www.elsevier.com/locate/ynbdi

Neurobiology of Disease 19 (2005) 301–311

Haloperidol treatment reverses behavioural and anatomical changes

in cocaine-dependent mice

C.L. Parish,a J. Drago,b D. Stanic,a E. Borrelli,c D.I. Finkelstein,b and M.K. Horneb,TaDepartment of Medicine, Monash University, Monash Medical Centre, Clayton 3168, AustraliabHoward Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville 3010, AustraliacInstitut de Genetique et de Biologie Moleculaire et Cellulaire, B.P.163. 67404 Illkirch Cedex, France

Received 3 August 2004; revised 9 October 2004; accepted 12 January 2005

Available online 19 February 2005

Abnormal dopamine (DA) transmission occurs in many pathological

conditions, including drug addiction. Previously, we showed DA D2

receptor (D2R) activation results in pruning of the axonal arbour of DA

neurones that innervate the dorsal striatum. Thus, we hypothesised

that long-term D2R stimulation through drugs of addiction should

cause arbour pruning of neurones that innervate the ventral striatum

and thus reduce DA release and contribute to craving. If so, D2R

blockade should return these arbours to normal size and may

overcome craving. We show that long-term treatment with a D2R

antagonist (haloperidol) reverses behavioural and anatomical effects of

cocaine dependence in mice, including relapse. This change in arbour

size reflects new synapse formation and our data suggest this must

occur in the presence of increased DA activity to reverse cocaine-

seeking behaviour. These findings hold significant implications for the

understanding and treatment of cocaine addiction.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Addiction; Dopamine; Cocaine; Haloperidol; Stereology;

Behaviour; HPLC

Introduction

Dopaminergic neurones in the ventral tegmental area (VTA)

and substantia nigra pars compacta (SNpc) project onto the nucleus

accumbens (NAc) and dorsal striatum, respectively, and have an

essential role in reinforcement of both natural rewards and those

associated with addictive drugs (Arroyo et al., 2000; Hyman and

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.01.009

Abbreviations: Ac, anterior commissure; C + H, cocaine and

haloperidol; DAB, diaminobenzidine; DAT, dopamine transporter; DOPAC,

dihydroxphenylacetic acid; D1R, D1 dopamine receptor; D2R, D2

dopamine receptor; D2(�/�), D2 dopamine receptor knockout; D3R, D3

dopamine receptor; D3(�/�), D3 dopamine receptor knockout; NAc,

nucleus accumbens; SNpc, substantia nigra pars compacta; TH, tyrosine

hydroxylase; TT, terminal tree; VTA, ventral tegmental area.

* Corresponding author.

E-mail address: m.horne@hfi.unimelb.edu.au (M.K. Horne).

Available online on ScienceDirect (www.sciencedirect.com).

Malenka, 2001; Wise, 1996). Factors that maintain addiction are

not fully understood. However, marked reduction of DA release

seems to be a common feature of drug withdrawal (Rossetti et al.,

1992), suggesting that long-term exposure to a drug suppresses

basal DA-mediated activity in order to dbalanceT chronic stimu-

lation by this drug. This reduction in DA release that follows

chronic drug use is not restricted to the acute withdrawal phase but

is present long after termination of chronic use (Diana et al., 1996).

Cocaine binds with high affinity to the DA transporter (DAT),

the membrane carrier responsible for transporting DA back into the

nerve terminal. In clearing DA from the synapse, DAT ultimately

determines the concentration of synaptic DA available for receptor

stimulation. Therefore, blockade of the DAT results in increased

synaptic DA and activation of DA receptors. Recently, we showed

in the dorsal striatum, that the D2R regulates DA axonal arbour,

and that direct or indirect D2 agonists (e.g., cocaine) results in

pruning whilst D2R antagonists (haloperidol) caused axonal

sprouting (Parish et al., 2002b). Cocaine-induced pruning of the

axonal arbour resulted in reduced density of DA terminals,

normalising post dose DA levels in the synaptic cleft. More

importantly though, striatal DA levels between cocaine doses

would be low and may thereby explain craving and lead to drug-

seeking behaviour. If a similar effect were present in the ventral

striatum and NAc, it may contribute to habituation to drug effect.

We therefore wished to establish whether drug-seeking beha-

viour was associated with reduced striatal dopamine as a con-

sequence of axonal arbour retraction. If so, then reversal of arbour

pruning with a D2R antagonist might reverse drug-seeking

behaviour despite ongoing cocaine intake. Our original observa-

tions of the effect of pharmacological manipulation of the D2R on

arbour size were made in the dorsal striatum. The effects of

addictive drugs are thought to be mediated by altered neuro-

transmission in the ventral striatum, NAc and amygdala, where the

D3R may be the more relevant autoreceptor (Broderick and

Piercey, 1998; Ellinwood et al., 2000). Hence, this study also

addresses whether a similar pruning of terminal arbours occurs in

the ventral striatum and NAc in response to cocaine and whether

the D3R has a significant role in regulating arbour size.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311302

Materials and methods

We examined 210 adult C57BL/6 male mice and 6 D2 and 6 D3

receptor deficient mice [D2(�/�) and D3(�/�), respectively]

(Accili et al., 1996; Baik et al., 1995). The heterozygous D2 and

D3 mice, originally in a hybrid C57/BL6 and 129/Sv genetic

background had been backcrossed for 5 generations into a C57/

BL6 background. All methods conformed to the Australian

National Health and Medical Research Council published code

of practice for animal research. Animals were administered the

following drug doses (alone or in combination, Supplementary

information Table 1): saline (0.25 ml), cocaine (25 mg/kg,

Southern Healthcare Network Pharmacy, Clayton, Australia) and

haloperidol (2.5 mg/kg, Serenace, Searle Laboratories, Australia).

Cocaine self-administration

Animals were housed individually and drug treatments were

delivered in the drinking water for up to 24 weeks, refer Sup-

plementary Fig. 1. To prevent overdosing, each animal was res-

tricted to 10 ml of fluid over 2 days and fluid consumption was

monitored daily to ensure animals were adequately hydrated and

receiving appropriate drug doses. Spigots delivering cocaine (or

cocaine + haloperidol) had a ddabT of peppermint oil on the top to

establish an association between cocaine and the odour. To estab-

lish whether co-treatment with haloperidol would restore arbour

size and ameliorate cocaine-seeking behaviour, animals previously

treated with cocaine for 8 weeks treatment were subsequently

treated with cocaine + haloperidol (C + H) for a further 8 weeks.

To assess the effect of co-treatment of cocaine with haloperidol on

propensity to relapse, daily drug treatment was withdrawn after 8

weeks in a subset of these animals and cocaine preference was

reassessed after a further 8 weeks receiving just water. Cocaine

preference was assessed at the end of each treatment regimen by

depriving animals of fluids for 8 h and then providing a choice of

two drinking spigots, cocaine (+peppermint) or water, for 24 h

and determining the volume consumed from each spigot. Animals

were then returned to their previous drinking source for 24 h and

retested to confirm preferences, with mean values for the two tests

for each animal being compared between groups.

Identification of DA terminals and neurones

Terminal arbour size of SNpc and VTA neurones was assessed

in animals following a number of treatment regimes as well as in

D2 and D3(�/�) animals. Cocaine was administered at the same

time daily via i.p. injection, and haloperidol was administered via

drinking water. Treatment was maintained for 2 months before

killing animals for neuroanatomical analysis. Immunohistochemi-

cal reactivity against DAT was used to identify DA varicosities

within the striatum and NAc to determine terminal density (rat anti-

DAT, 1:3000, Chemicon). DA cell bodies in the SNpc and VTA

were identified using tyrosine hydroxylase (TH) immunohisto-

chemistry (mouse anti-TH, 1:1000, Boehringer Mannheim) as

previously described (Parish et al., 2001).

Fractionator design for estimating the number of SNpc and VTA

neurones

The total number of SNpc and VTA cells, and the proportion of

cells that were TH immunoreactive were estimated using a

fractionator sampling design (Finkelstein et al., 2000; West et al.,

1991). Boundaries of the SNpc were delineated on neutral red

stained sections as previously described (Parish et al., 2001). The

VTA, which lies rostro-medially to the SNpc was distinguished

from the SNpc by its smaller (13 Am), less densely packed cells.

Figs. 1A and C illustrate the boundaries of the SNpc and VTA.

In each of the sections sampled, SNpc or VTA neurones were

counted, using the nuclei of stained cells as the counting unit

according to optical dissector rules (Gundersen et al., 1988).

Neutral Red or tyrosine hydroxylase counts of SNpc neurones were

made on alternate coronal sections. A 45 Am � 35 Am counting

frame, placed at 170 Am � 170 Am intervals was used to count

SNpc neurones (Parish et al., 2001). TH-ir VTA neurones were

counted at the following intervals (x = 170 Am, y = 170 Am) and

with a 35 Am � 25 Am counting frame.

After all sections from an SNpc or VTA were analyzed, the

fraction of the area of the sections sampled was calculated (West et

al., 1991, 1996). The area sampling fraction is obtained by dividing

the area of the counting frame by the area of the distance between

sampling regions, i.e., x and y intervals. As detailed above for the

SNpc, the x and y intervals in sections were both 170 Amand the area

of the counting frame was 1575 Am2. Therefore, the area sampling

fraction is 1575/(140� 140) = 0.0804. The total number of neurones

in the SNpc was estimated by multiplying the number of neurones

counted within the sampled regions with the reciprocals of the

fraction of the sectional area sampled and the fraction of the section

thickness sampled.

Fractionator design for estimating density of DAT-ir varicosities

The most rostral 2.5 mm of the striatum was sectioned and

examined. DAT-ir terminal density was determined in the dorsal

striatum, ventral striatum and nucleus accumbens. Ideally, arbour

size would be obtained by dividing the total number of DAT-ir

terminals in the dorsal CPu (obtained by multiplying DAT-ir

density by volume of dorsal CPu) by the actual number of TH-

ir neurones counted in the SNpc. However, the precise volume of

the dorsal tier innervated by SNpc neurones cannot be delineated.

The volume of the CPu is the same in all animals, and we assume

that the volume of the dorsal tier innervated by SNpc neurones is

the same in all groups. As previously shown (Parish et al., 2001),

when volume is constant, density will be proportional to total

number of terminals. Thus, the average terminal arbour size will be

density divided by cell number, multiplied by K (where K is a

constant). However, K is unknown, so density divided by cell

number provides an accurate but proportional representation of

average tree size. Thus, the important step is to precisely define a

sampling region of known and constant volume for determining

density of DAT terminals in the CPu. A similar argument is

advanced for the ventral striatum and NAc. The dorsal striatum

sampling region was defined as the most dorsal 400 Am of the

striatum. The ventral striatal region was an area 800 Am wide by

400 Am high lying dorsal to the dorsal surface of the anterior

commissure. The nucleus accumbens region was a 100-Am zone

surrounding the anterior commissure showing DAT labelling,

beginning at the level where striatum was first visible to a point

1000 Am caudal, refer to Figs. 1B and D (selections areas based on

Franklin and Paxinos, 1997).

DAT positive terminals were identified as predominantly round

swellings in association with axonal processes. Total terminal

numbers (DAT number) were estimated as described for counts of

Fig. 1. (A) Illustration of the boundaries of the SNpc (dark grey) and VTA (light grey); 3.2 mm posterior to bregma. (B) Schematic representation of the

sampling regions of the dorsal striatum (dashed lines), ventral striatum (striped) and nucleus accumbens (grey region), refer to Materials and methods section

for detailed delineations. Ctx: cortex, Hp: Hippocampus, cc: corpus callosum, Lv: lateral ventricle. Adapted from Franklin and Paxinos (1997). (C) Micrograph

of TH-ir VTA and SNpc neurones, scale bar = 250 Am. (D) DAT staining in the striatum and NAc from a wildtype mouse. (E) DAT-labelled terminals in the

dorsal striatum, scale bar = 20 Am.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311 303

SNpc neurones (above). The striatum and NAc was sectioned at 16

Am thickness with 240 Am between sections. Again, the entire z-

dimension was sampled. Counts were made at regular predeter-

mined intervals (dorsal striatum, x = 170 Am, y = 170 Am; ventral

striatum, x = 150 Am, y = 120 Am and NAc, x = 65 Am, y = 65 Am)

using a counting frame of known area (5 Am � 4 Am = 20 Am2).

Refer to Parish et al. (2001) for detailed methods. Terminal density

was expressed as the number of terminals per volume of region

sampled (Am3). Coefficients of error (CE) and variance (CV) were

calculated as estimates of precision, and values less than 0.1 were

accepted (Braendgaard et al., 1990; West and Gundersen, 1990;

West et al., 1991).

As previously described, terminal tree size (TT) was estimated

by dividing terminal density in the CPu or NAc by the number of

SNpc or VTA neurones, respectively (Finkelstein et al., 2000;

Parish et al., 2001, 2002a,b). Implicit is the assumption that the

SNpc predominantly innervates the dorsal striatum and the VTA

innervates the ventral striatum and NAc with little overlap between

the arbours of the two midbrain projections within these three areas

(Fallon and Moore, 1978; Gerfen et al., 1987).

Neurochemical analysis

After 8 weeks of daily drug treatment (saline, cocaine,

haloperidol and cocaine + haloperidol), animals were killed and

striatal DA activity was measured using high-performance liquid

chromatography (HPLC) as previously described (Herges and

Taylor, 1999; Parish et al., 2001).

Behavioural assessment

Motor activity

Measurements of motor activity or sedation that may result

from long-term drug administration (Supplementary Table 1) were

made at the onset of treatment and after 4 and 8 weeks of daily

drug treatment (saline, cocaine, haloperidol and cocaine +

haloperidol). On each occasion, animals were observed for 2 h

prior to drug administration and, on the following day for 2 h after

drug administration. Behavioural assessments were performed

using a rapid time-sampling behavioural checklist described in

detail elsewhere (Clifford et al., 1998; Ross et al., 2000).

Assessment of anxiety levels

An elevated plus-maze and light–dark paradigm was used to

assess anxiety and agitation (Clifford et al., 1998; Crawley and

Goodwin, 1980; Cruz et al., 1994). Animals were tested for levels

of anxiety in both the addicted state and 24 h after withdrawal of the

drug. The design and use of the elevated plus maze followed the

descriptions of others (Brioni et al., 1993; Pellow et al., 1985) with a

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311304

modification of small walls (1 cm) on the open arms of the maze.

Animals were habituated to the testing room prior to testing. The

following variables were scored: (i) time spent in the open and

enclosed arms; and (ii) number of entries into open and closed arms.

Entry into an arm was defined as the entry of both front feet of the

mouse into the arm; an exit was defined by the exit of both

forelimbs from the arm. Plus-maze behaviour was assessed by

direct observation over a 5-min period. The light–dark test

consisted of a rectangular darkened glass chamber (12 � 20 � 20

cm) with a small entrance connecting with a lit chamber (24� 20�20 cm). Animals were placed in the light portion of the chamber

facing away from the entrance and the amount of time spent in

illuminated and dark areas (and number of entrances) was recorded.

Results

Self-administration

Each day, animals were administered combinations of water,

cocaine and haloperidol for 8, 12, 16 or 24 weeks (Supplementary

Fig. 1) to assess the effects of these treatments on the subsequent

preference for cocaine and to compare this preference with terminal

arbour size in the NAc, ventral and dorsal striatum. After 8, 12, 16

or 24 weeks of pre-treatment, preference for cocaine was tested by

providing animals a choice of either cocaine in water or water

alone.

Fig. 2. Preferences of mice for drinking either cocaine or water following vario

differences in drinking preferences of animals treated for 8 weeks with water, halo

preferred cocaine to water. This preference for cocaine persisted (and increased to

haloperidol had no preference for cocaine and if anything seemed to have a predilec

this and subsequent figures indicates co-administration of cocaine and haloperido

Animals pre-treated with water or haloperidol tended to prefer

water to cocaine whereas animals receiving cocaine pre-treatment

showed a marked preference for cocaine (67%, with these mice

drinking up to 8.2 ml cocaine-treated water daily). However, when

haloperidol was co-administered with cocaine from the outset of

treatment, a preference for cocaine did not develop (Fig. 2). Water

was also preferred when 8 weeks of cocaine treatment (which is

sufficient to establish cocaine preference) was followed by 8 weeks

of co-treatment with haloperidol. Thus, haloperidol co-treatment

attenuated cocaine preference, even if haloperidol co-administra-

tion was commenced after cocaine preference was established.

These effects appeared to be long lasting because animals

continued to prefer water 8 weeks after co-treatment was ceased,

even though prior to co-treatment, the animals preferred cocaine. In

contrast, animals continued to prefer cocaine to water when they

were deprived of cocaine for 8 weeks (following 8 weeks of

treatment, Fig. 2).

Assessment of treatments on DA SNpc and VTA terminal arbours

The number of SNpc and VTA neurones were counted in all

animals as previously described (Parish et al., 2001). None of the

drug treatments altered neuronal numbers (Figs. 3A–C). Density of

DAT-ir terminals and TT was estimated in the dorsal and ventral

striatum and NAc. The terminal density and TT size in the dorsal

striatum of wildtype mice was 12.3 � 10�3 and 20.5 � 10�7,

respectively. Density and TT size was greater in the NAc (15.8 �

us treatment regimens (mean F SEM). Note that there was no significant

peridol or cocaine + haloperidol. Animals that received cocaine for 8 weeks

91%) after 8 weeks of drug withdrawal. Co-administration of cocaine and

tion for water (see cocaine + haloperidol followed by water alone). C + H in

l. *P b 0.05.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311 305

10�3 and 28.4 � 10�7, respectively) than in the dorsal striatum and

even greater within the ventral striatum (17.0 � 10�3 and 30.5 �10�7, respectively) with the same trends seen following all

treatments (see supporting information, Table 2).

Treatment with haloperidol significantly increased arbours in all

three regions: dorsal striatum (36%), ventral striatum (16%) and

NAc (12%) whereas cocaine treatment pruned terminal arbours in

all three regions: dorsal striatum (21%), ventral striatum (13%) and

NAc (16%), Fig. 3. Because the effects of cocaine and/or

haloperidol treatments were of similar proportions in all three

regions (e.g., cocaine decreased TT size by approximately 15% in

the dorsal striatum, ventral striatum and NAc), for the rest of the

study, we made measurements from one region, assuming that the

effects would be of similar proportions in the other two regions.

The dorsal striatum was chosen because it most readily delineated

and comparisons were available from previous studies. Eight

weeks after withdrawal of either cocaine or haloperidol, terminal

tree size is normal, implying that the effect of these drugs on

terminal arbour has bwashed outT by 8 weeks (Fig. 3G, and

Supplementary information Table 2). Eight weeks of cocaine

followed by 8 weeks of haloperidol increased tree size by 12%,

presumably as a consequence of the unopposed actions of

haloperidol. When cocaine was followed by 4 weeks of haloper-

idol, arbours were slightly retracted (2.5%) compared to wildtype

animals, presumably because the effects of cocaine retraction had

not been fully reversed by haloperidol-induced D2R blockade

(Fig. 3G).

Thus, when haloperidol was co-administered with cocaine,

even after cocaine addiction was established, arbour size returns to

normal. Whilst this might suggest a correlation between tree size

and a propensity to prefer cocaine, it should be noted that terminal

tree size returned to normal in animals deprived of cocaine for 8

weeks (after addiction was established). Despite normalisation of

the terminal tree (3G), these animals maintained a marked

preference for cocaine, indicating memory and propensity to

relapse (Fig. 2).

Determination of striatal DA activity

Basal levels of DA, DOPAC and DA activity (ratio of DOPAC

to DA) in the striatum of mice treated for 8 weeks with saline,

cocaine, haloperidol and cocaine + haloperidol were determined.

There was a trend toward increased DA activity following

haloperidol treatment and decreased activity following cocaine

treatment (Fig. 4). However, DOPAC levels and DA activity

increased significantly following cocaine + haloperidol treatment,

reflecting increased DA synthesis and release and the combined

effects of the two drugs: a lack of D2R autoreceptor feedback in the

presence of diminished reuptake. From separate sets of animals, we

used DA activity and terminal density to estimate the effect of the

various treatments on the average DA activity of individual

terminals. DA turnover per terminal in haloperidol animals was

significantly less than in saline-treated animals (as terminal arbours

but not DA activity was significantly increased). Following

cocaine treatment, DA activity remained unchanged but TT size

was significantly reduced and consequently DA activity per

terminal was increased. As a result of co-administration of cocaine

and haloperidol, DA activity per terminal was almost three times

greater than following cocaine treatment alone. This was because

DA activity was increased significantly in the presence of only

modest increase in terminal density, Fig. 4D.

Assessment of motor behaviour and anxiety

Behaviours were assessed (including locomotion, sniffing and

grooming) to demonstrate that the drug doses were sufficient to

affect not only the neuroanatomy but also behaviour. Similar

patterns were observed with all behaviours (i.e., sniffing, rearing

and locomotion), and locomotion was used as an exemplar of these

trends. In control animals, locomotion was unchanged by treatment

throughout the 8 weeks (Figs. 5A and B). Locomotion was reduced

prior to haloperidol treatment, with even further reduction after

treatment: the effect of diminishing over 8 weeks, possibly related

to TT growth. Locomotion before treatment did not change over 8

weeks of cocaine treatment but dramatically increased after

treatment. This post-treatment increase in locomotion slowly

diminished over 8 weeks, possibly related to TT pruning. By

contrast, combined haloperidol and cocaine treatment resulted in a

similar increase in locomotion produced by cocaine but without the

subsequent diminution at 8 weeks, possibly related suppressed TT

pruning in the face of suppressed reuptake. These results indicated

that the drug doses administered not only caused anatomical

alterations but were also sufficient to affect behaviour.

Anxiety-like behaviour was greater in cocaine-treated mice

exposed to the light–dark paradigm or placed in the elevated plus

maze than in control animals. When cocaine was withdrawn for

24 h, the level of anxiety-like behaviour increased further, reflected

by fewer open arm entries in the plus maze and light chamber of

the light–dark paradigm (Figs. 5C–F). When cocaine treatment was

combined with haloperidol, either from the outset or after 8 weeks

of cocaine alone, the level of anxiety-like behaviour was no

different to non-cocaine-treated mice. Similarly, after cocaine had

been withdrawn for 8 weeks, anxiety measures were similar to

controls. It is noteworthy that terminal arbour size was normal

under each of these circumstances (Fig. 3G).

Assessment of the effect of D2R and D3R in regulation of arbour

size

As SNpc, VTA or DAT terminal density counts for C57BL/6

mice, D2(+/+) and D3(+/+) (wildtype littermates) were not

significantly different, in this study C57BL/6 mice were used as

controls. The total number of SNpc neurones was significantly

reduced in the D2(�/�) and D3(�/�) mice (22.5% and 7.9%,

respectively), as were tyrosine hydroxylase immunoreactive (TH-

ir) VTA neurones (25% and 14%, respectively). The proportion of

TH-ir SNpc cells was also reduced in the D2(�/�) (75% compared

to 90% seen in wildtype) (Fig. 3, supporting information). As

described in the Materials and methods section, a value propor-

tional to terminal tree (TT) size was determined by dividing density

by number of cells in the relevant mesencephalic nucleus (Parish

et al., 2001).

Terminal density and TT size tended to increase from dorsal

striatum to NAc and to be greatest in the ventral striatum in all

three genetic strains (Fig. 3). In each of the three regions, TTs were

larger in both mutants than in wild types, although the TTs were

largest in the D2(�/�) mice.

Discussion

This work shows that co-administration of haloperidol and

cocaine prevents the marked preference for cocaine that usually

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311306

follows its long-term use. Haloperidol’s effect is present whether

commenced at the outset of cocaine treatment or added to cocaine

at a time when addiction is already well established (Fig. 2). We

aimed to establish whether drug-seeking behaviour was related to

arbour size and if so, whether the reversal of arbour pruning with a

D2R antagonist might reverse drug-seeking behaviour even though

Fig. 4. Histograms showing percentage changes (compared to saline) in (A) DA concentration, (B) DOPAC concentration, (C) dopamine turnover and (D)

dopamine turnover per terminal density of haloperidol, cocaine and cocaine + haloperidol treated mice; mean F SEM. Cocaine + haloperidol treated animals

showed significantly increased DOPAC levels and DA activity. The effect on DOPAC levels and DA activity of other treatment, whilst showing consistent

trends, was not significant. Statistical significance (*P b 0.05) was determined by ANOVA with Tukey post hoc tests.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311 307

cocaine was still in use. This hypothesis was based on a previous

observation that activation of the D2R autoreceptor led to pruning

of the TT of nigrostriatal neurones, whereas receptor blockade

induced sprouting (Parish et al., 2001, 2002b). As reported

previously, cocaine treatment pruned terminal arbours by 20%,

whereas arbour size increased by 34% following haloperidol

treatment (Fig. 3). In keeping with the idea that these effects on TT

size are effected through autoreceptor activation, TT size was

unchanged following co-administration of these two drugs. The

effect of either drug on tree size disappeared by 8 weeks after

cessation of treatment (Fig. 3).

The D2/D3 autoreceptor acts as a feedback mechanism for

maintaining neurotransmitter release by regulating the firing rate

of DA neurones, DA synthesis as well as DA release (Meador-

Woodruff et al., 1994; Wolf and Roth, 1990). We previously

proposed that as part of a mechanism for maintaining synaptic

DA, reduced activation of the D2 autoreceptor not only

increased DA synthesis and release, but also increased terminal

Fig. 3. (A–C) The effect of various drug treatments (expressed as a percentage chan

TH-ir SNpc neurones and (C) TH-ir VTA neurones, meanF SE. Significant change

D3(�/�) mice. (D–F) The effect of various drug treatments (expressed as a percent

CPu, (E) ventral CPu and (F) nucleus accumbens. Panel (G) illustrates the effects

after an initial 8 weeks of cocaine treatment and the effects of 8-week withdrawa

difference was seen from wildtype in any of these groups. Treatments are for 8 w

density in the striatum so as to maintain DA output (Parish et

al., 2001). Thus, initially, cocaine treatment increases synaptic

DA levels, which also increases activation of the D2 autor-

eceptor. This initially results in increased post-synaptic DA

receptor activation and hyperactivity (0 weeks, Fig. 5B), but in

time, through pre-synaptic D2R activation, DA synthesis and

release is down-regulated and also the TT is pruned, leading

eventually to reduced basal levels of striatal DA activity. Indeed,

striatal DA activity was modestly reduced (when measured 24 h

after the last cocaine dose) compared to normal, even though

(and in keeping with the proposed mechanism) DA activity per

terminal was increased (Fig. 4). Similarly, haloperidol treatment

leads to decreased autoreceptor activation and hence increased

TT size, thus increasing total DA activity in the striatum in the

face of a modest reduction in DA activity per terminal. The

combined activity of cocaine and haloperidol resulted in an

increase in both striatal DA activity and DA activity per

terminal.

ge from untreated wildtype) on the number of (A) neurones in the SNpc, (B)

s (*) were observed in the numbers of neurones counted in the D2(�/�) andage change from untreated wildtype) on terminal tree size (TT) in (D) dorsal

on TT size of administering haloperidol for 4 (C8_H4) or 8 weeks (C8_H8)

l of cocaine (C8_R8) or haloperidol (H8_R8) after 8 weeks. No significant

eeks (except G). C = cocaine, H = haloperidol, R = rest (i.e., withdrawal).

Fig. 5. Motor activity (locomotion) (A) before, and (B) after drug administration (each bar is the aggregate of observations for the 2-h period, mean F SEM).

bBeforeQ drug administration had no significant effect on locomotion however. After administration of haloperidol, behaviour was significantly reduced whilst

cocaine and cocaine + haloperidol resulted in significant increases in locomotion. After 8 weeks of cocaine treatment, activity was significantly less than at 0 or

4 weeks. (C) Time spent in the open arms (white bar) and closed arms (black) of the elevated plus maze during treatment and following a 24-h drug withdrawal

(open arm: white bar with strips; closed arm: black bar with strips). (D) Number of entrances into the open and closed arms of the elevated plus maze before

and after drug withdrawal. (E) Time spent in the light (white bar) and dark (black) chambers of the light–dark paradigm during treatment and after 24 h of drug

withdrawal (striped bars). (F) Number of passages through the entrance between the chambers of dark–light test box during drug treatment (black bars) and

after 24-h withdrawal (white bars). Note, long-term cocaine administration caused increase anxiety and this anxiety was significantly increased by 24-h drug

withdrawal. C + H co-administration restored anxiety to normal as did 8 weeks of drug withdrawal. *P b 0.05.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311308

Changes in TT size, as reported here, could contribute to the

development of tolerance that follows extended cocaine use

(Kalivas and Stewart, 1991). The behavioural changes (Fig. 5)

show trends that support the concept that TT size may provide an

anatomical substrate for some of the behavioural effects of chronic

cocaine use. Cocaine treatment alone or combined with haloperidol

markedly increases bafterQ treatment locomotion (Fig. 5B). This

effect is somewhat attenuated after 8 weeks of cocaine treatment,

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311 309

by which time TT size is reduced, but not if haloperidol is added,

when tree size does not change.

One of the questions addressed in this study was whether

preference for cocaine related to changes in tree size. Haloperidol

co-administered with cocaine overcomes the usual marked

preference for cocaine that follows long-term use, even when

haloperidol was co-administered after 8 weeks of cocaine alone.

Even though animals were previously addicted, the effect was

maintained long after cessation of haloperidol administration.

Whilst the initial impression is that cocaine preference correlates

with tree size (Fig. 3), it is not the whole explanation because

cocaine preference persists after 8 weeks of abstinence, even

though TT size returns to normal (Fig. 5). Drug-seeking behaviour

and craving may be a consequence of learned mechanisms (Hyman

and Malenka, 2001). This concept is based on the notion that the

strength of the glutamatergic cortico-striatal synapse is modified by

dopaminergic influences, an idea that has been difficult to confirm

directly, but has a body of evidence to support it (Hyman and

Malenka, 2001). Cocaine elicits changes in the relative ratios of

NMDA and AMPA receptors (Thomas et al., 2001) and rewards

can potentiate cortico-striatal synapses when dopamine is released

in response to those rewards (Reynolds et al., 2001). LTP at the

cortico-striatal synapse appears dependent on dopamine because it

is suppressed by D1R blockade, and cannot be elicited in mice with

lesioned SNpc (Centonze et al., 2001). D1 receptor activation

depolarises spiny neurones and promotes their vigorous spiking by

enhancing L-type Ca+ currents (Nicola et al., 2000). Whilst this

will result in diminished sensitivity of the spiny neurones to weak,

transitory cortical inputs, it will enhance their response to strong,

maintained cortical synaptic inputs (Hernandez-Lopez et al., 1997).

Whether this reflects D1 or D5 receptor involvement is unclear

from these experiments, as D1R blockade cannot discriminate

between these DA receptor subtypes. Experiments in D1(�/�)mice have shown that mice lacking the D1R develop place

preference in response to cocaine (Miner et al., 1995). The

retraction of the terminal tree and subsequent normalisation after

cocaine withdrawal implies sprouting and new synapse formation.

Sprouting of DA nigrostriatal neurones in other contexts is

associated with changes in structure, function and location of

DA terminals (Finkelstein et al., 2000; Stanic et al., 2003).

Synapses reformed as a consequence of injury-induced sprouting

are large, with increased numbers of vesicles and mitochondria,

have abnormal DA regulation with a marked reduction in DAT

function and form more proximal synaptic contacts onto spiny

neurones (Finkelstein et al., 2000; Stanic et al., 2003). Others have

also found structural modification of the dendritic structure after

denervation or haloperidol treatment (Meredith et al., 2000;

Rodriguez and Pickel, 1999). Whilst it is not known whether

similar changes are present with the remodelling that follows

cocaine withdrawal, the density of dendritic spines and the

incidence of spines with multiple heads increase on medium spiny

neurones of the NAc following long-term cocaine self-adminis-

tration: changes that persist up to 1 month after drug withdrawal

(Pierce and Kalivas, 1997; Robinson and Kolb, 1997; Robinson et

al., 2001). It seems plausible that reduction of DA synapses during

chronic drug use and their remodelling after cessation of drug use

could affect plasticity at the cortico-striatal synapse and predispose

to both craving and a long-term risk of relapse into drug-using

behaviour. It is intriguing therefore as to why cocaine preference

was removed by co-treatment with haloperidol. One important

factor is that re-innervation associated with cocaine withdrawal

occurs with a striatum depleted of DA, with both DA concentration

and DA activity reduced, whereas when haloperidol is co-

administered, there are normal DA concentrations and abnormally

high DA activity in the striatum (Fig. 4). It is possible that if

synapses are formed in the presence of increased synaptic DA (i.e.,

C + H co-administration), they can induce plastic changes in the

cortico-striatal synapse, perhaps by inducing L-type Ca+ currents

or direct D1R stimulation. These effects may not occur when

synapses are reformed in the presence of low DA concentrations

usually associated with cocaine withdrawal. Thus, the effects on

post-synaptic targets could be vastly different in the two circum-

stances, with remodelling in the presence of abundant dopamine

leading to learning of behaviour that does seek cocaine.

Our previous findings related to the projection of DA nigral

neurones onto the dorsal striatum and used D2(�/�) mice to

establish that this receptor was important in this mechanism.

However, the effects of addictive drugs are thought to be

influenced by transmission in the ventral striatum, nucleus

accumbens and amygdala where the D3R may be an important

autoreceptor (Broderick and Piercey, 1998; Ellinwood et al., 2000).

Our findings show the effects of cocaine and haloperidol on

terminal arbours are similar in the accumbens, ventral striatum and

dorsal striatum. Furthermore, the role of the D3R is more modest

but otherwise similar to that of the D2R. The effects of haloperidol

and cocaine on arbour size could therefore potentially be

influenced by either receptor.

We have not attempted to discriminate between the relative

roles of the D2R and D3R in terms of mediating the effects of

haloperidol and cocaine co-administration. Our main objective,

with respect to the use of the D3(�/�), was to demonstrate that

both D2R and D3R are involved in regulation of arbour size and

that the phenomenon is relevant to all dopaminergic neurones.

The evidence for a selective role for D3R in cocaine-seeking

behaviour is conflicting (Pilla et al., 1999; Vorel et al., 2002)

and whilst D2R has been implicated in a propensity toward

addiction, the role of selective D2R antagonism is also unclear.

If sprouting and remodelling of new synapses does play an

important part in determining drug-seeking behaviour, then co-

treatment may need to extend for very long periods. Following

SNpc lesions, re-innervation of the dorsal striatum takes 8 weeks

in mice (Parish et al., 2001, 2002a), 16 weeks in rats

(Finkelstein et al., 2000) and 6 months in monkey (unpublished

data): it may therefore take 12 months in humans. Whether the

time for re-innervation following cocaine is similar to SNpc

lesions is not yet known. Furthermore, the very high levels of

DA turnover associated with cocaine and haloperidol (Fig. 4)

may produce affective states that cannot be tolerated for that

duration, leading to non-compliance. It has previously been

noted that due to side effects, brisperidone is unlikely to find

broad acceptance with the treatment-seeking populationQ (Gra-

bowski et al., 2000). Our findings suggest a possible mechanism,

anatomical substrate and potential therapeutic target, therefore

opening up possibilities for further studies that may prove fruitful

for the treatment of addictive behaviour.

Acknowledgments

Supported by grants from the Australian National Health and

Medical Research Council (NH and MRC). John Drago is an NH

and MRC Practitioner Fellow.

C.L. Parish et al. / Neurobiology of Disease 19 (2005) 301–311310

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.nbd.2005.01.009.

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