-tral (F1,36 = 22.91, P < 0.0001) as we11 as the cace of fuenuc1eus accumbens (F¡,36 = 21.93, P < 0.0001) post-hoc

tests revealed that fue level of DA D2 receptors wassignificantly higher in fue F344 compared to LEW rats(Table 2).

The levels of DA D3 receptors were estimated using[3H]-7-0H-DPAT as a ligand and data were similar toprevious reports [5,14,19]. The highest specific labelling isobserved in fue nucleus accumbens and olfactory tuberclewith very low levels in fue striatum (Table 3 and Fig. 2). ATwo-way ANOV A revealed a significant effect of strain(F1.36 = 19.78, P < 0.0001) as well as an interaction ofstrain by brain region (F5,36 = 6.366, p < 0.0001). Morespecifically, post-hoc tests reveal that LEW rats have asignificantly lower levels of DA D3 receptor sites in fueshell afilie nucleus accumbens (F1,35 = 30.49, P < 0.0001)and olfactory tubercle (F1,36 = 29.18, p < 0.0001), whencompared with F344 rats (Table 3 and Fig. 2). In fuestriatum and in fue cace of nucleus accumbens DA D3receptor levels did not differ between strains.

DA transporters, as measured by [3H]WIN-35428 bind-ing, are distributed throughout fue dorsal and ventral stria-

Fig. 2. Digitized photomicrographs of D3 receptor binding in fue brainsof Fischer 344 and Lewis rats. Coronal sections were incubated, asdescribed in Material and Methods, with 2 nM [3H]-7-0H-DPAT. Resultsfrom fue quantitative analysis of D3 receptor binding are presented inTable 3.

tum in a dorso-to-ventral gradient (Table 4). A Two-wayANOV A revealed that fueTe was a significant effect ofstrain (F¡,36 = 77.92, p < 0.0001) but not an interaction ofstrain by brain region (FS,36 = 1.462, p = 0.2128). There-tole, DA transporter is significantly decreased in LEW

Table 3Quantitative evaluation oí D3 DA receptors by autoradiography in subre-gions oí fue striatum

Table 4Quantitative evaluation oí doparnine transporter by autoradiographysubregions oí the striaturn

Dorsolateral striatumDorsomedial striatumVentral striatumAccumbens coreAccumbens shellOlfactory tubercleIslands of Calleja

Dorsolateral striatumDorsomedial striatumVentral striatumAccumbens coreAccumbens shellOlfactory tubercle

Receptor levels are expressed in fmoljmg of wet tissue and represent fuemean:t S.E.M. from four animals per group of pooled values obtainedfrom four sections on one slide for each animal. Striatal subregions aredefined according to Paxinos and Watson's Atlas [23]. * * p < 0.0001,

compared with Fischer 344 rats.

Receptor levels are expressed in fmoljmg of wet tissue and represent fuemean:tS.E.M. from four animals per group of pooled values obtainedfrom four sections on one slide for each animal. Striatal subregions aredefined according to Paxinos and Watson's Atlas [23]. * * p < 0.0001,

compared with Fischer 344 rats.

:1:0.4:1:0.3:1:0.6:1:0.4:1:0.4* *:1:0.4* *

:1:2.4

:1: 1.5 * *

:1: 1.6* *

:1:2.2* *

:1:0.7* *

:1:0.8* *

:1: 1.8* *

G. Flores et al. / Brain Research 814 (1998) 34-4038

Fischer 344

Lewis

Fig. 3. Digitized photomicrographs of fue levels of dopamine transporterbinding in fue striatum and nucleus accumbens of Fischer 344 and Lewisrats. Coronal sections were incubated, as described in material andmethods, in fue presence of ID nM rH]WIN-35428. Results from fuequantitative analysis of (3H]WIN-35428 binding are presented in Table 4.

compared to F344 rats in all regions tested (Table 4 andFig. 3).

4. Discussion

[21] in fue striata1 region of LEW and F344 rats. This isconsistent with OUT resu1ts on DA D1-1ike receptors sincein al1 subregions analysed F344 and LEW rats did notdiffer in [3H]SCH-23390 binding. However, we found thatLEW rats showed 10wer 1evels of DA D2-1ike receptors, asmeasured by [3H]YM-09151-2 binding, in fue striatum andnucleus accumbens compared with F344 rats. These appar-ent discrepancies may relate to use of different radioli-gands (spiperone vs. YM-09151-2) and techniques (homo-genates vs. autoradiography).

As previously stated, fue locomotor response to noveltyor acute injections of psychostimulants, like metham-phetamine and cocaine, is increased in LEW rats [3,8].Since stress results in a marked release of DA in fuenucleus accumbens [1,29] and DA transporters are vital inregulating access to DA [11], it is reasonable to suggestthat fue reduced levels of DA transporters in fue nucleusaccumbens of LEW rats may be associated with fue ob-served increased locomotor response to fue stress of anovel environment. Interestingly, one of the behaviouraleffects of knocking out the DA transporter gene is in-creased novelty-induced locomotion [11]. The increasedlocomotion produced by methamphetamine and cocaine inLEW rats may algo be due to reduced amounts of fue DAtransporter. Both cocaine and methamphetamine inhibitDA transport, which results in greater levels of synapticDA, leading to an enhanced behavioural effect. Therefore,it is proposed that fue lower amounts of DA transporter infue nucleus accumbens of LEW rats relates to their greaterlocomotor response to cocaine or methamphetamine.

A similar argument could be advanced for am-phetamine. However, two results may suggest otherwise.First, and most importantly, amphetamine has been shownto produce an attenuated locomotor response in LEWcompared to F344 rats. Second, amphetamine has recentlybeen shown to rely on fue DA transporter to release DAfrom the neuron, into fue synapse [15]. Therefore, it maybe possible that fue reduced locomotion produced by injec-tions of amphetamine in LEW vs. F344 rats, could resultfrom lower density of DA transporters in neurons of LEWrats.

Alterations in DA D3 receptors levels may algo berelevant when examining differences in locomotor re-sponse to stimulants. It has been reported that systemicadministration of D3 agonists produce a decrease in loco-motor activity [30]. Therefore, decreased levels of DA D3receptors in the nucleus accumbens of LEW rats couldresult in attenuated inhibition of locomotion, resulting in agreater locomotor response to amphetamine. Most likely,the reduced levels of DA D3 and DA transporter in LEWrats together lead to fue enhanced dopamine-related be-havioural effects of LEW compared to F344 rats.

Another behavioural difference that can relate to theresults of this study is that of increased stereotypes inLEW rats [3]. Again the important factor is fue lowerlevels of DA transporters in LEW rats; however, in this

The main finding of fue study is that LEW rats havelower DA transporter levels in fue striatum and nucleusaccumbens compared to F344 rats. Additionally, LEW ratshave lower levels of DA D3 receptors in limbic subre-gions, such as fue nucleus accumbens and olfactory tuber-cle. Finally, LEW rats have lower amounts of DA D2-1ikereceptors throughout fue striatum and in fue core of fuenucleus accumbens. The differences in transporter andreceptor levels could suggest alterations in the geneticelements that regulate DA transporter and receptor expres-sion, possibly explaining fue differences in fue behaviourof these two strains. However, genetically-induced be-havioural differences, which lead to compensation in fuemesolimbic dopamine system, can not be ruled out.

Previous reports did not find any differences in fueamount of DA Dl-like, D2-1ike receptors [9] or D2 rnRNA

G. Flores et al. / Brain Research 814 (1998) 34-40 39

is supported by fue FRSQ, while LKS and RQ areChercheur-boursier and Chercheur-boursier de mérite ex-ceptionel of fue FRSQ.

References

case fue region responsible is most likely fue striatum.Interestingly, DA D2-like receptors are expressed in loweramounts in the striatum of LEW rats compared to F344.This alteration in DA D2 receptor density may be acompensatory mechanism related to increased synaptic DAlevels. In DA transporter knockout mice, a similar situa-tion occurs with DA D2 receptors seemingly attempting tocompensate for increased synaptic DA activity [11].

Genetic factors have been shown to playa critical ro1ein addiction, and the study of genetic animal s models, suchas comparing the propensity of F344 and LEW rats toself-administer drugs, has improved OUT understanding ofgenetic contributions to addiction [7,8]. As mentioned pre-viously, LEW rats demonstrate a predisposition to developdrug addiction while F344 rats exhibit a resistance[6,8,26-28]. OUT data may offer some explanation for fueobserved differences between F344 and LEW rats in theirpredisposition to self-administer stimulants. In this regard,fue changes in DA transporter levels seen between F344and LEW rats are interesting since DA levels within fuenucleus accumbens are intimately linked to drug reinforce-ment [31].

It has also recently been shown that neonatal ventralhippocampallesion induces an enhanced response to stress[5,20] which is thought to be related at least in part to adecrease in DA D3 receptor levels in limbic subregionssuch as fue nucleus accumbens [5]. Neonatal ventral hip-pocampallesion of LEW rats fail to produce any effect onlocomotion while F344 rats exhibit enhanced spontaneousand amphetamine-induce locomotion [20]. The results ofthis study suggest that since LEW rats already have re-duced amounts of DA D3 receptors a further decrease inDA D3 receptors due to neonatal ventral hippocampallesions may not occur.

This study provides further characterisation of inherentdifferences that exist between LEW and F344 rats withrespect to fue mesolimbic DA system. Furthermore, OUTresults suggest that fue increased novelty, metham-phetamine and cocaine-induced locomotion and decreasedamphetamine-induced locomotion of LEW rats could bedue to decreased levels of DA transporters. Finally, theresults suggest that the reduced DA transporter and DA D3receptor amounts in fue nucleus accumbens of LEW ratscan contribute to fue susceptibility to addiction and insen-sitivity to neonatal ventral hippocampal lesion of LEWrats, respectively.

Acknowledgements

This study was supported in part by grants from fueFonds de la Recherche en Santé du Québec (FRSQ). Wethank Dr. Jean-Guy Chabot for help in computer analysisof autoradiographic data and Dr. Joseph Rochford for helpin fue statistical analysis. GF is a postdoctoral fellow of theConsejo Nacional de Ciencia y Tecnologia, Mexico. GKW

í.§b

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