Withdrawal from repeated amphetamine administrationreduces NMDAR1 expression in the rat substantia nigra,nucleus accumbens and medial prefrontal cortex

Wenxiao Lu, Lisa M. Monteggia1 and Marina E. WolfDepartment of Neuroscience, FUHS/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064±3095, USA1Department of Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, USA

Keywords: behavioural sensitization, dopamine, glutamate receptors, ventral tegmental area

Abstract

Glutamate plays a critical role in neuroadaptations induced by drugs of abuse. This study determined whether expression of theNMDAR1 subunit of the NMDA receptor is altered by repeated amphetamine administration. We quanti®ed NMDAR1 mRNA (using insitu hybridization with 35S-labelled oligonucleotide probes) and immunolabelling (using immunocytochemistry with 35S-labelledsecondary antibodies) in rat ventral midbrain, nucleus accumbens and prefrontal cortex after 3 or 14 days of withdrawal from ®ve dailyinjections of saline or amphetamine sulphate (5 mg/kg/day). No changes in NMDAR1 expression were observed after 3 days ofwithdrawal, whereas signi®cant decreases were observed in all regions after 14 days. NMDAR1 mRNA levels in midbrain were toolow for reliable quanti®cation, but immunolabelling was decreased signi®cantly in intermediate and caudal portions of the substantianigra. This may indicate a reduction in excitatory drive to substantia nigra dopaminergic neurons. In the nucleus accumbens, therewere signi®cant decreases in NMDAR1 mRNA levels (74.8 6 7.7% of control, P < 0.05) and immunolabelling (76.7 6 4.4%, P < 0.05).This may account for previously-reported decreases in the electrophysiological responsiveness of nucleus accumbens neurons toNMDA after chronic amphetamine treatment, and contribute to dysregulation of goal-directed behaviour. In prefrontal cortex, therewas a signi®cant decrease in NMDAR1 mRNA levels (76.1 6 7.1%, P < 0.05) and a trend towards decreased immunolabelling(89.5 6 7.0%). This may indicate decreased neuronal excitability within prefrontal cortex. A resultant decrease in activity of excitatoryprefrontal cortical projections to nucleus accumbens or midbrain could synergize with local decreases in NMDAR1 to further reduceneuronal excitability in these latter regions.

Introduction

The repeated administration of amphetamine or cocaine to rats results

in profound behavioural adaptations, some of which may model

addiction-related behavioural changes in humans (Robinson &

Berridge, 1993). Psychostimulant-induced behavioural changes are

closely associated with a complex cascade of cellular changes in the

mesocorticolimbic and nigrostriatal dopamine (DA) systems (White

& Kalivas, 1998). It is now well established that many of these

behavioural and cellular adaptations require glutamate transmission

for their induction, suggesting mechanistic similarities to other forms

of plasticity (Wolf, 1998). More recently, attention has been focused

on how glutamate transmission itself may be altered by repeated

psychostimulant administration.

Recent studies indicate that glutamate receptor expression under-

goes progressive changes during the withdrawal period. At very early

withdrawals from cocaine (16±18 h), increased levels of GluR1 and

NMDAR1 in the ventral tegmental area have been reported

(Fitzgerald et al., 1996), although GluR1 is not increased 16±18 h

after discontinuing repeated amphetamine (see Discussion). In

prefrontal cortex, GluR1 is increased 3 days after discontinuing

amphetamine administration but returns to normal by 14 days. In

nucleus accumbens, GluR1 and GluR2 are unchanged after 3 days of

withdrawal but decreased after 14 days (Lu et al., 1997; Lu & Wolf,

1999). Electrophysiological changes that parallel these changes in a-

amino-3-hydroxy-5-methy-lisoxazole-4-propionate (AMPA) receptor

subunit expression have been found in both prefrontal cortex and

nucleus accumbens (White et al., 1995a; Peterson et al., 1998; White

et al., 1999).

The present study focuses on N-methyl-D-aspartate (NMDA)

receptors. Electrophysiological studies show that NMDA receptors

play an important role in regulating the ®ring rate and pattern of

midbrain DA neurons (White, 1996; Overton & Clark, 1997)

whereas DA- and NMDA-receptor-mediated signals interact to

regulate the output of principal neurons in DA-innervated regions

(Cepeda & Levine, 1998). NMDA receptors are also implicated in

synaptic plasticity involving glutamatergic afferents to striatum

(Calabresi et al., 1996) and nucleus accumbens (Pennartz et al.,

1993; Kombian & Malenka, 1994). NMDA receptors are

composed of at least one NMDAR1 subunit in combination with

one or more NMDAR2 subunits (NMDAR2A-D). Consistent with

obligatory inclusion of NMDAR1, this subunit is expressed

throughout the brain. The NR2 subunits exhibit more restricted

regional distributions and their differential inclusion in the

oligomeric receptor is thought to underlie functional diversity of

NMDA receptors in different brain regions (Hollmann &

Heinemann, 1994; Michaelis, 1998).

Correspondence: Dr Marina E Wolf, as above.E-mail: wolfm@®nchcms.edu

Received 15 December 1998, revised 23 April 1999, accepted 26 April 1999

European Journal of Neuroscience, Vol. 11, pp. 3167±3177, 1999 ã European Neuroscience Association

There are many mechanisms by which neuronal activity can

modulate NMDA receptor function, including regulation of phos-

phorylation (Hall & Soderling, 1997), traf®cking (Rao & Craig,

1997) and splicing (Ra®ki et al., 1998). Another mechanism that is

well-documented involves transcriptional control of the expression of

individual subunits. For example, NMDAR1 mRNA levels in the rat

brain are altered in a regionally selective manner by seizures (Pratt

et al., 1993; Jensen et al., 1997; Lason et al., 1997; Liang & Jones,

1997; Ra®ki et al., 1998), long-term treatment with antipsychotic

drugs (Fitzgerald et al., 1995; Meshul et al., 1996; Riva et al., 1997;

Chen et al., 1998) and chronic ethanol ingestion (Ortiz et al., 1995;

Snell et al., 1996).

The purpose of the present study was to determine whether

repeated amphetamine administration alters levels of NMDAR1

mRNA or immunolabelling at three levels of the mesocorticolimbic

system: the ventral midbrain, the nucleus accumbens and the medial

prefrontal cortex. Because the NMDAR1 subunit is required for the

formation of functional channels (above), changes in NMDAR1

expression may provide a useful index of amphetamine-induced

changes in the number of functional NMDA receptors. An abstract

describing some of this work has been published previously (Lu et al.,

1996b).

Materials and methods

Animals and drug treatment

Male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA), weighing

200±225 g at the beginning of the experiments, were used in these

studies. All procedures were performed in strict accordance with the

National Institutes of Health `Guide for the Care and Use of

Laboratory Animals' and were approved by the Institutional Animal

Care and Use Committee of the Chicago Medical School. Rats were

handled for three to four days before drug or vehicle treatment began

and then injected intraperitoneally with amphetamine sulphate (5 mg/

kg/day) or saline (1 mL/kg/day) for 5 days in home cages. This

regimen produces robust behavioural sensitization (Wolf & Jeziorski,

1993; Wolf et al., 1994). The same regimen was used in previous

studies on the effect of repeated amphetamine on AMPA receptor

subunit expression (Lu et al., 1997; Lu & Wolf, 1999) and

electrophysiological responsiveness of ventral tegmental area

(VTA), nucleus accumbens and prefrontal cortex neurons to

glutamate agonists (White et al., 1995a; Zhang et al., 1997;

Peterson et al., 1998; White et al., 1999). Rats were perfused 3 or

14 days after the last injection. Four pretreatment groups were thus

generated: amphetamine + 3-day withdrawal; vehicle + 3-day with-

drawal, amphetamine + 14-day withdrawal; and vehicle + 14-day

withdrawal. Each group consisted of eight to 10 rats.

Rat brain tissue preparation

Because of the large number of rats involved in the study, perfusions

were staggered over 3 consecutive days. All rats were perfused

between 09.00 and 13.00 h. To minimize variability, a pair of rats

(one from the amphetamine group and one from the vehicle group)

was always perfused simultaneously. Rats were anaesthetized with

pentobarbital and perfused with 200 mL of ice-cold saline, followed

by 400 mL of ®xative solution containing 4% paraformaldehyde

(Sigma-Aldrich, St Louis, MO, USA), 1.5% sucrose and 0.1 M

phosphate buffer at pH 7.2 (PB). After perfusion, the brains were

immediately removed and immersed in the above ®xative solution for

another hour. Brains were then immersed sequentially in solutions

containing 0.1 M PB, 0.1% sodium azide and either 10, 20 or 30%

sucrose. Sections (40 mm) were cut frozen on a sliding microtome.

Forebrain sections (containing prefrontal cortex and nucleus

accumbens) were sequentially placed into 12 wells of a cell culture

plate, whereas midbrain sections were sequentially placed into six

wells. Thus, for prefrontal cortex and nucleus accumbens, each

section group (one well) contained sections that sampled the entire

rostral-caudal extent of that brain region at 480-mm intervals (two or

three coronal sections for prefrontal cortex and three or four sections

for nucleus accumbens). For the midbrain, each section group

contained six or seven sections that sampled the midbrain at 240 mm

intervals. One section group was used for in situ hybridization with

each probe or immunocytochemistry with each antibody. Sections

were stored free-¯oating in cryoprotectant solution [30% sucrose,

30% ethylene glycol (Fisher Scienti®c, Pittsburgh, PA, USA) and

0.1 M PB (pH 7.2)] (deOlmos et al., 1978) at ±20 °C.

In situ hybridization histochemistry

We used a quantitative method developed in our laboratory. Because

of the ribonuclease-resistant nature of this method, it results in high

levels of speci®c hybridization and enhanced reproducibility, and is

therefore suitable for between-group comparisons of mRNA levels

(Lu et al., 1996a). Brie¯y, sections stored in cryoprotectant solution

were transferred, using a paint brush, into a plastic net (Brain

Laboratories, Boston, MA, USA) in a glass dish. Sections were rinsed

in 50% formamide (EM Science, Gibbstown, NJ, USA) and 4 3standard saline citrate (SSC) (1 3 SSC equals 150 mM sodium

chloride and 15 mM sodium citrate, pH 7.2) twice for at least 30 min

each time at room temperature with gentle agitation. Then, sections

were transferred into 1 mL of hybridization buffer in 2 mL tubes.

Hybridization buffer consisted of 50% formamide, 4 3 SSC, 0.02%

polyvinylpyrrolidone (Fisher), 0.02% ®coll (Fisher), 0.02% bovine

serum albumin (Sigma), 100 mg/mL denatured salmon DNA (Sigma),

250 mg/mL yeast RNA (Fisher), 50 mM dithiothreitol (Fisher), 10%

dextran sulphate (Fisher) and 35S-labelled NMDAR1 oligodeoxynu-

cleotide probes (10 3 106 c.p.m./mL). The tubes were incubated at

37 °C for 20 h, with continuous agitation. After overnight incubation,

sections were transferred into a net in a glass dish, and rinsed

sequentially in 2 3 SSC, 1 3 SSC and 0.5 3 SSC, once in each buffer

(10 min per rinse), at room temperature. Then, sections were rinsed

four times (30 min per rinse) in 0.5 3 SSC at 54 °C (14±15 °C lower

than the melting temperature of the probes). All four pretreatment

groups were processed simultaneously for in situ hybridization, using

the same labelled probes and the same hybridization buffer (see Lu

et al., 1996a for details). Finally, sections were mounted onto gelatin-

coated slides and dried overnight. NMDAR1 expression was

examined using two oligodeoxynucleotide probes (DuPont,

Wilmington, DE, USA), complementary to nucleotides 375±420

and 1011±1056 of rat NMDAR1. The two probes were used

simultaneously in order to enhance the signal. The probes were

labelled at the 3¢-terminal with 35S-dATP and terminal transferase,

and puri®ed by ethanol precipitation. Both probes recognize all

NMDAR1 isoforms. The speci®city of these oligodeoxynucleotide

probes for NMDAR1 was veri®ed by the manufacturer using

Northern blots. In addition, we performed control experiments (not

shown) to verify that a high concentration (10 nM) of each of the

unlabeled probes for NMDAR1, when added to hybridization buffer

containing the corresponding 35S-labelled probe (0.35 nM), reduced

speci®c hybridization to background levels.

Immunocytochemistry

Sections were transferred from the cryoprotectant solution into a net

in a dish containing rinsing buffer. Sections were rinsed in 0.1 M PB

(pH 7.2) for 4 3 10 min and in 0.1 M PB containing 0.3% Triton X-

3168 M. E. Wolf et al.

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

100 (PBT) for 4 3 10 min. After incubating in 10% horse serum (Life

Technologies, Grand Island, NY, USA) in PBT for 30 min to block

background staining, sections were transferred into wells of cell

culture plates containing primary antibodies in 10% horse serum and

PBT, and incubated overnight at 4 °C with continuous agitation. The

concentration of monoclonal anti-NMDAR1 antibody (Pharmingen,

San Diego, CA, USA) was 0.5 mg/mL for all brain regions. After

rinsing in PBT for 4 3 10 min and in PBT containing 10% horse

serum for 10 min, sections were incubated with 35S-labelled

antimouse IgG antibody (1 : 300 dilution for all brain regions;

Amersham, Arlington Heights, IL, USA) at room temperature for 2 h

with continuous agitation. Finally, sections were rinsed in PBT

(4 3 10 min) and in PB (4 3 10 min), and then mounted onto gelatin-

coated microslides. Sections from amphetamine and vehicle pretreat-

ment groups were processed simultaneously throughout all steps of

the immunocytochemical procedure. All sections were mounted on

the same day in random order, to avoid differential loss of signals

during storage in PB prior to mounting. The speci®city of the

antibody for NMDAR1 was veri®ed by the manufacturer using

Western blots and immunocytochemistry in rat brain, monkey brain

and transfected cells. In addition, we veri®ed that speci®c staining

was abolished when the primary antibody was incubated in a boiling

water bath for 10 min and that signals from 35S-labelled antimouse

IgG secondary antibody were abolished by 10% rabbit serum or by

boiling the antibody.

Autoradiography and image analysis

Sections were exposed to BioMax-MR ®lms (Kodak, Rochester, NY,

USA) with 14C-standard microscale strips (Amersham) for 3 days for

in situ hybridization and 8 days for immunocytochemistry. Sections

from amphetamine and vehicle pretreatment groups with the same

withdrawal time were exposed to the same ®lm, to avoid possible

differences between ®lms. Films were developed with GBX

developer (Kodak) for 4 min and ®xed with rapid ®xer (Kodak).

Autoradiographs on ®lms were scanned using a Power Macintosh G3

and an AppleOne scanner (Macintosh, Cupertino, CA, USA). The

scanner can resolve 256 grey levels from white to black. Boundaries

of scanned regions are shown in Fig. 1. For analysis of the prefrontal

cortex, two or three coronal sections between Bregma 2.7±3.7 mm

(Paxinos & Watson, 1986) were scanned, on right and left sides, for

each rat in each pretreatment group. The regions scanned were

restricted to the medial precentral, the dorsal anterior cingulate and

the prelimbic cortices as de®ned by Sesack et al. (1989). All layers of

prefrontal cortex were scanned (Fig. 1A). For the nucleus accumbens,

three or four sections between Bregma 0.7 and 2.2 mm (Paxinos &

Watson, 1986) were scanned for each rat in each pretreatment group.

Within each section, the boundary of the entire nucleus accumbens

was de®ned according to Paxinos & Watson (1986), including both

core and shell subregions of the nucleus accumbens, but carefully

excluding some surrounding areas with high signals, such as the

islands of Calleja and the olfactory tubercle. The entire region of the

nucleus accumbens, de®ned in this manner, was scanned on both left

and right sides of each section. For analysis of subregions of the

nucleus accumbens, the core and shell were scanned separately. Due

to the lack of a clear boundary between the core and the shell

subregions, a transitional zone between the core and the shell was

avoided in scanning autoradiographs. In addition, because it is

dif®cult to divide the rostral and caudal poles of the nucleus

accumbens into core and shell, these subregions were scanned only in

those sections between Bregma 1.0±2.0 mm (two or three coronal

sections) (Paxinos & Watson, 1986). Thus, the total area for the core

and shell subregions is smaller than that scanned for the entire

nucleus accumbens (Fig. 1B). For midbrain, coronal sections were

divided into three portions according to Paxinos & Watson (1986): a

rostral portion (interaural 3.7±4.5 mm, two or three coronal sections

in each section group), an intermediate portion (interaural 3.2±

3.7 mm, two sections per section group) and a caudal portion

(interaural 2.7±3.2 mm, two sections per section group). Thus, a total

of six or seven coronal sections between interaural 2.7 and 4.5 mm

were examined for each rat, on both right and left sides. A transitional

area exists between the substantia nigra and the VTA, where the

medial substantia nigra and ventrolateral VTA are gradually merged.

Thus, three areas (substantia nigra, transitional area and VTA) were

quantitatively examined at each of the three rostral-caudal levels

described above, for a total of nine midbrain subregions: rostral,

intermediate and caudal substantia nigra; rostral, intermediate and

caudal transitional area; and rostral, intermediate and caudal VTA

(Fig. 1C). This strategy for analysing the midbrain has been used in

our previous study of DA transporter mRNA expression (Lu & Wolf,

1997). The region scanned for the substantia nigra includes both the

pars reticulata and pars compacta regions. It is dif®cult to determine

the border between these regions on autoradiographs, where only

silver grains are visible, not stained cells.

NIH Image computer software was used for quantitative analysis of

autoradiographs. Within each region, there are areas that should not

be included in the analysis of speci®c signals, such as white matter

areas located within these structures (e.g. the anterior commissure),

blood vessels and areas where the section was damaged. To separate

such areas from those with speci®c signals, the threshold function of

the NIH Image program was employed with a cutoff value. The cutoff

value was determined by making background measurements in

surrounding white matter regions and was de®ned as the mean of

these background measurements plus two standard deviations (to set

FIG. 1. Diagram illustrating the borders of scanned brain regions. (A) medialprefrontal cortex (PFC). (B) Nucleus accumbens (NAc) and its subregions,core and shell. (C) Subregions of the ventral midbrain: substantia nigra (SN),ventral tegmental area (VTA) and a transitional area between the two (TA).

Amphetamine and NMDAR1 3169

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

the cutoff level at a point that would be > 95% of all background

measurements). The areas that exhibited values lower than this cutoff

were de®ned as background. In regions with values greater than the

cutoff, the speci®c signal was de®ned as the total signal minus the

mean background signal. Data were expressed as nCi/g dry tissue

weight, determined using 14C-standard microscales.

Data analysis

Statistical comparison of data from amphetamine and vehicle groups

was performed using a two-tailed Student's t-test. For ®gures, data for

amphetamine pretreatment groups are expressed as percentage of the

corresponding control group (e.g. `amphetamine + 3-day withdrawal'

rats are compared with `vehicle + 3-day withdrawal' rats).

Results

Distribution of NMDAR1 mRNA and immunolabelling

For each rat in each pretreatment group, we determined average

levels of labelling in the nucleus accumbens or prefrontal cortex

by scanning both right and left sides of several coronal sections

spanning the rostral-caudal extent of these regions. For the

midbrain, we analysed nine subregions (VTA, substantia nigra and

a transitional area, each at rostral, intermediate and caudal levels)

to minimize the possibility of an effect in one subregion being

masked by lack of effect in others. Boundaries of scanned regions

are illustrated in Fig. 1. To reduce variability, different groups

were processed simultaneously to as great an extent as possible,

including drug injections, perfusions, sectioning, immunocyto-

chemical staining, in situ hybridization, section mounting and so

on (for details, see Materials and methods). Our sampling and

processing methods enabled accurate and reproducible measure-

ments, as demonstrated by small standard errors in all experi-

mental groups (see Figs 3±5).

Representative autoradiographs from control rats are presented in

Fig. 2 to illustrate the distribution of NMDAR1 mRNA and

NMDAR1 immunolabelling in the nucleus accumbens, prefrontal

cortex and midbrain. NMDAR1 mRNA and NMDAR1 immunolabel-

ling were expressed at moderate levels throughout the nucleus

accumbens, with no apparent differences between core and shell

subregions. In the prefrontal cortex, moderate levels of NMDAR1

mRNA were observed in all layers except the most super®cial.

NMDAR1 immunolabelling exhibited a similar pattern of distribu-

tion. In the midbrain, NMDAR1 immunolabelling was present

throughout the VTA, transitional area and both pars compacta and

zona reticulata regions of the substantia nigra. Our results are

consistent with those reported previously in rat for nucleus

accumbens (Petralia et al., 1994; Standaert et al., 1994; Sato et al.,

1995), prefrontal cortex (Sato et al., 1995; Rudolf et al., 1996), and

midbrain (Petralia et al., 1994; Standaert et al., 1994; Sato et al.,

1995).

Effect of amphetamine on NMDAR1 expression in the ventralmidbrain

Figure 3 shows the effect of repeated saline or amphetamine

administration on NMDAR1 immunolabelling in nine subregions of

the rat midbrain at 3- and 14-day withdrawal times. The only

signi®cant effect was a decrease in NMDAR1 immunolabelling at the

14-day withdrawal time in intermediate and caudal subregions of the

substantia nigra (83.5 6 3.4 and 85.0 6 3.7% of vehicle group values,

respectively, P < 0.05). There was a trend towards decreased

NMDAR1 immunolabelling in intermediate and caudal subregions

of the VTA and transitional area at the 14-day withdrawal time, but it

did not reach statistical signi®cance. At the mRNA level, we detected

a signal that was higher than background but too small for reliable

quanti®cation using our in situ hybridization method (data not

shown).

Effect of amphetamine on NMDAR1 expression in the nucleusaccumbens

For all experiments, we analysed the entire nucleus accumbens as

well as core and shell subregions (see Materials and methods). Data

from in situ hybridization and immunocytochemical experiments are

presented in Fig. 4. Levels of NMDAR1 mRNA and immunolabelling

in the nucleus accumbens and its subregions did not differ

signi®cantly between amphetamine and saline pretreatment groups

at the 3-day withdrawal time. However, after 14 days of withdrawal,

NMDAR1 mRNA levels in the entire nucleus accumbens were

signi®cantly reduced in the amphetamine pretreatment group

(74.8 6 7.7% of vehicle group levels, P < 0.05). A signi®cant

reduction was also observed when core or shell subregions were

scanned separately (80.2 6 7.0 and 75.6 6 6.7% of vehicle group

levels, respectively, P < 0.05). Corresponding decreases in NMDAR1

immunolabelling at the 14-day withdrawal time were observed in the

entire nucleus accumbens (76.7 6 4.4%, P < 0.05), the core

(74.7 6 4.2%, P < 0.05), and the shell (73.0 6 5.4%, P < 0.05).

Effect of amphetamine on NMDAR1 expression in the medialprefrontal cortex

In situ hybridization and immunocytochemical data are presented in

Fig. 5. NMDAR1 mRNA levels did not differ signi®cantly between

vehicle and amphetamine groups at the 3-day withdrawal time, but

were signi®cantly reduced in the amphetamine group at the 14-day

withdrawal time (76.1 6 7.1% of vehicle group, P < 0.05). NMDAR1

immunolabelling was not signi®cantly altered at the 3-day with-

drawal time. After 14 days of withdrawal, there was a trend towards a

decrease in NMDAR1 immunolabelling (89.5 6 7.0%) but it failed to

reach statistical signi®cance.

Discussion

Summary

No changes in NMDAR1 expression were found in any region after

3 days of withdrawal from repeated amphetamine. However, after

14 days of withdrawal, signi®cant decreases in NMDAR1 levels were

found in the nucleus accumbens, the medial prefrontal cortex, and

intermediate and caudal portions of the substantia nigra.

Relationship to amphetamine-induced behavioural changes

One of the most striking behavioural changes produced by repeated

amphetamine administration is behavioural sensitization, de®ned as

the progressive augmentation of behavioural responses to psychos-

timulants that occurs during and following their repeated adminis-

tration (Robinson & Becker, 1986). Sensitization provides a model

for the intensi®cation of drug craving that is a hallmark of addiction

(Robinson & Berridge, 1993). After regimens similar to the present

one, sensitization can be demonstrated immediately after disconti-

nuation of drug administration but intensi®es with length of

withdrawal (Li & Wolf, 1997 and references therein). Different

cellular mechanisms appear to contribute to sensitization at different

withdrawal times (see White & Kalivas, 1998). Thus, the decrease in

NMDAR1 expression observed at the 14-day time may be involved in

later phases of the consolidation or maintenance of sensitization. It is

more dif®cult to determine whether these alterations might contribute

to a withdrawal syndrome, because withdrawal syndromes have not

3170 M. E. Wolf et al.

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

been well-characterized after regimens similar to the present one.

However, after a more aggressive escalating dose regimen, a

withdrawal syndrome characterized by decreased nocturnal locomo-

tion and a decrease in the ef®cacy of intracranial self-stimulation

reward can be demonstrated. These behavioural changes last at least

7 days following discontinuation of drug administration but are

normalized by 28 days of withdrawal (Paulson et al., 1991; Munn &

Wise, 1992; Paulson & Robinson, 1996). It is conceivable that the

observed decreases in NMDAR1 contribute to a withdrawal

syndrome, particularly if their onset occurs somewhere in between

the 3- and 14-day withdrawal times examined in our study. Possible

relationships of altered NMDAR1 expression to both sensitization

and withdrawal syndromes are discussed below for midbrain, nucleus

accumbens and prefrontal cortex.

NMDAR1 in the ventral midbrain

Several lines of evidence suggest that an increase in excitatory drive

to midbrain DA neurons may be a necessary step in the cascade

leading to sensitization (Clark & Overton, 1998; Wolf, 1998). Thus

there has been considerable interest in the possibility that psychos-

timulants regulate glutamate receptor expression in the midbrain.

There have been reports of increased levels of GluR1 and NMDAR1

FIG. 2. Representative autoradiographs illustrating localization of NMDAR1 mRNA and immunolabelling in the rat prefrontal cortex (top), nucleus accumbens(middle) and ventral midbrain (bottom). Levels of NMDAR1 mRNA were determined using a quantitative method of in situ hybridization with 35S-labelledoligonucleotide probes, while autoradiographic immunocytochemistry with 35S-labelled secondary antibodies was used to quantitatively examine NMDAR1immunolabelling (see Materials and methods section).

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Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

levels in the VTA but not substantia nigra of rats killed 16±18 h after

discontinuation of chronic ethanol, cocaine or stress paradigms (Ortiz

et al., 1995; Fitzgerald et al., 1996). In contrast, we have not observed

signi®cant changes in GluR1 levels in substantia nigra or VTA after

withdrawal from repeated amphetamine (16±18 h, 3-day and 14-day

time-points were examined) (Monteggia et al., 1997; and W. Lu,

L. M. Monteggia & M. E. Wolf, unpublished results), suggesting

differences between cocaine and amphetamine. In the present study,

we found that NMDAR1 immunolabelling in the midbrain was not

altered after 3 days of withdrawal from amphetamine, suggesting that

the increase in NMDAR1 observed by Ortiz et al. (1995) and

Fitzgerald et al. (1996) after cocaine, ethanol or stress either does not

occur after amphetamine administration or that it occurs but is very

short-lived. After 14 days of withdrawal, NMDAR1 immunolabelling

was signi®cantly decreased in intermediate and caudal regions of the

substantia nigra. There was also a trend towards decreased NMDAR1

in intermediate and caudal portions of the VTA and transitional area.

It remains possible that these latter changes are functionally

signi®cant and that failure to achieve statistical signi®cance re¯ects

lower levels of NMDAR1 expression in the VTA and transitional area

compared with the substantia nigra (present results; Sato et al., 1995;

Paquet et al., 1997).

NMDAR1 is expressed throughout the rat substantia nigra and

VTA (Petralia et al., 1994; Standaert et al., 1994; Sato et al., 1995),

and an electron microscopic study in squirrel monkey found that all

DA neurons in these regions were immunoreactive for NMDAR1

(Paquet et al., 1997). It is thus possible that decreased NMDAR1

expression occurred within DA neurons of the substantia nigra. While

electrophysiological approaches could be used to test this possibility,

the appropriate studies have not been performed in the substantia

nigra. In the VTA, however, we have used extracellular recording to

examine the sensitivity of VTA DA neurons to glutamate agonists

after the same amphetamine regimen and withdrawal times used for

our receptor expression studies (White et al., 1995a; Zhang et al.,

1997). VTA DA neurons showed enhanced responsiveness to

iontophoretic glutamate and AMPA at the 3-day withdrawal time,

with both responses normalizing by 14 days. However, there was no

change in sensitivity to NMDA at either withdrawal time, consistent

with the present ®nding of no change in NMDAR1 expression within

the VTA. Another study examined the responsiveness of VTA and

substantia nigra DA neurons to electrical stimulation of the prefrontal

cortex after an amphetamine regimen similar to that used in the

FIG. 3. Effect of repeated amphetamine administration on NMDAR1 im-munolabelling in the rat midbrain. Levels of NMDAR1 immunolabelling werecompared between vehicle- and amphetamine-pretreatment groups after 3 and14 days of withdrawal. Autoradiographs were analysed quantitatively usingNIH Image software. For each rat, nine subregions of the ventral midbrain(substantia nigra, ventral tegmental area and a transitional area; each at rostral,intermediate and caudal levels) were analysed by scanning both right and leftsides of two or three coronal sections at various rostral-caudal levels (seeMaterials and methods section). The bars represent the mean 6 SEM of suchdeterminations from nine rats in each pretreatment group. In this and allsubsequent ®gures, data are presented as percentage of the appropriate vehiclecontrol group, i.e. the `amphetamine + 3-day withdrawal' group is comparedwith the `vehicle + 3-day withdrawal' group. Groups were compared using thetwo-tailed Student's t-test. *P < 0.05.

FIG. 4. Effect of repeated amphetamine administration on the expression ofNMDAR1 mRNA and NMDAR1 immunolabelling in the rat nucleusaccumbens (NAc). Vehicle and amphetamine pretreatment groups werecompared after 3 and 14 days of withdrawal. For each rat in each pretreatmentgroup, the average levels of NMDAR1 mRNA (A) and NMDAR1immunolabelling (B) in the entire NAc and its subregions, core and shell,were determined by scanning both right and left sides of several coronalsections spanning the rostral-caudal extent of the NAc (see Materials andmethods for details of analysis). The bars represent the mean 6 SEM of suchdeterminations from eight to 10 rats in each pretreatment group. *P < 0.05,two-tailed Student's t-test.

3172 M. E. Wolf et al.

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

present study and found an increase in the likelihood of excitatory

responses on withdrawal day 10 (Tong et al., 1995). In that these

results indicate a potentiation of excitatory drive to substantia nigra

DA neurons, they appear to con¯ict with the present ®ndings of

decreased NMDAR1 expression, particularly because the prefrontal

cortex excites midbrain DA neurons at least in part through NMDA

receptor activation (Tong et al., 1996). However, Tong et al. (1995)

only recorded from DA neurons in the medial substantia nigra (0.4±

1.3 mm lateral to midline) so the results are dif®cult to compare with

those from our receptor expression studies, which sampled the entire

substantia nigra.

If decreased NMDAR1 expression did occur within substantia

nigra DA neurons, it might suggest that NMDA receptor-mediated

excitatory drive to these neurons declines sometime between 3

and 14 days of withdrawal. This, in turn, might lead to decreased

®ring rates and decreased DA release from their terminals in the

striatum. Because decreases in extracellular DA following with-

drawal may contribute to dysphoric effects that lead to drug

craving (Koob et al., 1997), many studies have examined basal

extracellular DA levels in striatum and nucleus accumbens after

withdrawal from psychostimulants. While some found decreased

DA levels in nucleus accumbens after cocaine or amphetamine

withdrawal (Parsons et al., 1991; Robertson et al., 1991; Imperato

et al., 1992; Rossetti et al., 1992; Weiss et al., 1992), others did

not (Segal & Kuczenski, 1992a,b;Crippens et al., 1993; Kalivas &

Duffy, 1993; Wolf et al., 1993; Crippens & Robinson, 1994;

Hooks et al., 1994; Heidbreder et al., 1996). The discrepancies are

dif®cult to attribute to different drug regimens or withdrawal

times (e.g. Crippens & Robinson, 1994), but may re¯ect

differences between striatal subregions. Paulson & Robinson

(1996) used an escalating dose regimen of amphetamine that

results in a withdrawal syndrome characterized by nocturnal

hypoactivity at early withdrawals (3 or 7 days) but not at a later

withdrawal (28 days). At the early withdrawal times, basal

extracellular levels of DA and its metabolites were decreased in

the dorsolateral caudate but not the nucleus accumbens. Our

results, showing a signi®cant decrease in NMDAR1 in the

substantia nigra but not the VTA, are consistent with the idea

that certain changes in DA transmission during withdrawal from

repeated amphetamine administration may be more pronounced in

the nigrostriatal DA system than the mesolimbic DA system.

While most NMDAR1 immunolabelling in the substantia nigra is

associated with postsynaptic densities of asymmetric synapses

established on dendritic shafts and spines, some presynaptic labelling

is observed, often on presumed glutamatergic terminals (Paquet et al.,

1997). Our results could indicate a decreased number of such NMDA

autoreceptors. Because NMDA autoreceptors facilitate glutamate

release in other brain regions (Young & Bradford, 1991; Bustos et al.,

1992), their loss might dampen excitatory drive within the substantia

nigra. NMDAR1 expression was also decreased in prefrontal cortex

(below), so it is possible that prefrontal cortex neurons projecting to

substantia nigra exhibit reduced NMDAR1 expression both at cell

body and nerve terminal levels. Finally, it is possible that NMDAR1

expression is altered on terminals of GABAergic striatal projection

neurons, because all of these neurons express NMDAR1 mRNA

(Standaert et al., 1994, 1999).

NMDAR1 in the nucleus accumbens

NMDAR1 expression was unchanged in the nucleus accumbens after

3 days of withdrawal from repeated amphetamine administration.

Similarly, another study found no change in NMDAR1 subunit levels

in the nucleus accumbens 16±18 h after discontinuation of repeated

cocaine administration (Fitzgerald et al., 1996). However, after

14 days of withdrawal, we observed signi®cant decreases in

NMDAR1 mRNA and NMDAR1 immunolabelling in both core

and shell subregions. NMDAR1 immunolabelling is present post-

synaptically in medium spiny neurons (Gracy & Pickel, 1996; Gracy

et al., 1997) and is frequently coexpressed with DA receptors (Ariano

et al., 1997). However, electron microscopic studies in the nucleus

accumbens shell indicate that NMDAR1 immunolabelling is more

frequently observed in axons and axon terminals, including

glutamatergic and DA terminals (Gracy et al., 1997). This raises the

question of whether the observed decrease in NMDAR1 occurred in

presynaptic or postsynaptic receptor populations. Because decreases

in mRNA and immunolabelling were of similar magnitude (74.8 and

76.7% of control, respectively), it seems most likely that decreased

NMDAR1 expression occurred in principal neurons of the nucleus

accumbens (which would contain both mRNA and protein) rather

than nerve terminals.

In previous studies, we examined the electrophysiological respon-

siveness of nucleus accumbens neurons recorded from rats treated

with this amphetamine regimen or with repeated cocaine. Nucleus

accumbens neurons recorded from amphetamine- or cocaine-treated

rats were subsensitive to the excitatory effects of iontophoretic

glutamate, AMPA and NMDA at both the 3- and 14-day withdrawal

times (White et al., 1995a; White et al., 1999). Subsensitivity at the

early withdrawal time may re¯ect decreased Na+ currents in nucleus

accumbens neurons (Zhang et al., 1998). However, at the 14-day

withdrawal time, subsensitivity to AMPA may be attributable to

decreases in the GluR1 and GluR2 expression in the nucleus

accumbens that we have observed in previous studies using this

regimen and withdrawal time (Lu et al., 1997; Lu & Wolf, 1999)

whereas subsensitivity to NMDA may re¯ect the decreased

NMDAR1 expression observed in the present study.

Although cortically-evoked EPSPs in striatal and nucleus accum-

bens neurons are mediated primarily by AMPA receptors (e.g.

Pennartz et al., 1990, 1991; Hu & White, 1996), the contribution of

NMDA receptors increases as the cell membrane becomes more

depolarised (Kita, 1996). Thus, the observed decrease in NMDAR1

expression would have its greatest impact during convergent

activation of nucleus accumbens neurons by multiple excitatory

FIG. 5. Effect of repeated amphetamine administration on the expression ofNMDAR1 mRNA and NMDAR1 immunolabelling in the rat medial prefrontalcortex (PFC). Vehicle and amphetamine pretreatment groups were comparedafter 3 and 14 days of withdrawal. For each rat in each pretreatment group, theaverage levels of NMDAR1 mRNA (A) and NMDAR1 immunolabelling (B)were determined by scanning all layers of the PFC on both right and left sidesof several coronal sections spanning the rostral-caudal extent of the PFC (seeMaterials and methods for details of analysis). The bars represent the mean6 SEM of such determinations from eight to 10 rats in each pretreatmentgroup. *P < 0.05, two-tailed Student's t-test.

Amphetamine and NMDAR1 3173

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3167±3177

inputs (O'Donnell & Grace, 1995) and would likely in¯uence NMDA

receptor-dependent plasticity in the corticostriatal pathway (Pennartz

et al., 1993; Kombian & Malenka, 1994; Calabresi et al., 1996).

Under normal circumstances, modulation of glutamate transmission

by DA may serve to regulate the signal-to-noise ratio in nucleus

accumbens (Kiyatkin & Rebec, 1996). One type of neuromodulatory

effect involves potentiation of NMDA receptor-mediated responses in

striatal neurons by D1 receptor stimulation (Cepeda & Levine, 1998).

Decreased NMDAR1 expression, combined with sensitization-related

enhancement of D1 receptor function (Henry & White, 1991), may

profoundly alter such interactions, leading to dysregulation of

dopaminergic modulation of goal-directed behaviour (see Salamone

et al., 1997).

NMDAR1 in the medial prefrontal cortex

In the prefrontal cortex, we found a signi®cant decrease in NMDAR1

mRNA after 14 days of withdrawal, accompanied by a statistically

nonsigni®cant reduction in NMDAR1 immunolabelling. Both

NMDAR1 mRNA and NMDAR1 protein are located in cortical

pyramidal cells, while only NMDAR1 protein is present in neuropil

(Conti et al., 1994; Petralia et al., 1994; Rudolf et al., 1996). It is thus

possible that a decrease in NMDAR1 protein levels in pyramidal cells

could have been masked by no change or an increase in presynaptic

NMDAR1 levels. Alternatively, these results may suggest a role for

post-transcriptional control of NMDAR1 synthesis (Sucher et al.,

1993 and references therein).

Both NMDA and AMPA receptors contribute to excitatory

postsynaptic potentials in prefrontal cortex pyramidal neurons

(Hirsch & CreÂpel, 1990; Law-Tho et al., 1994; Arvanov & Wang,

1997). Indirect evidence indicates that NMDA receptors also

modulate glutamate release in prefrontal cortex (Arvanov & Wang,

1997; Moghaddam et al., 1997). As for DA±glutamate interactions,

D1 agonists decrease NMDA and AMPA components of glutamate

transmission in pyramidal cells of the rat prefrontal cortex (Law-Tho

et al., 1994), whereas neurochemical studies indicate that NMDA

receptors exert a tonic inhibitory control over DA release that is likely

mediated through interneurons (Hata et al., 1990; Wedzony et al.,

1993; Hondo et al., 1994; Nishijima et al., 1994; Kashiwa et al., 1995;

Jedema & Moghaddam, 1996).

Many ®ndings suggest that the prefrontal cortex, an important

source of excitatory amino acid projections to VTA and nucleus

accumbens (Sesack & Pickel, 1992), is critical to sensitization.

Kindling of the prefrontal cortex produces sensitization (Schenk &

Snow, 1994), lesion studies indicate that prefrontal cortex projections

provide the glutamatergic tone in VTA that is required for induction

of sensitization (Wolf et al., 1995; Cador et al., 1997; Tzschentke &

Schmidt, 1998; Li et al., 1999), and the responsiveness of midbrain

DA neurons to prefrontal cortex stimulation is altered in sensitized

rats (Tong et al., 1995). Alterations in both glutamate and DA

transmission in the prefrontal cortex may contribute to sensitization.

After 3 days of withdrawal from amphetamine, we have found

increased GluR1 mRNA and immunolabelling in prefrontal cortex

(Lu et al., 1997; Lu & Wolf, 1999) and a corresponding increase in

responsiveness of prefrontal cortex neurons to the excitatory effects

of glutamate (Peterson et al., 1998). Enhanced activation of prefrontal

cortex neurons via AMPA receptors, combined with a decreased

inhibitory in¯uence of DA in the prefrontal cortex (Sorg & Kalivas,

1993; White et al., 1995b; Sorg et al., 1997; Peterson et al., 1998),

might increase the activity of excitatory prefrontal cortex projections

to VTA and thereby contribute to the transient increase in DA cell

activity that is believed to represent an obligatory step in the

sensitization cascade (Zhang et al., 1997; Clark & Overton, 1998;

Henry et al., 1998; Wolf, 1998).

What then is the functional signi®cance of decreased NMDAR1

expression in the prefrontal cortex after 14 days of withdrawal?

Assuming it occurs in pyramidal neurons, one prediction is that the

activity of prefrontal cortex projections to the midbrain might be

decreased due to a reduction in NMDA receptor-mediated excitation

of prefrontal cortex neurons. Thus, excitatory drive to midbrain DA

neurons may be decreased directly, via reductions in NMDAR1

expression in substantia nigra (above), as well as indirectly, via

reductions in afferent activity. Similarly, decreased activity of

prefrontal cortex projections to the nucleus accumbens may

exacerbate the effects of local decreases in NMDAR1 in this region.

Finally, prefrontal cortex neurons in sensitized rats might be less

likely to undergo adaptations requiring NMDA receptor-dependent

long-term potentiation (e.g. Jay et al., 1995).

Conclusions

Our results demonstrate that decreased NMDAR1 expression is

one mechanism by which repeated stimulant administration alters

neurotransmission within reward-related neuronal circuits. Because

NMDAR1 is required for the formation of functional channels,

changes in NMDAR1 expression may provide an index of

changes in the number of functional NMDA receptors.

Decreased NMDAR1 expression in nucleus accumbens could

account for previously reported decreases in electrophysiological

responsiveness to glutamate in sensitized rats and for dysregula-

tion of goal-directed behaviour. Decreased NMDAR1 expression

in the substantia nigra may reduce excitatory drive to substantia

nigra DA neurons. Decreased NMDAR1 in prefrontal cortex may

reduce excitability of prefrontal cortex neurons as well as their

targets. For example, decreased activity of excitatory prefrontal

cortex projections to the nucleus accumbens or midbrain could

synergize with local decreases in NMDAR1 to further reduce the

excitability of neurons in these regions. Such changes may

contribute to the broad restructuring of reward-related circuitry

that appears to underlie the persistence of drug dependence and

vulnerability to relapse (Koob et al., 1998).

Acknowledgements

We thank Chang-Jiang Xue and Christy Stine for assisting with someprocedures. This work was supported by USPHS grant DA09621 to M.E.W.

Abbreviations

AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate; DA, dopamine;NMDA, N-methyl-D-aspartate; PB, phosphate buffer PBT, PB containing0.3% Triton X-100; VTA, ventral tegmental area.

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