Host Brain Regulation of DopaminergicGrafts Function: Role of the Serotonergic

and Noradrenergic Systems inAmphetamine-Induced Responses

ANA MUNOZ, JANNETTE RODRIGUEZ-PALLARES, MARIA JOSE GUERRA, ANDJOSE LUIS LABANDEIRA-GARCIA*

Laboratory of Neuroanatomy and Experimental Neurology, Department of Morphological Sciences,Faculty of Medicine, University of Santiago de Compostela, Spain

KEY WORDS striatum; transplants; dopamine; serotonin; adrenergic; Parkinson;Fos; Prazosin

ABSTRACT The indirect dopaminergic (DA) agonist amphetamine has frequentlybeen used to study functional responses of DA grafted neurons. However, it is not knownif striatal responses, primarily related to DA release by the grafted neurons, aremodulated by the host striatal afferents. We investigated the changes in amphetamine-induced rotational behavior and striatal expression of Fos in DA-denervated and graftedrats subjected to serotonergic denervation and/or treatment with the �1-adrenergicreceptor antagonist Prazosin. Acute serotonergic lesions with p-chlorophenylalaninesuppressed the expression of Fos induced by 1 mg/kg of amphetamine in both the graftedand the contralateral striatum. Chronic serotonergic denervation with 5,7-dihy-droxytryptamine induced a significant reduction in Fos expression in both the graftedand nongrafted striata and a nonsignificant reduction in the contraversive rotation. InDA-innervated striata, Prazosin significantly reduced the expression of Fos but only inthe presence of serotonergic innervation. However, Prazosin did not decrease the ex-pression of Fos induced by grafts located in striata not subjected to serotonergic dener-vation. The present results suggest functional integration of transplanted DA neuronsand major host striatal afferent systems, particularly the serotonergic system, in mod-ulating responses of the host striatal neurons. However, indirect effects exerted by thenoradrenergic system on the normal striatum were not observed in the DA-denervatedand grafted striata. Synapse 47:66–76, 2003. © 2002 Wiley-Liss, Inc.

INTRODUCTION

Fetal dopaminergic (DA) neurons grafted in previ-ously DA-depleted adult striatum survive, partially re-innervate the host striatum, and reverse a number oflesion-induced changes (Lopez-Martin et al., 1999;Nikkhah et al., 1993; Rozas and Labandeira-Garcia,1997). It appears that an alternative source of DAneurons other than primary fetal tissue must be foundin the near future for treatment of Parkinson’s diseaseby nigral grafting (Dunnett et al., 2001; Rodriguez-Pallares et al., 2001). However, independent of thesource of the DA neurons, the degree of functionalintegration of the grafted neurons in the host tissueand the mechanisms involved in their functional effectsmust be completely clarified.

In rats subjected to unilateral DA denervation andthen grafting of DA neurons, administration of theindirect DA agonist amphetamine (AMPH) has been

frequently used to study functional responses of thegrafted neurons. It is well known that AMPH inducesgraft-derived DA release (Strecker et al., 1987; Zetter-strom et al., 1986), contraversive rotation (i.e., in DA-denervated rats showing ipsiversive rotation beforegrafting; Brundin et al., 1987; Lopez-Martin et al.,1999), and induction of Fos in the striatal neuronssurrounding the graft (i.e., in DA-denervated neuronsshowing no AMPH-induced Fos expression beforegrafting; Abrous et al., 1992; Cenci et al., 1992; Laban-

Contract grant sponsors: XUGA and the Spanish DGESIC (PGC).

*Correspondence to: Prof. Jose Luis Labandeira-Garcıa, Facultad de Medicina,Dept. Ciencias Morfologicas, 15782 – Santiago de Compostela, Spain.E-mail: cmlaband@usc.es

Received 29 April 2002; Accepted 23 July 2002

DOI 10.1002/syn.10152

Published online 00 Month in Wiley InterScience (www.interscience.wiley.com).

SYNAPSE 47:66–76 (2003)

© 2002 WILEY-LISS, INC.

deira-Garcia et al., 1996; Lopez-Martin et al., 1999).These changes have been attributed to AMPH-inducedrelease of DA by the grafted neurons, because it isclassically considered that AMPH acts by promotingincreases in extracellular DA in brain regions rich inDA nerve terminals (Carboni et al., 1989; Kuczenskiand Segal, 1989; Uretsky and Snodgrass, 1977). How-ever, a number of studies suggest that DA release isnecessary but by itself not sufficient to produce thecharacteristic effects of AMPH on the striatum andthat other neurochemical systems are also involved.

It has been shown that that AMPH alters noradren-ergic (NA) transmission (Florin et al., 1994; Ramirezand Wang, 1986) and that manipulation of the NAsystem influences AMPH-induced behavior (Archer etal., 1986). The stimulation of cortical �1-adrenergicreceptors is involved in the locomotor-activating effectsof AMPH (Blanc et al., 1994; Darracq et al., 1998) andtransection of the corticostriatal afferents reducesAMPH-induced striatal Fos expression and rotationalbehavior in rats (Cenci and Bjorklund, 1993; Lopez-Martin et al., 1998). It has been recently observed notonly that the NA system is important in AMPH-inducedresponses (i.e., locomotor hyperactivity and striatal ex-pression of Fos), but also that the serotonergic (5-HT)system is necessary for mediating AMPH-induced NAstimulation (Genova and Hyman, 1998; Guerra et al.,1998; Munoz and Labandeira-Garcia, unpubl. obs.). Ithas also been shown that the firing of the raphe 5-HTneurons and their release of 5-HT are subjected to NAfacilitatory influence mediated by �1-adrenergic recep-tors (Baraban and Aghajanian, 1980; Hjorth et al., 1995).

It would be interesting to know if well-known striatalresponses primarily related to the release of DA by thegrafted neurons—such as the expression of Fos by thestriatal neurons surrounding the graft (i.e., in the re-innervation area) and contraversive rotational behav-ior—are modulated by host striatal afferents. It hasbeen observed that transection of corticostriatal affer-ents reduces AMPH-induced contraversive rotationand expression of Fos in grafted striata (Cenci andBjorklund, 1994), as previously observed in the normalstriatum (Cenci and Bjorklund, 1993). However, it isnot known if graft-derived responses are modulated byother major host neurochemical systems such as theserotonergic and noradrenergic systems.

In the study reported here, we investigated thechanges in rotational behavior and striatal expressionof Fos induced by AMPH in DA-denervated and graftedrats subjected to acute or chronic serotonergic dener-vation and/or treatment with the �1-adrenergic recep-tor antagonist Prazosin.

MATERIALS AND METHODSExperimental design

Adult female Sprague-Dawley rats (Letica, Barce-lona, Spain) each weighing about 200 g at the begin-

ning of the experiment were used. All experimentswere carried out in accordance with the EuropeanCommunities Council Directive of 24 November 1986(86/609/EEC). Rats were divided into four groups (A–D). Most of the rats (groups A–C) were subjected tomaximal unilateral DA denervation with 6-hydroxydo-pamine (6-OHDA) and 1 month later received fetalmesencephalic grafts in the denervated striatum. Aftera survival period of 3 months, the rats were dividedinto the following groups. Rats in Group A were in-jected intraperitoneally (i.p.) with Prazosin (0.5 mg/kg,n � 5) or saline (n � 5), and 30 min later withD-Amphetamine (Sigma, St. Louis, MO; 1 mg/kg). Ratsin Group B were subjected to 5-HT lesions with DL-p-chlorophenylalanine (pCPA, see below), then injectedwith Prazosin (0.5 mg/kg, n � 5) or saline (n � 5), and30 min later with D-Amphetamine (1 mg/kg). Rats inGroup C were subjected to serotonergic denervationwith 5,7-dihydroxytryptamine (5,7-DHT, see below)and at least 1 month later injected with Prazosin (0.5mg/kg, n � 5) or saline (n � 5), and 30 min later withD-Amphetamine (1 mg/kg). In Group D, normal rats(n � 10; Group D1) and 6-OHDA-lesioned and non-grafted rats (n � 10; Group D2) were injected withsaline (n � 5 � 5) or Prazosin (n � 5 � 5) and 30 minlater with D-Amphetamine.

6-Hydroxydopamine lesionand transplantation surgery

All surgery was performed under equithesin anes-thesia (3 ml/kg i.p.). Unilateral lesions of the DA sys-tem were performed by injection into the right medialforebrain bundle of 12 �g of 6-OHDA HBr (Sigma) in 4�l of sterile saline containing 0.2% ascorbic acid. Thestereotaxic coordinates were 3.7 mm posterior tobregma, �1.6 mm lateral to midline, 8.8 mm ventral tothe skull at the midline, in the flat skull position (Paxi-nos and Watson, 1986). The solution was injected usinga 5-�l Hamilton syringe coupled to a motorized injector(Stoelting, Wood Dale, IL) at 0.1 �l/min and the can-nula was left in situ for 2 min after injection. Thirtyminutes prior to surgery, rats received desipramine(Sigma, 25 mg/kg i.p.) to prevent uptake of 6-OHDA bynoradrenergic terminals. The efficacy of the lesion wasevaluated using a rotometer (see below): rats showingmore than 200 net contraversive turns per hour afterinjection of 0.05 mg/kg of apomorphine (i.e., maximallylesioned rats; see Hudson et al., 1993) were used andthe lesion confirmed by subsequent immunohistochem-istry (see below).

For nigral grafts, rats received intrastriatal injec-tions of cell suspensions prepared from fetal ventralmesencephalon at 13–14 days of gestation. The piecesof ventral mesencephalon were dissected out and incu-bated in 0.1% trypsin (Sigma), 0.05%DNase (Sigma),and DMEM (Gibco, Grand Island, NY) for 20 min at37°C. The tissue was then rinsed in DNase/DMEM and

HOST REGULATION OF DA GRAFT FUNCTION 67

mechanically dissociated to produce a milky cell sus-pension. This cell suspension was centrifuged at 600rpm for 5 min and the supernatant was carefully re-moved and resuspended in 0.05% DNase/DMEM to thefinal volume required. A total of approximately onemillion viable cells (100,000–150,000 cells/�l) were ad-ministered to each rat using a 10-�l Hamilton syringeat three injection sites (approximately 3 � 2.5 �l): 1)A � 1.8, L � �2.2; V � 4.5; 2) A � 0.6, L � �2.0, V �4.5; 3) A � 0.6, L � �3.2, V � 4.5. The cell viabilityprior to transplantation was 90–95% (estimated byacridine orange/ethidium bromide). For details, seeNikkhah et al. (1993) and Dunnett and Bjorklund(1997).

Lesion of the serotonergic system

Lesion of the serotonergic (5-HT) system was per-formed by injection of DL-p-chlorophenylalaninemethyl ester hydrochloride (pCPA; Sigma; acute lesion,Group-B rats) or 5,7-dihydroxytryptamine (5,7-DHT;Sigma; chronic lesion, Group-C rats). The pCPA (300mg/kg i.p.) was administered 24 h and 1 h before injec-tion of AMPH. The 5,7-DHT was stereotaxically in-jected into the medial raphe and into the dorsal raphenuclei (AP � �7.8; L � 0; V � 8.7 and 6.7, respectively;tooth bar at �5). Each injection consisted of 6 �g of5,7-DHT (weight of free base) dissolved in 3 �l of sterilesaline containing 0.2% ascorbic acid. The solution wasinjected using a 5-�l Hamilton syringe coupled to amotorized injector (Stoelting) at 0.1 �l/min and thecanula was left in situ for 5 min between the twoinjections and after the second injection. Thirty min-utes prior to surgery, rats received desipramine to pre-vent damage to the noradrenergic system. Lesionswere confirmed in Cresyl Violet sections and in sectionsimmunostained for 5-HT (see below).

Behavioral testing

Drug-induced rotation was tested in a bank of eightautomated rotometer bowls (Rota-count 8, ColumbusInstruments, Columbus, OH, USA), which monitor full(360°) body turns in either direction. For each rat, thenet rotation asymmetry score was calculated by sub-tracting the total number of full turns to the left (i.e.,contralateral to the lesion) from the total number of fullturns to the right (i.e., ipsilateral to the lesion) over thetest period.

The rats were acclimatized to the Rota-count for atleast 15 min before drug treatment; turning behaviorwas monitored for 90 min after the injection of D-amphetamine. The results are presented as means �SEM and statistical differences were tested using two-way ANOVA (lesion � Prazosin) followed by post-hocTukey tests (P � 0.05).

Immunohistochemistry

After behavioral testing, the animals were killed bychloral hydrate overdose and then perfused first with0.9% saline and then with cold 4% paraformaldehydein 0.1 M phosphate buffer, pH 7.4. The brains wereremoved and subsequently washed and cryoprotectedin the same buffer containing 20% sucrose and finallycut into 40 �m sections on a freezing microtome.

Series of free-floating sections were processed forFos, 5-HT, and tyrosine hydroxylase (TH) immunohis-tochemistry as follows. After incubation for 1 min 10%normal serum (rabbit serum for Fos and swine serumfor 5-HT and TH) with 0.25% Triton-X-100 in 0.02 Mpotassium phosphate-buffered saline containing 1% bo-vine serum albumin (KPBS-BSA), sections were incu-bated overnight at room temperature with the corre-sponding primary antibody: sheep polyclonal anti-Fosantiserum directed against the N-terminal region ofthe c-fos protein molecule (1:1,000 in KPBS-BSA con-taining 1% normal rabbit serum, 0.25% Triton-X-100,and 0.1% sodium azide; antiserum OA-11-824A, Geno-sys Biotechnologies, UK), rabbit polyclonal antiserumto 5-HT (1:1,500 in KPBS-BSA containing 2% normalswine serum and 0.25% Triton-X-100; Incstar, Stillwa-ter, MN, USA), or rabbit polyclonal antiserum to TH(1:500 in KPBS-BSA containing 2% normal swine se-rum and 0.25% Triton-X-100; Pel-Freez, Rogers, AR,USA). The sections were subsequently incubated firstfor 60 min with the corresponding biotinylated second-ary antibody (Vector, Burlingame, CA, USA; diluted1:200) and then for 90 min with an avidin-biotin-per-oxidase complex (ABC, Vector; diluted 1:100 in KPBS,containing 0.25% Triton-X-100 for Fos). Finally, thelabeling was visualized by treatment with 0.04% hy-drogen peroxidase and 0.05% 3-3 diaminobenzidine(DAB, Sigma).

Quantification of Fos

Fos immunoreactive nuclei were counted with theaid of NIH-Image 1.55 image analysis software (WayneRasband, NIMH) on a Macintosh personal computercoupled to a videocamera (CCD-72, MTI) connected to aNikon Optiphot 2 microscope with a 20� Nikon Apo-plan objective. The thresholding options of the imageanalysis program (size of the particle and optical den-sity) were set so that only cells with unequivocallypositive nuclei were counted; the background was ig-nored. The area to be analyzed was then encircled andthe Fos-positive nuclei counted automatically to obtainthe number of nuclei/mm2. The nuclei were countedblind to the treatment of the animals. We took thosesections containing the nucleus accumbens (at leastthree sections) and those sections containing the cen-tral striatum (at least three sections) from those seriesof each rat processed for Fos and the density (nuclei/mm2) was estimated in the dorsomedial striatum, dor-solateral striatum, and nucleus accumbens.

68 A. MUNOZ ET AL.

The results are presented as means � SEM andstatistical differences were tested using three-wayANOVA (lesion � Prazosin � striatal region), being thethird factor with repeated measures (striatal region:reinnervation area, dorsomedial striatum, lateral stri-atum, and nucleus accumbens), and then by two-wayANOVA (lesion � Prazosin) of each striatal region fol-lowed by post-hoc Tukey tests (P � 0.05). Finally, theeffect of rotation on Fos expression was assessed byintroducing the rotation data as a covariable.

RESULTSNormal rats and rats with unilateral DA

denervation (Group D)

Normal (i.e., nonlesioned; Group D1) rats showedintense striatal TH-immunoreactivity (TH-ir), and se-rotonergic (5-HT) immunohistochemistry revealed that5-HT terminals densely innervated both striata (Fig.1A,C). Injection of AMPH induced intense striatal ex-pression of Fos. The Fos-ir nuclei were not homoge-neously distributed. In the caudal striatum, most of thenuclei were located dorsally and in the central androstral striatum Fos-ir were most densely concentratedin the medial striatal region (Fig. 2). See Labandeira-Garcia et al. (1994) and Lopez-Martin et al. (1999) fordetails.

In rats subjected to maximal DA denervation with6-OHDA (Group D2), TH-ir was practically absent fromthe DA-denervated striatum (Fig. 1D) and there wasloss of TH-ir neurons in the ipsilateral substantianigra. The Fos immunohistochemistry revealed nonsig-nificant AMPH-induced Fos expression in the DA-de-nervated striatum and a significant decrease in Fosexpression in the striatum contralateral to the DA le-sion (P � 0.001 in all striatal regions). Administrationof Prazosin induced a significant decrease in Fos ex-pression in normal rats (P � 0.001 in all striatal re-gions) and in striata contralateral to the DA lesion (P �0.008 in the dorsomedial striatum, P � 0.003 in thenucleus accumbens; Fig. 2). Evaluation of AMPH-in-duced rotational behavior in rats with unilateral DAdenervation (i.e., DA-denervated and nongrafted rats)revealed intense rotation towards the denervated side(i.e., 489 � 71 ipsiversive turns).

Rats with unilateral DA denervationand nigral grafts (Group A)

In rats subjected to DA denervation and graftingthere were numerous TH-ir neurons within the graftand the TH-ir area (i.e., reinnervation area) surround-ing each graft (Fig. 3A,B). Serotonergic immunohisto-chemistry revealed a dense network of 5-HT-ir fibers inthe DA denervated striatum and within the graft. Inaddition, a variable number of 5-HT-ir neurons wereobserved within the grafts. The Fos immunohistochem-istry revealed an intense AMPH-induced expression of

Fos in the area surrounding each graft (i.e., the rein-nervation area; Fig. 4). In the contralateral (i.e., non-DA-denervated) striatum there were dense TH- and

Fig. 1. Serotonin (A,B) and tyrosine hydroxylase (C,D) immuno-reactivity in nonlesioned rats (A,C) and in rats with bilateral seroto-nergic denervation by 5,7-DHT (B) or unilateral dopaminergic dener-vation (D), showing that immunoreactivity has disappeared from thelesioned striata. Scale bar � 1 mm.

HOST REGULATION OF DA GRAFT FUNCTION 69

5-HT-ir. However, the density of Fos-ir nuclei waslower than that observed in the reinnervation area(F1,8 � 13.431, P � 0.006 in the dorsomedial striatum;F1,8 � 29.212, P � 0.001 in the dorsolateral striatum;F1,8 � 20.954, P � 0.002 in the nucleus accumbens) orin normal (i.e., non-DA-denervated; Group D1) rats(F1,9 � 7.746, P � 0.021 in the dorsomedial striatum)injected with the same doses of AMPH (Figs. 2, 4). Theevaluation of the AMPH-induced rotational behaviorrevealed that the intense ipsiversive rotation observedafter the 6-OHDA lesion changed into intense contra-versive (i.e., towards the nondenervated striatum) ro-tation after grafting (Fig. 5).

Rats grafted and subjected serotonergic lesion(Groups B and C)

The data on Fos expression showed statistically sig-nificant interaction between serotonergic lesion andstriatal region (F6,51 � 21.025, P � 0.001). Therefore,the effect of the lesion (serotonergic lesion by pCPA or5,7-DHT) depends on the striatal area studied.

Rats subjected to acute serotonergic lesion withpCPA (Group B) showed bilateral loss of 5-HT-ir in thestriatum, including the striatal area surrounding thegrafts. After injection of 1 mg/kg of AMPH, only a fewFos-ir nuclei were observed in the grafted and non-grafted striata (Figs. 4, 6D). Therefore, rats pretreatedwith Prazosin did not show any further reduction inAMPH-induced expression of Fos in the grafted or thecontralateral striata. Rats lesioned with pCPA alsoshowed contraversive rotation, although it was signif-icantly lower than that observed in control rats not

treated with pCPA (P � 0.002). The contraversive ro-tation of rats pretreated with Prazosin was also lowerthan in control rats (Group A, P � 0.001), although itwas not statistically different from that in rats treatedwith pCPA alone (Fig. 5).

Rats subjected to chronic serotonergic lesion with5,7-DHT (Group C) showed bilateral loss of 5-HT-ir inthe striatum. However, there was a region of graft-derived 5-HT-ir surrounding each graft and a variablenumber of 5-HT-ir neurons within the grafts (Fig.3C,D). After injection of AMPH, there was considerableexpression of Fos surrounding each graft (i.e., in thereinnervation area) and in the contralateral striatum.However, both striata showed a significantly lowerdensity of Fos-ir nuclei than that observed in thegrafted rats not subjected to 5,7-DHT denervation (P �0.001 in the reinnervation area, P � 0.003 in the dor-somedial striatum and P � 0.001 in the dorsolateralstriatum and nucleus accumbens; Figs. 4, 6A,C). Pre-treatment with Prazosin did not induce any furtherreduction in Fos expression (Fig. 4). After 5,7-DHTlesion or 5,7-DHT lesion and pretreatment with Prazo-sin, the contraversive rotational behavior was appar-ently less intense than in grafted rats not subjected to5,7-DHT denervation, although the difference was notstatistically significant (Fig. 5).

Rats treated with Prazosin (Groups A–D)

After treatment with Prazosin, there was a signifi-cantly lower density of Fos-ir nuclei induced by AMPHin normal rats (Group D1; Fig. 2; see above) and in thestriatum contralateral to the DA lesion (Group D2; Fig.2; see above), or in the striatum contralateral to thegraft (Group A; Fig. 4; P � 0.001 in dorsomedial anddorsolateral striatum, P � 0.019 in accumbens) than instriata of the corresponding rats not treated with Pra-zosin.

In the striatum contralateral to the grafts (GroupsA–C) there was a statistically significant interactionbetween serotonergic lesion and Prazosin administra-tion (F2,17 � 6.391, P � 0.009 in the dorsomedial stri-atum; F2,17 � 8.479, P � 0.003 in the dorsolateralstriatum). Therefore, serotonin depletion produced sig-nificant reduction in Fos expression and Prazosin sig-nificantly reduced the expression of Fos in the presenceof serotonergic innervation. However, pretreatmentwith Prazosin did not induce any statistically signifi-cant decrease in Fos expression in rats subjected to5-HT denervation by 5,7-DHT or pCPA injection (Fig.4). In addition, it is interesting to note that there wasno significant Prazosin-induced reduction in Fos ex-pression in the reinnervation area surrounding thegraft, even in the presence of serotonergic innervation(i.e., Group-A controls; Fig. 4).

The contraversive rotational behavior induced by 1mg/kg of AMPH was apparently less intense in ratspretreated with Prazosin, but the difference was not

Fig. 2. Density of Fos-immunoreactive nuclei/mm2 (mean � SEM)induced by 1 mg/kg of amphetamine in the dorsomedial and dorsolat-eral regions of the central striatum and in the nucleus accumbens innonlesioned rats (NL; Group D1) and in the nondenervated striatumof rats with unilateral dopaminergic lesion (UL; nongrafted rats,Group D2), that were pretreated with saline or Prazosin. Means withdifferent letters (a–d) differ significantly. Three-way ANOVA andpost-hoc Tukey test; P � 0.05.

70 A. MUNOZ ET AL.

statistically significant (Fig. 5). The effect of the rota-tion on Fos expression was also tested and the resultsshowed that the rotation does not change the effects ofthe serotonergic lesion and Prazosin administration onthe striatal expression of Fos (P � 0.606).

DISCUSSIONDopaminergic system

In rats with unilateral DA denervation, AMPH in-duces Fos expression in the DA-innervated but not inthe DA-denervated striatum, as well as intense rota-tion towards the denervated side. Grafting of DA neu-rons not only induces contraversive rotation afterAMPH injection, but also intense Fos expression in thestriatal region surrounding the grafts (i.e., the reinner-vation area; Abrous et al., 1992; Brundin et al., 1987;Cenci et al., 1992; Labandeira-Garcia et al., 1996;Lopez-Martin et al., 1999). The density of Fos-ir nuclei

in the reinnervation area is higher than in the con-tralateral striatum and it has been suggested thatgrafts overcompensate the rotational asymmetry andFos expression (Abrous et al., 1992; Cenci et al., 1992).However, the expression of Fos in the contralateralstriatum cannot be used as a control value becausethere is a significantly lower density of Fos-ir nucleithan in normal rats (i.e., rats not subjected to unilat-eral DA denervation). We suggest that this apparentlygraft-induced hyperexpression of Fos and overcompen-sated rotation are also related to the reduced responseto AMPH in the contralateral striatum, probably as aresult of compensatory changes following unilateralDA-denervation (Caruncho et al., 1997; Labandeira-Garcia et al., 1996; Lindefors and Ungerstedt, 1990).

It is classically considered that AMPH acts by pro-moting dose- and time-dependent increases in extracel-lular DA in regions of the brain rich in DA nerve

Fig. 3. Tyrosine hydroxylase (TH; A,B) and serotonin (5-HT; C,D)immunoreactivity (ir) in rats subjected to unilateral dopaminergicdenervation followed by nigral grafts (g). A: Intense TH-ir in thereinnervation area surrounding the grafts (g) and in the contralateral(i.e., nondenervated) striatum. C: After bilateral serotonergic dener-

vation with 5,7-DHT there was a bilateral loss of 5-HT-ir, excepting aregion of graft-derived 5-HT-ir surrounding each graft (g). Highermagnifications of grafts (g) in A and C are shown in B and D,revealing numerous TH (B) and 5-HT (D) immunoreactive neurons(arrows). Scale bars � 1 mm (A,C), 200 �m (B,D).

HOST REGULATION OF DA GRAFT FUNCTION 71

terminals (Dubocovich and Zahniser, 1985; Kuczenskiand Segal, 1989; Uretsky and Snodgrass, 1977). How-ever, a number of studies suggest that the NA systemis involved in the AMPH-induced responses (Archer etal., 1986; Florin et al., 1994; Ramirez and Wang, 1986)and that the corticostriatal (Darracq et al., 1998; Cenciand Bjorklund, 1993) and serotonergic (Genova andHyman, 1998; Guerra et al., 1998) systems modulatethe striatal response to AMPH. Administration ofAMPH therefore appears to be a useful tool for study-ing the degree of functional integration of the graftedneurons.

Corticostriatal system

Regarding the role of the corticostriatal system inthe striatal responses to AMPH, it is interesting to notethat AMPH induces release of NA in the prefrontalcortex (Florin et al., 1994). It has also recently beenreported that stimulation of cortical �1-adrenergic re-ceptors is necessary for the locomotor-activating effectsof AMPH and it was suggested that this stimulationmight either modify glutamic acid release in the stria-tum or act indirectly via DA neurons to increase DArelease (Darracq et al., 1998). It has also been observedthat transection of corticostriatal afferents to DA-in-nervated striata reduces AMPH-induced striatal Fosexpression and rotational behavior (Cenci and Bjork-lund, 1993; Lopez-Martin et al., 1998). Similarly, tran-section of corticostriatal afferents to DA-denervatedand grafted striata reduces the apparent hyperexpres-sion of Fos and the contraversive rotation (Cenci andBjorklund, 1994). Thus, it was suggested that this ap-parent overcompensation may be due to abnormalfunctional interaction between the host corticostriatalinput and the new DA innervation provided by thegraft (Cenci and Bjorklund, 1994). However, it is alsopossible that interaction between the graft-derived DA-innervation and corticostriatal (and serotonergic, inthe present study) terminals are necessary for a nor-mal striatal response and corticostriatal (or serotoner-gic, in the present study) denervation reduces the re-sponse of the striatal cells in the grafted striatum tolevels similar to those observed in the contralateralstriatum (see above).

Serotonergic system

Regarding the role of the serotonergic system in thestriatal responses to AMPH, we have recently observedthat in rats lesion of the 5-HT system significantlyreduces the locomotor hyperactivity and striatal ex-pression of Fos induced by AMPH and that the NAsystem is also involved in these responses. In addition,the 5-HT system appears to be necessary for mediatingthe effects of the AMPH-induced NA stimulation(Genova and Hyman, 1998; Guerra et al., 1998; Munozand Labandeira-Garcia, unpublished observation). Thepresent study shows that the host 5-HT system modu-lates the effects of the graft on the host striatal neu-rons, as previously observed for the corticostriatal sys-tem (Cenci and Bjorklund, 1994).

Administration of pCPA suppressed the expressionof Fos induced by 1 mg/kg of AMPH in both the graftedand in the contralateral striatum. In rats subjected tochronic serotonergic lesions (i.e., 5,7-DHT lesions), weobserved a reduction in Fos expression both in thegrafted and in the nongrafted striatum. However, theeffects were not as intense as those observed followingacute (i.e., pCPA) lesions. This cannot be attributedmerely to the presence of graft-derived 5-HT innerva-tion, which is affected by pCPA and not by the 5,7-DHT

Fig. 4. Density of Fos-immunoreactive nuclei/mm2 (mean � SEM)induced by 1 mg/kg of amphetamine in the reinnervation area sur-rounding nigral grafts and in the contralateral side (i.e., nondener-vated striatum) of control rats (i.e., with DA graft and no 5-HT lesion,Group A) and rats with grafts and pCPA lesion (Group B) or 5,7-DHTlesion (Group C), pretreated with saline or Prazosin. Means withdifferent letters (a–d) differ significantly. Three-way ANOVA andpost-hoc Tukey test; P � 0.05.

Fig. 5. Rotation scores over the 90-min session of control rats (i.e.,rats with DA graft and no 5-HT lesion, Group A) and rats with graftsand pCPA lesion (Group B) or 5,7-DHT lesion (Group C), pretreatedwith saline or Prazosin. Negative scores indicate contraversive turns.Means with different letters (a or b) differ significantly. Two-wayANOVA and post-hoc Tukey test; P � 0.05.

72 A. MUNOZ ET AL.

Fig. 6. Striatal expression of Fos induced by 1 mg/kg of amphet-amine in rats with unilateral dopaminergic denervation and nigralgrafts (g). Amphetamine induced intense expression of Fos-positivenuclei (dots) in the striatal area surrounding the grafts (A). Highermagnification of the graft in A is shown in B; asterisks indicate the

graft–host border. In rats subjected to serotonergic denervation with5,7-DHT there was a significant reduction in density of Fos-positivenuclei surrounding the grafts (C) and Fos expression was practicallysuppressed after pCPA administration (D). Scale bars � 300 �m(A,C,D) and 200 �m (B). cc, corpus callosum.

HOST REGULATION OF DA GRAFT FUNCTION 73

lesion, given that the effect of pCPA on Fos expressionwas also more intense than that of 5,7-DHT lesions inthe contralateral nongrafted striatum and in normalrats (unpubl. obs.). This may be related to compensa-tory mechanisms developed to counteract the effects ofa chronic lesion, since similar effects on Fos expressionwere observed when comparing acute and chronic le-sions of the dopaminergic or the corticostriatal system(Guerra et al., 1998; Liste et al., 1995, 1997; Lopez-Martin et al., 1998, Vargo and Marshall, 1996).

In rats subjected to bilateral lesion of the serotoner-gic system with 5,7-DHT, we observed a slight andnonsignificant reduction in contraversive rotation. Thissuggests that the host 5-HT system interacts with bothhost dopaminergic innervation (i.e., left side) and thegrafted dopaminergic innervation (right side). Smallnonsignificant changes in the rotational behavior maybe related to additional factors, including graft-derived5-HT innervation. Administration of pCPA induced amore marked and statistically significant reduction.This may be related to an additional effect of pCPA onthe grafted 5-HT neurons. However, it may also berelated to a marked decrease in the locomotor activityobserved in rats after pCPA administration (Guerra etal., 1998; Munoz and Labandeira-Garcia, unpubl. obs.).A marked reduction in locomotor activity may lead to areduction in the total number of net turns, which maynot totally reflect a reduction in the asymmetry be-tween the right and the left striatum (see Pycock,1980).

A dense network of 5-HT-ir fibers from the host andthe grafted 5-HT neurons innervated the DA-dener-vated striata and the nigral grafts. It has been shownthat DA denervation induces serotonergic sproutingand rapid hyperinnervation of the ipsilateral striatumin neonates and slow and long-term hyperinnervationin adults (Descarries et al., 1992; Guerra et al., 1997;Rozas et al., 1998; Stachowiak et al., 1984; Zhou et al.,1991). We have previously shown that this hyperinner-vation cannot be prevented or reverted by nigral grafts(Guerra et al., 1997). Similarly, it has been shown thatafter pretreatment of the fetal tissue with 5,7-DHT toeliminate the 5-HT neurons, the host 5-HT fibersdensely innervated the grafts implanted in newbornrats but scarcely (�80%) innervated grafts implantedin adults (Mounir et al., 1994; Pierret et al., 1998).Therefore, the above-mentioned results suggest thatthe host 5-HT terminals interact with the grafted DAneurons mainly in the reinnervation area surroundingthe graft, presumably as the DA and 5-HT terminals inthe normal striatum interact.

Noradrenergic system

Although the striatum is not significantly innervatedby NA afferents (Aston-Jones et al., 1995), it has beensuggested that the stimulatory effects of AMPH on theNA system may be transmitted to the striatal neurons

through the corticostriatal (Blanc et al., 1994; Darracqet al., 1998) and, particularly, the serotonergic system(Genova and Hyman, 1998; Guerra et al., 1998), whichare subjected to NA facilitatory influence. In the non-grafted striatum (i.e., the striatum innervated by thehost substantia nigra), administration of Prazosin sig-nificantly reduced the expression of Fos but only in thepresence of serotonergic innervation. Prazosin did notinduce any additional reduction in striatal Fos expres-sion in 5-HT denervated striata. Similarly, in thegrafted and 5-HT denervated striata, Prazosin did notinduce any further reduction in Fos expression. Giventhat pCPA almost suppressed the expression of Fosinduced by 1 mg/kg of AMPH, administration of Pra-zosin cannot induce any significant reduction in Fosexpression. However, in additional experiments (un-publ. obs.), we injected pCPA-lesioned rats with 5mg/kg of AMPH, which induced a higher number ofFos-ir nuclei in both the reinnervation area surround-ing the grafts (127 � 22 nuclei/mm2) and the contralat-eral striatum (74 � 21 nuclei/mm2 in the dorsomedialregion). Again, these rats showed no significant reduc-tion in Fos expression after pretreatment with Prazo-sin.

Interestingly, contrary to what is observed in thecontralateral (i.e., nongrafted) striatum or in normalrats, Prazosin did not decrease the expression of Fos inthe reinnervation area of grafts located in striata notsubjected to 5-HT denervation. There are several pos-sible ways of explaining why, in contrast to what weobserved in DA-innervated striata, the stimulatory ef-fect of AMPH on the NA system may not significantlyaffect the graft-derived expression of Fos. First, it hasbeen observed that nigral DA neurons exert an inhib-itory control on the raphe 5-HT neurons (Ferre et al.,1994), and this suggest that a DA lesion may lead tohyperactivity of 5-HT neurons, which may further re-duce the activity of the corresponding NA neurons(Haddjeri et al., 1997). Indeed, DA denervation inducesserotonergic sprouting and hyperinnervation of the ip-silateral striatum (see above). The hyperactivity of the5-HT system and possibly hypoactivity of the corre-sponding NA neurons may reduce the effects of Prazo-sin on the expression of Fos in the DA-denervated andgrafted striatum. Second, the 5-HT neurons of the graft(i.e., the graft-derived 5-HT innervation) are not sub-jected to NA input, although their effect on the expres-sion of Fos in the reinnervation area is not clear (seeabove). Third, DA neurons in the nigra are subjected toNA excitatory influence through �1-adrenergic recep-tors (Lategan et al., 1990) and grafted DA neurons arenot subjected to this influence.

CONCLUSION

The present results suggest that there is functionalintegration of the transplanted DA neurons and majorhost striatal afferent systems, particularly the seroto-

74 A. MUNOZ ET AL.

nergic system, in modulating the responses of the hoststriatal neurons. However, indirect effects exerted bythe NA system on the normal striatum were not ob-served in DA-denervated and grafted striata.

ACKNOWLEDGMENT

We thank Prof. J.L. Otero-Cepeda (Department ofStatistics, University of Santiago de Compostela,Spain) for critical review of the statistical analysis.

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