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. Author manuscript; available in PMC: 2013 Feb 28.
Published in final edited form as: J Neurosci Res. 2012 Apr;90(4):895–904. doi: 10.1002/jnr.22821

Distinct Roles of Dopamine D3 Receptors in Modulating Methamphetamine-Induced Behavioral Sensitization and Ultrastructural Plasticity in the Shell of the Nucleus Accumbens

Jie Zhu 1,2, Yanjiong Chen 3, Na Zhao 1,2, Guofen Cao 1,2, Yonghui Dang 1,2, Wei Han 1,2, Ming Xu 4, Teng Chen 1,2,*
PMCID: PMC3584342  NIHMSID: NIHMS442170  PMID: 22420045

Abstract

Persistent changes in behavior and psychological function that occur as a consequence of exposure to drugs of abuse are thought to be mediated by the structural plasticity of specific neural circuits such as the brain’s dopamine (DA) system. Changes in dendritic morphology in the nucleus accumbens (NAc) accompany druginduced enduring behavioral and molecular changes, yet ultrastructural changes in synapses following repeated exposure to drugs have not been well studied. The current study examines the role of DA D3 receptors in modulating locomotor activity induced by both acute and repeated methamphetamine (METH) administration and accompanying ultrastructural plasticity in the shell of NAc in mice. We found that D3 receptor mutant (D3−/−) mice exhibited attenuated acute locomotor responses as well as the development of behavioral sensitization to METH compared with wild-type mice. In the absence of obvious neurotoxic effects, METH induced similar increases in synaptic density in the shell of NAc in both wild-type and D3−/− mice. These results suggest that D3 receptors modulate locomotor responses to both acute and repeated METH treatment. In contrast, the D3 receptor is not obviously involved in modulating baseline or METH-induced ultrastructural changes in the NAc shell.

Keywords: dopamine D3 receptor, methamphetamine, behavioral sensitization, ultrastructural plasticity, nucleus accumbens shell

An essential feature of drug addiction is that individuals experience persistent compulsive drug seeking and taking despite adverse physical or psychosocial consequences (Wise, 2000; Dackis and O’Brien, 2005; Kalivas and Volkow, 2005; Hyman et al., 2006; Kauer and Malenka, 2007; Kalivas and O’Brien, 2008; Koob and Volkow, 2010). It has been hypothesized that long-term changes such as structural plasticity that occur within the brain’s reward circuitry are at least partially responsible for the long-lasting behavioral changes (Robinson and Berridge, 2000). One commonly used behavioral paradigm for studying drug-induced persistent changes is behavioral sensitization, which is a progressive increase in behavioral responsivity following intermittent repeated administration of drugs of abuse such as methamphetamine (METH; Robinson and Berridge, 2000, 2003). It is thought that the nucleus accumbens (NAc) shell is particularly important for the development of behavioral sensitization to stimulants (Ito et al., 2004).

Enduring changes in behavior and psychological function that occur as a consequence of exposure to drugs of abuse are also thought to be mediated by the reorganization of synaptic connections in brain’s reward circuitry, such as the NAc (Robinson and Kolb, 2004). A useful approach to examine the impact of abused drugs on synaptic reorganization is the use of Golgistained material to quantify changes in dendrites and the density of dendritic spines in different brain regions. With this approach, it has been found that repeated exposure to amphetamine or cocaine alters the number of dendrites and the density of dendritic spines of neurons in the NAc and cortex (Robinson and Kolb, 1997, 1999; Robinson et al., 2001; Norrholm et al., 2003; Zhang et al., 2006; Ren et al., 2010). In these cases, the structure of dendrites or the density of spines on neurons was quantified by measuring total dendritic lengths, total numbers of dendritic branches, and spine density (Robinson and Kolb, 1999; Zhang et al., 2006; Singer et al., 2009; Ren et al., 2010). These measures, however, do not provide a direct measure of potential changes in synapses. Without ultrastructural studies directly on synapses, it remains unknown whether changes in dendrites and spines are accompanied by alterations in synaptic contacts, although this is likely to be the case.

The mesocorticolimbic dopamine (DA) system mediates the effects of drugs of abuse (Koob and Volkow, 2010). D3 receptors are expressed mainly in the NAc, and activation of these receptors is negatively linked to the cAMP production (Missale et al., 1998). Previous pharmacological studies demonstrated that D3 receptors may play an important role in mediating druginduced reward and positive reinforcing effects (Heidbreder et al., 2005) and cocaine-seeking behavior (Pilla et al., 1999). Using D3 receptor mutant mice, we showed that this receptor is also involved in consolidation (Kong et al., 2011) and extinction (Chen and Xu, 2010) of reward learning induced by cocaine or amphetamine (Xu et al., 1997). We and others also showed that D3 receptor modulate acute and repeated stimulantinduced locomotor activity (Richtand et al., 2003; McNamara et al., 2006; Prichard et al., 2007; Chen et al., 2007; Liu et al., 2009). Taken together, these results suggest that D3 receptors contribute to the development of drug-induced behavior.

The current study used normal and D3 receptor mutant (D3−/−) mice to evaluate the role of this receptor in METH-induced acute locomotion and behavioral sensitization. We then used electron microscopy (EM) to quantify synaptic density directly to determine the ultrastructural plasticity in the NAc shell associated with METH-induced behavioral sensitization in mice. Our results suggest that D3 receptors play distinct roles in modulating METH-induced behavioral sensitization and ultrastructural plasticity in the shell of the NAc.

MATERIALS AND METHODS

Animals

We obtained D3−/− mice generated byXu et al. (1997), and the mice were back-crossed from the 129Sv/C57BL6J genetic background to the C57BL6J background for three generations. Homozygous mutant and wild-type littermates were produced by crossing D3 receptor heterozygous mutant mice and were genotyped by polymerase chain reaction (PCR). Four oligonucleotide primers were used in PCRs with genomic DNA isolated from tails of wild-type and D3−/− mice. The sequences of these primers are 5′-AGCAAGGCGAGATGACAGGA, 5′-CAAGATGGATTGCACGCAGG, 5′-GCTCACCACTAGGTAGTTG, and 5′-ACCTCTGAGCCAGATAAGC. D3−/− mice exhibit apparent normal development. We previously showed that DA-containing systems of the D3−/− brains appear to be normal, and the mutation of the D3 receptor gene did not significantly affect the expression of D1 class receptors or other D2 class receptors (Xu et al., 1997).

Male mice were housed under a 12-hr light/dark cycle and were housed in groups of two with food and water provided ad libitum. Both temperature and humidity of the housing room were controlled. All animal protocols used were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. All efforts were made to minimize the number of animals used and the distress to the animals. We used a total of 50 each of adult male D3−/− and wild-type mice (aged 7–8 weeks) weighing about 20–25 g in the current study.

Drugs

METH hydrochloride was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, People’s Rebulic of China), and was dissolved in 0.9% physiological saline. The volume of intraperitoneal (i.p.) injection was 10 ml/kg.

Behavioral Analyses

The overall scheme for the behavioral studies is shown in Figure 1. Each mouse was handled for 3 min once daily for 7 consecutive days before all treatments were carried out (days 1–7). For acute and behavioral sensitization measurement, mice were habituated to the activity chambers (43 × 43 × 43 cm) for 2 days (days 8 and 9). All groups of mice were injected i.p. once daily with saline on days 8 and 9 and their horizontal locomotor activities were determined by a Smart Video Tracking System (version 2.5; Palnlab Technology for Bioresearch, Spain) for 60 min before and after the injections.

Fig. 1.

Fig. 1

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Treatment schedules. Days 1–7: Mice were handled for 3 min once daily to habituate them to the experimenter. Days 8, 9: Mice were injected i.p. with 10 ml/kg saline once daily, and their horizontal locomotor activities were measured for 60 min before and after the injections to habituate them to the open field and drug treatment. Days 10–14, days 17–21, days 24–28, days 31–35, and day 64: For the 2 mg/kg METH and saline groups, mice were given once daily injections of either METH or saline, and their horizontal locomotor activities were determined for 60 min before and after the injections. Days 15 and 16, days 22 and 23, days 29 and 30, and days 36–63: Mice were left undisturbed.

For acute studies, mice were habituated again for 1 hr, followed by an injection of either METH (at the dose of 0.2, 0.6, or 2 mg/kg) or saline (n = 6–8 mice each). Their horizontal locomotor activities were determined for 60 min after the injections (day 10). For behavioral sensitization studies, wild-type and D3−/− mice were then divided into two groups, a 2 mg/kg METH and a saline group (n = 17 on days 8–17 and n = 8 on days 18–64 per group). Mice were treated as shown in Figure 1 (days 10–64). The treatment regimens were based on previous studies (Robinson and Kolb, 1997, 1999; Robinson et al., 2001; Singer et al., 2009), with some modifications. Briefly, mice were given once daily injections of METH or saline for 5 consecutive days followed by 2 injection-free days, and this procedure was repeated for a total of four times. Mice were then given a 4-week withdrawal period to allow potential changes in morphology to occur (Robinson and Kolb, 1997, 1999; Robinson et al., 2001; Singer et al., 2009). On day 64, mice were given a challenge injection of either 2 mg/kg METH or saline. Horizontal locomotor activities were determined on all drug treatment days for 60 min before and after the injections. Injections were performed during the light phase of the light/dark cycle.

A factorial-designed ANOVA was used to analyze the effects of acute METH and genotype on locomotor activities, and an SNK-q test was used to analyze the multiple comparison. Two-way repeated-measures (two-way RM) ANOVA with a post hoc multiple-comparisons test was used to analyze the effects of time, METH, and genotype on behavioral sensitization and the differences in the entire observation time or at each time point among different mouse groups.

Brain Sample Preparation for EM and Data Analysis

For EM analysis, six mice from each of the four treatment mouse groups were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) on day 65, 24 hr after the challenge treatment with METH or saline. This withdrawal time was chosen to avoid potential changes associated with acute injections (Zhang et al., 2005). The brain tissues were fixed by vascular perfusion through the left ventricle of the heart with sequential delivery of 50 ml saline and 60 ml 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4; Sesack and Pickel, 1990). Brains were then removed, and bilateral NAc shells were dissected out on an ice-cold brain matrix immediately (Paxinos and Franklin, 2001; see Fig. 4). These tissues were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M PB for 30 min and then postfixed in 1% osmium tetroxide in 0.1 M PB, dehydrated, and flat-embedded in epon (19% EM bedding media-812; 36% dodecenyl succinic anhydride; 44% methyl nadic anhydride; 1% benyzldimethylamine). Brain samples were then cut into 50-nm coronal ultrathin sections through the NAc shell with an ultramicrotome (LKB-V).

Fig. 4.

Fig. 4

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Coronal brain section representing relative sites of dissected NAc shell (hatched area). NAcsh, nucleus accumbens shell; NAcc, nucleus accumbens core; ac, anterior commissure; CPu, caudate putamen. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Forty-eight ultrathin sections of the NAc shell (two sections from each mouse from four treatment groups) were used for electron microscopic (H-7650) analysis. Ultrathin sections of NAc shell were stuck using a copper screen with a mesh of 300, and three mesh squares were randomly selected from each NAc shell region. These regions were analyzed at magnifications of ×25,000, and synapses were analyzed in a total tissue area of 70,650 µm2 per mouse (3 × 23,550 µm2) in the NAc shell. Equal tissue areas were examined from each mouse in all groups (n = 6). Synapses were considered to be identified when 1) they contained three or more synaptic vesicles in their presynaptic element and 2) there were obvious compact layer in their postsynaptic element (Jones and Calverley, 1991; Peters et al., 1991). All EM analysis was carried out blindly in the current study.

Synaptic density is presented as the total number of identified synapses in all of the three selected regions from each mouse. A factorial-designed ANOVA was used to analyze the effects of METH and genotype, and an SNK-q test was used to analyze the multiple comparisons. Multiple linear regression was used to analyze the effect of METH on wild-type and D3−/− mice after controlling for the corresponding saline group. Dependent variable was synaptic density, and the covariant was defined as follows: genotype was treated as the binary variable (wild-type = 1, D3−/− = 0); synaptic density in saline groups was treated as the continuous variable.

Effects of Repeated METH Injections on the NAc

Both rapid Golgi staining and Nissl staining were performed to determine potential METH-induced degeneration of neurons and nerve terminals. For rapid Golgi staining, two mice from each of the four treatment groups were decapitated quickly 24 hr after the challenge treatment with METH or saline on day 65, and their brain samples were processed as previously described (Gundappa and Desiraju, 1988; Shankaranarayana Rao et al., 2001). The stained brain tissues were then cut into 200-µm sections. Brain areas were identified at ×100 magnification, and neurons from each hemisphere of the NAc shell were viewed with a microscope (DM 3000) at magnifications of ×200 for neurons and nervous processes and ×400 for spines. At least 10 neurons were evaluated for each region in each hemisphere per mouse to establish the surface integrity of general cytoarchitectonic characteristics of neurons, nervous processes, and spines in the NAc shell. Two dendritic segments were randomly selected from each NAc shell, and spines were counted in a length of 5 µm per segment at magnifications of ×400. Spine density was presented as the number of identified spines in each selected dendritic segment. A factorial-designed ANOVA was used to analyze the effects of METH and genotype, and an SNK-q test was used to analyze the multiple comparisons.

For Nissl staining, we injected four groups of mice (wild-type and D3−/−, METH at 2 mg/kg and saline, n = 2 mice per group) in their home cage using the injection timeline described in Figure 1. On day 65, mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) 24 hr after the challenge treatment with METH or saline. Their brain tissues were fixed by vascular perfusion through the left ventricle of the heart with sequential delivery of 50 ml saline and 60 ml 4% paraformaldehyde in 0.1 M PB (pH 7.4). Brains were then removed and fixed with 4% paraformaldehyde in 0.1 M PB overnight. Subsequently, the brains were cut into 30-µm coronal vibratome sections through the NAc shell with a freezing microtome (CM 1850), and then the slices were stained with cresyl violet. Brain areas were identified at ×100 magnification, and neurons from each hemisphere of the NAc shell were viewed with a microscope (DM 3000) at magnifications of ×200 to establish the general cytoarchitectonic characteristics of neurons. Two regions were randomly selected from each NAc shell, and neurons were counted in a tissue area of 625 µm2 per region at a magnification of ×200. Neuron density is presented as the number of identified neurons in each selected region. A factorial-designed ANOVA was used to analyze the effects of METH and genotype, and an SNK-q test was used to analyze the multiple comparisons.

RESULTS

D3 Receptors Modulate METH-Induced Acute Locomotion and Behavioral Sensitization

The effect of D3 receptors on METH-induced acute locomotor activity in mice is presented in Figures 2 and 3A (day 10). A factorial-designed ANOVA found significant main effects of dose (F3,48 = 17.978, P < 0.05) and genotype (F1,48 = 14.669, P < 0.05), but not their interaction (F3,48 = 1.553, P > 0.05). For wild-type mice, locomotor activities stimulated by the 0.6 mg/kg (P < 0.05) and 2 mg/kg (P < 0.05) METH doses were significantly greater than locomotion following saline treatment. Locomotor activity following the 0.2 mg/kg METH dose did not differ significantly from saline treatment (P > 0.05). For D3−/− mice, locomotor activities stimulated by the 2 mg/kg (P < 0.05) METH doses were significantly greater than locomotion following saline treatment. Locomotor activities following the 0.2 mg/kg and 0.6 mg/kg METH dose did not differ significantly from saline treatment. Comparisons between wild-type and D3−/− mice found significant differences only at the 2 mg/kg METH dose (P < 0.05; Fig. 2).

Fig. 2.

Fig. 2

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DA D3 receptors modulate METH-induced acute locomotion. Acute locomotor responses of wild-type and D3−/− mice to i.p. injections of different doses of METH (n = 6 at the 0.2 mg/kg and 0.6 mg/kg dose, n = 8 at the 2 mg/kg dose) or saline (n = 8 each) were meausred for 60 min. Values are presented as mean ± SEM. *P < 0.05 compared with same-dose wild-type mice; +P < 0.05 compared with saline. D3+/+: wild-type.

Fig. 3.

Fig. 3

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DA D3 receptors modulate METH-induced behavioral sensitization. We recorded locomotor activities of four groups of mice on days 8–17 (A; n = 17 each), days 18–21 (B; n = 8 each), days 24–28 (C; n = 8 each), days 31–35 (D; n = 8 each), and day 64 (E; n = 8 each) 60 min before and after saline or METH (2 mg/kg) injections. Values are presented as mean ± SEM. *P < 0.01 compared with same-dose wild-type mice; @P < 0.05 compared with same-dose wild-type mice; #P < 0.01 compared with day 10; +P < 0.01 compared with the same genotype in saline mice. D3+/+, wild-type.

To investigate the role of D3 receptors in METH-induced behavioral sensitization, we used two groups each of D3−/− and wild-type mice and the procedure shown in Figure 1. There were no significant differences in baseline activity or time course after saline injections between D3−/− and wild-type mice, and repeated saline treatment did not induce appreciable changes in locomotor activity in either wild-type or D3−/− mice (n = 17 or 8 mice each; Fig. 3). Although repeated intermittent METH injections at the 2 mg/kg dose induced behavioral sensitization in both wild-type and D3−/− mice (Fig. 3; #P < 0.01 compared with day 10), there was a significant main effect of genotype (Fig. 3; F1,28 = 14.352, P < 0.01), treatment (F1,28 = 515.062, P < 0.001), and time (F22,616 = 41.241, P < 0.001) and the interactions of genotype and treatment (F1,28 = 5.934, P < 0.05) and treatment and time (F22,616 = 53.762, P < 0.001). Multiple comparisons for analysis of the differences among groups at each time point showed that D3−/− mice exhibited significantly attenuated responses to METH at the 2 mg/kg dose compared with wild-type mice at all METH injection days (Fig. 3; *P < 0.01 or @P < 0.05), including the challenge injection on day 64 (Fig. 3E; @P < 0.05). These results suggest that D3 receptors regulate both the acute locomotor response and the development of behavioral sensitization to METH at the 2 mg/kg dose in the injection paradigms that we used but not the baseline locomotor activity of mice. Additionally, METH-induced sensitization can persist for at least 1 month after the discontinuation of daily METH treatment in both wild-type and D3−/− mice (Fig. 3; #P < 0.01 compared with day 10).

Repeated Exposure to METH Similarly Increases Synaptic Density in the NAc Shell in Both D3−/− and Wild-Type Mice

To investigate whether METH-induced enduring behavioral sensitization is accompanied by alterations in synaptic contacts, we performed ultrastructural studies on synapses in the shell of NAc after the treatment regimens shown in Figure 1 (Fig. 4). A factorial-designed ANOVA found significant effects of METH (F1,20 = 22.413, P < 0.05) but not genotype (F1,20 = 0.008, P > 0.05) or their interaction (F1,20 = 0.341, P > 0.05). Repeated intermittent METH injections led to an increase in synaptic density on medium spiny neurons in the NAc shell compared with saline injections in both wild-type (Fig. 5; 159 ± 14 vs. 105 ± 41; *P < 0.05) and D3−/− mice (165 ± 38 vs. 97 ± 24; *P < 0.05). There were no significantly baseline differences or differences in synaptic density after repeated intermittent METH injections between D3−/− and wild-type mice (Fig. 5E). Multiple linear regression showed no significant differential effects of METH on wild-type and D3−/− mice after controlling for their corresponding saline group (t = −0.656, P > 0.05). These results indicate that repeated intermittent METH induced reorganization of synaptic connections in the NAc shell, which may contribute to the long-lasting behavioral sensitization in both wild-type and D3−/− mice. Moreover, D3 receptor is not obviously involved in modulating baseline or METH-induced changes in synaptic density in the NAc shell.

Fig. 5.

Fig. 5

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Repeated intermittent METH injections led to a similar increase in synaptic density in the NAc shell compared with saline injections in both wild-type and D3−/− mice. A–D: Representative synapses (arrows) from wild-type saline (A), wild-type METH 2 mg/kg (B), D3−/− saline (C), and D3−/− METH 2 mg/kg (D) groups are shown. Axons and axonal terminals form synapses with spines (white arrows) or dendrite (black arrows). E: Quantification of synaptic density in the four groups of mice (n = 6 mice each). Data represent mean ± SEM total number of synapses per 70,650 µm2 of NAc shell per mouse. *P < 0.05 compared with saline control group. S, dendritic spines; D, dendrites; v, vesicles; m, mitochondrion. D3+/+: wild-type. Scale bars = 0.5 µm.

Repeated Exposure to METH Has No Detectable Effects on the General Structure of Neurons, Nervous Processes, and Spines in the NAc Shell in Mice

To examine potential neurotoxic effect of repeated METH treatment, we processed the NAc of mice for rapid Golgi staining and Nissl’s staining 24 hr after the last treatment with METH or saline on day 65. There were no significant changes in the surface integrity of general cytoarchitectonic characteristics of neurons, nervous processes, or spines in the NAc shell in the METH group or the saline group (Figs. 6, 7). Neuron counts in the Nissl-stained material showed no significant effects of METH (F1,12 = 0.267, P > 0.05) or genotype (F1,12 = 1.455, P > 0.05) and their interaction (F1,12 = 1.455, P > 0.05; Fig. 6B). There was no detectable neuronal loss in the METH group compared with the saline group (Fig. 6B; P > 0.05). Spine counts from the Golgi material showed no significant effects of METH (F1,12 = 0.686, P > 0.05) or genotype (F1,12 = 0.686, P > 0.05) or their interaction (F1,12 = 0.171, P > 0.05; Fig. 7C). There was no significant quantitative loss in spine densities in the METH group compared with the saline group (Fig. 7C; P > 0.05). These results suggest that repeated exposure to METH at this dose had no significant neurotoxicity effects on neurons of NAc shell in the treatment regimens that we used. Moreover, the ultrastructural changes in synaptic contacts detected in the current study were not significantly affected by METH-induced nerve terminal degeneration.

Fig. 6.

Fig. 6

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There was no detectable neuronal cell loss following repeated METH treatment compared with that after saline treatment. A: Representative photographs of Nissl staining of neurons in the NAc shell of each group (n = 2 each). B: Quantification of neuron density in the four groups of mice. Neuron density is presented as the number of identified neurons in each selected region. Two regions were randomly selected from each NAc shell, and neurons were counted in a tissue area of 625 µm2 per region at a magnification of ×200. Values are presented as mean ± SEM. D3+/+: wild-type. Scale bars = 25 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 7.

Fig. 7

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Repeated exposure to METH has no detectable toxic effects on the general structure of neurons, nervous processes, or spines in the NAc shell. Representative photos of rapid Golgi staining of neurons (A), nervous processes (A,B), and spines (B) in the NAc shell of each group. C: Quantification of spine density in the four groups of mice (n = 2 each). Spine density is presented as the number of identified spines in each selected dendritic segment. Two dendritic segments were randomly selected from each NAc shell, and spines were counted in a length of 5 µm per segment at a magnification of ×400. There were no significantly losses in spine density in the METH group compared with saline group. Values are presented as mean ± SEM. D3+/+: wild-type. Scale bars = 10 µm in A; 5 µm in B. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

DISCUSSION

METH Induces Behavioral Sensitization and Increases in Synaptic Densities in the NAc Shell in Mice

The persistent nature of drug addiction at the behavioral level suggests that repeated exposure to drugs of abuse may lead to long-term neuroplastic changes in related neuronal circuits, and dendrites as well as dendrtic spines are thought to be major elements of drug-induced structural plasticity (Robinson and Kolb, 2004; Wolf et al., 2004; Hyman et al., 2006; Kalivas and O’Brien, 2008). Structural plasticity in neurons of the NAc is thought to alter an individual’s responses to abused drugs, motivation for drugs, escalation of drug intake, and eventually compulsive use behavior (Everitt et al., 2001; Nestler, 2001; Robinson and Kolb, 2004; Koob and Le Moal, 2005; Kalivas and O’Brien, 2008). Previous studies, including ours, demonstrated that repeated amphetamine or cocaine exposure alters the number of dendrites and the density of dendritic spines of neurons in the NAc and cortex (Robinson and Kolb, 1997, 1999; Robinson et al., 2001; Norrholm et al., 2003, Zhang et al., 2006; Ren et al., 2010). Although the Golgi-impregnation method has been used extensively to evaluate morphological changes induced by abused drugs (Robinson and Kolb, 1997; Robinson et al., 2001; Kolb et al., 2003; Ferrario et al., 2005; Zhang et al., 2006; Pulipparacharuvil et al., 2008; Ren et al., 2010), Golgi-stained neurons are not necessarily representative of the total population of neurons, in that only a small percentage of neurons takes up the stain. Additionally, this approach does not provide detailed changes in synapses (Robinson and Kolb, 2004). We thus used a repeated intermittent administration regimen in the current study to investigate METH-induced behavioral sensitization and the EM method to quantify synaptic density directly to determine the accompanying ultrastructural plasticity in the NAc shell in mice. We found that repeated intermittent METH injections induced not only long-lasting behavioral sensitization (Fig. 3) but also ultrastructural changes in synaptic contacts (Fig. 5) in mice. Increased synaptic density was detectable 1 month after the discontinuation of METH treatment when the behavioral sensitization was present in both wild-type and D3−/− mice. Our results suggest that METH-induced reorganization of synaptic connections in the NAc shell correlates with and may contribute to behavioral sensitization produced by repeated intermittent administration of METH. To our knowledge, this is the first description of increased synaptic density induced by repeated exposure to METH in the context of behavioral sensitization. These data are consistent with the assumption that structural plasticity may underlie, at least partially, drug-induced persistent changes in behavior (Robinson and Kolb, 2004).

High doses of METH in binge-treatment regimens induce nerve terminal degeneration and neuronal apoptosis (Chapman et al., 2001; Riddle et al., 2006; Cadet and Krasnova, 2009), which may evoke morphological changes of nervous processes and spines and impact the structural plasticity of synaptic contacts. Typical neurotoxic METH regimens are 5–10 mg/kg given parenterally four to 10 times within 1–4 days (Seiden and Sabol, 1996; Quinton and Yamamoto, 2006; Cadet and Krasnova, 2009; Gouzoulis-Mayfrank and Daumann, 2009). Based on available literature, 2 mg/kg METH does not induce significant neurotoxicity in the brains of the mice. Nonetheless, we evalauted potential neurotoxic effects of METH used in the current treatment regimen. The NAc shell of mice were processed for rapid Golgi staining and Nissl staining 24 hr after the challenge treatment with METH or saline. There were no significantly neurotoxic effects on the general structure of neurons, nervous processes, or spines in the shell of NAc in either the METH or the saline groups (Figs. 6, 7). These results suggest that METH at the 2 mg/kg dose produced no significantly neurotoxicity in the treatment regimens that we used. Consequently, the ultrastructural changes in synaptic contacts that we detected were not obviously due to METH-induced neurotoxicity.

DA D3 Receptors Modulate METH-Induced Acute Locomotion and Behavioral Sensitization

D3 receptors are expressed mainly in the NAc. We found that, whereas METH induced increases in acute locomotion and behavioral sensitization in both wild-type and D3−/− mice, D3−/− mice were less sensitive to METH than wild-type mice (Figs. 2, 3). There were no significant differences in baseline activity between D3−/− and wild-type mice (Figs. 2, 3).Chen et al. (2007) reported that, at the same dose of METH, D3−/− mice exhibit higher levels of behavioral sensitization than wild-type mice. One possible explanation is that the mice used in our study have a genetic background different from that of the mice used in their study (129/Sv × C57BL/6 vs. C57BL/6). Alternatively, the D3−/− mice used had a different genetic mutation (Accili et al., 1996; Xu et al., 1997). Although the reasons for the difference remain unclear, our results suggest that D3 receptor is an important mediator for both the acute locomotor response and the development of behavioral sensitization to METH at the 2 mg/kg dose in the injection paradigms that we used.

Distinct Roles of D3 Receptors in METH-Induced Behavioral Sensitization and Ultrastructural Plasticity

In the current study, we measured ultrastructural plasticity induced by METH in the context of behavioral sensitization. We found that there were no significantly baseline differences or differences in synaptic density after repeated intermittent METH injections between D3−/− and wild-type mice (Fig. 5). One possible explanation is that the D3 receptor is not significantly involved in modulating baseline or METH-induced changes in overall synaptic density in the NAc shell in the context of behavioral sensitization. Different afferents of nerve terminals form synapses with distinct postsynaptic targets, such as dendrites, spines, and even different sites on the same spine (Sesack and Pickel, 1990). For example, glutamate afferent terminals form synapses primarily with spine heads, whereas dopaminergic afferent terminals make synapses mainly on the spine neck and shafts of dendrites (Sesack and Pickel, 1990). The current study did not differentiate synapses from different presynaptic origins and distinct postsynaptic targets. Consequently, whether the D3 receptor mutation affects specifically synapses with dopaminergic afferents remains unknown. Whether D3 receptors affect changes in synaptic density following METH-induced reward related learning also remains unclear. Although D3−/− mice exhibit significantly attenuated responses to METH at the 2 mg/kg dose compared with wild-type mice on the challenge injection day (Fig. 3E), this difference in the expression of behavioral sensitization between the two groups of mice is less significant (P = 0.034) than at most of the time points in the induction phase of behavioral sensitization. This provides a possible explanation for the distinct roles of D3 receptors in METH-induced behavioral sensitization and ultrastructural plasticity. Unknown compensatory changes also occur throughout development in D3 receptor mutant mice that may impact the current results. Future experiments are needed to investigate these unresolved possibilities. Our current results suggest that D3 receptors modulate locomotor responses to both acute and repeated METH treatment. In contrast, the D3 receptor is not obviously involved in modulating baseline or METH-induced ultrastructural changes in the NAc shell.

Acknowledgments

Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 30973365; Contract grant sponsor: Ministry of Science and Technology of China; Contract grant number: 2009 DFA 31080 (to T.C.).

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