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Keywords:

  • GluA1;
  • GluA2;
  • metabotropic glutamatergic receptors;
  • MTEP;
  • sensitization;
  • STEP61

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Ionotropic AMPA receptors (AMPAR) and metabotropic glutamate group I subtype 5 receptors (mGlu5) mediate neuronal and behavioral effects of abused drugs. mGlu5 stimulation increases expression of striatal-enriched tyrosine phosphatase isoform 61 (STEP61) which internalizes AMPARs. We determined the rat brain profile of these proteins using two different classes of abused drugs, opiates, and stimulants. STEP61 levels, and cellular distribution/expression of AMPAR subunits (GluA1, GluA2) and mGlu5, were evaluated via a protein cross-linking assay in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP) harvested 1 day after acute, or fourteen days after repeated morphine (8 mg/kg) or methamphetamine (1 mg/kg) (treatments producing behavioral sensitization). Acute morphine decreased GluA1 and GluA2 surface expression in mPFC and GluA1 in NAc. Fourteen days after repeated morphine or methamphetamine, mGlu5 surface expression increased in VP. In mPFC, mGlu5 were unaltered; however, after methamphetamine, STEP61 levels decreased and GluA2 surface expression increased. Pre-treatment with a mGlu5-selective negative allosteric modulator, blocked methamphetamine-induced behavioral sensitization and changes in mPFC GluA2 and STEP61. These data reveal (i) region-specific distinctions in glutamate receptor trafficking between acute and repeated treatments of morphine and methamphetamine, and (ii) that mGlu5 is necessary for methamphetamine-induced alterations in mPFC GluA2 and STEP61.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

GluA1

AMPA glutamate receptor subunit 1

GluA2

AMPA glutamate receptor subunit 2

mGlu5

metabotropic glutamate receptor group 1 subtype 5

mPFC

medial prefrontal cortex

NAc

nucleus accumbens

STEP61

striatal-enriched tyrosine phosphatase isoform 61

VP

ventral pallidum

Repeated administration of opiates and psychomotor stimulants enhances motor activity beyond that induced by a single injection, a phenomenon termed behavioral sensitization. Sensitization can persist long after the cessation of drug administration. The circuitry that underlies behavioral sensitization includes the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP). The mPFC is implicated in the development (Wolf et al. 1995; Pierce et al. 1998; Cador et al. 1999; Hao et al. 2007), while the NAc mediates maintenance and expression (Pierce and Kalivas 1997; Vanderschuren and Kalivas 2000) of sensitization. Our laboratory revealed that the VP is critical for the development, maintenance, and expression of sensitization (Johnson and Napier 2000; McDaid et al. 2005, 2006a; Mickiewicz et al. 2009).

Behavioral sensitization is thought to reflect neuroadaptations that often involve glutamatergic transmission. The NAc receives glutamatergic input from the mPFC (Christie et al. 1985), amygdala, and hippocampus (Mulder et al. 1998). The VP also receives glutamatergic projections from the mPFC (Fuller et al. 1987; Sesack et al. 1989) and amygdala (Russchen and Price 1984; Fuller et al. 1987; Carnes et al. 1990), in addition to glutamatergic inputs from the subthalamic nucleus (Kita and Kitai 1987; Turner et al. 2001). Repeated treatments of psychomotor stimulants increase glutamate overflow in the mPFC (Qi et al. 2009), NAc (Pierce et al. 1996) and VP (Chen et al. 2001) following acute stimulant administration. Opiate-induced reinstatement of drug-seeking also increases extracellular glutamate in the NAc (LaLumiere and Kalivas 2008). VP neurons exhibit enhanced sensitivity to local glutamate application following withdrawal from repeated morphine (McDaid et al. 2006a). These studies illustrate the complexity of the glutamatergic role in neuronal consequences of abused drugs, including the neuroplasticity that is associated with repeated drug exposure.

Regulation of surface glutamate receptors is one mechanism that drives neuroplasticity. Increases in AMPA receptor (AMPAR) surface expression enhance synaptic strength promoting the development and maintenance of behavioral sensitization (Wolf et al. 2004; Kauer and Malenka 2007). One day after repeated, sensitizing treatments of cocaine, there is little change in the cellular location or levels of AMPAR subunits GluA1, GluA2, and group I, subtype 5 metabotropic glutamate receptors (mGlu5) in the mPFC and NAc (Ghasemzadeh et al. 2009a,b). After 21 days, there is an up-regulation of these receptors in synaptosomal NAc and mPFC membrane fractions, and an enhanced NAc surface expression of GluA1 and GluA2/3 (Boudreau and Wolf 2005; Ghasemzadeh et al. 2009a,b). In contrast, extended withdrawal from repeated amphetamine does not alter GluA1 or GluA2 in the NAc (Nelson et al. 2009), indicating that glutamate receptor trafficking induced by cocaine is not recapitulated by amphetamine. Less is known about methamphetamine. We have revealed that intracellular mGlu5 increase four days after repeated methamphetamine (Herrold et al. 2011) and that 1 day following repeated morphine, surface expression of GluA1 decreases in the mPFC (Mickiewicz and Napier 2011). It appears that various drugs can differentially regulate cellular distribution of ionotropic and metabotropic glutamate receptors in mPFC and NAc; effects in the VP are not yet characterized. This study provides the direct comparison needed to help clarify the particular overlaps and distinctions.

The membrane-associated protein, striatal-enriched protein tyrosine phosphatase isoform 61 (STEP61) is implicated as a molecular mechanism that underlies deficits associated with several neuropsychiatric disorders (Johnson and Lombroso 2012). In vitro and in vivo studies have demonstrated that mGlu5 regulate the functional state of GluA1 and GluA2 whereby activated mGlu5 enhance expression of STEP61; STEP61 dephosphorylates and subsequently internalizes AMPAR subunits (Snyder et al. 2001; Zhang et al. 2008). Thus, we hypothesized that in vivo, STEP61 and surface AMPAR subunit expression are inversely related. STEP61 is expressed in striatal and cortical neurons (Boulanger et al. 1995; Bult et al. 1996). Blockade of STEP61 reduces the development of amphetamine-induced behavioral sensitization (Tashev et al. 2009). We further hypothesized that if mGlu5 regulate sensitization processes and AMPAR subunit surface expression through STEP61, then these proteins should co-vary in brain regions that are involved in sensitization.

The aims of this study were to determine (i) if mGlu5 and AMPAR surface expression was differentially regulated by morphine and methamphetamine, (ii) if this regulation differed in a brain region-selective manner, (iii) if AMPAR changes co-varied with mGlu5 surface expression and STEP61 levels, and (iv) if trafficking processes differed between induction and maintenance of behavioral sensitization. We used an ex vivo cross-linking assay that allowed for assessments of receptor surface expression (Boudreau and Wolf 2005; Boudreau et al. 2007, 2012; Conrad et al. 2008; Nelson et al. 2009; Mickiewicz and Napier 2011). To provide a spatial snapshot of maintenance, we harvested the mPFC, NAc, and VP from rats 14 days after the last drug administration, a time frame when we have observed changes in these brain regions in both morphine- and methamphetamine-treated rats (McDaid et al. 2005, 2006a,b; Mickiewicz et al. 2009). To compare maintenance with the induction process, we also assayed samples taken 1 day after a single drug treatment (i.e., within the inter-dosing interval of the repeated treatment protocol).

The outcomes filled several knowledge gaps, including (i) direct comparisons of GluA1, GluA2, and mGlu5 surface expression within three brain regions involved in addictions, between two classes of abused drugs, opiates (morphine) and psychomotor stimulants (methamphetamine), using a behaviorally relevant treatment protocols, including those that are known to induce neuroplasticity, (ii) determination of the role of mGlu5 in behavioral and neuronal sensitization, and (iii) determination of a mechanism for mGlu5 regulation of drug-induced sensitization through regulation of AMPAR subunit and STEP61 protein expression.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Animals

Male Sprague–Dawley rats weighing 200–225 g upon arrival (Harlan, Indianapolis, IN, USA) were housed in pairs under environmentally controlled conditions (7:00AM/7:00PM light/dark cycle, temperature maintained at 23–25°C) with ad libitum access to rat chow and water. The rats were habituated to vivarium conditions for at least 1 week prior to experimentation. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, USA) and were approved by the University Institutional Animal Care and Use Committee.

Drugs

Morphine sulfate, obtained from the National Institute on Drug Abuse (Bethesda, MD, USA), was dissolved in 0.9% saline to yield a dose of 8 mg/mL/kg as the salt. Methamphetamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline to yield a dose of 1 mg/mL/kg as the salt. These doses are sufficient to induce behavioral sensitization (Ohmori et al. 1995; Mickiewicz et al. 2009). Control rats received the saline vehicle (1 mL/kg). Saline, morphine, and methamphetamine injections were given subcutaneously (sc). The negative allosteric modulator (NAM) of mGlu5, 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP; Tocris Bioscience, Ellisville, MO, USA) (Cosford et al. 2003) was used to ascertain if these receptors were necessary for sensitization. MTEP was dissolved in sterile water containing 20% w/v 2-hydroxypropyl-β-cyclodextrin (Sigma) to yield a dose of 5 mg/mL/kg as the salt. This dose is sufficient to block drug-induced sensitization (Dravolina et al. 2006). MTEP and its vehicle were injected intraperitoneally (ip).

Behavioral assessments and treatment protocols

Rats were transported across the hall from the housing room to the test room at least 30 min prior to the start of each experiment. The test room was dimly lit (5–10 foot candles) with white noise continuously present. Motor activity data were collected using Plexiglas activity chambers (25 cm × 30 cm × 30 cm) equipped with two sets of photobeams (AccuScan Instruments, Inc., Columbus, OH, USA) that allowed behavioral quantification in three-dimensional space, including indices of horizontal activity (locomotion and other sequential motor activity in the horizontal plane), vertical activity (up-and-down movements of the upper body, e.g., rearing and climbing on chamber walls), vertical time (the amount of time spent in behaviors involving a vertical position), and stereotypic activity (repetitive, localized movements captured within single second epochs). Two days prior to initiating treatments, the rats were habituated to the activity boxes for 1 h, given sc saline injections, and motor activity was monitored for 2 h post-injection. Motor activity data obtained on the second day were used as a measure of baseline activity. Drug treatments began 1 day after baseline collection (day 1); rats were randomly assigned to receive saline, morphine, or methamphetamine. After the 1 h habituation period, the rats were given a sc injected treatment (saline, morphine, or methamphetamine) and then immediately placed back into the motor box for 3 h of behavioral monitoring. For the acute treatment protocol, rats (= 32) were killed the following day (day 2) via rapid decapitation to collect tissue for biochemical analysis (see below). For the repeated treatment protocol (= 64 rats), the day 1 protocol was repeated for two more days (days 1–3). For the next 14 days, the rats were left in the home cage and no treatment was administered (i.e., withdrawal). A subset of rats (= 32) was tested on withdrawal day (WD) 14 for expression of sensitization after an acute treatment of morphine or methamphetamine. Other rats (= 32) were not given a drug challenge and were killed via rapid decapitation on WD 14 to collect tissue for biochemical analyses.

An additional group of rats was used to assess the effects of antagonizing mGlu5 on sensitization. These rats were given the mGlu5 NAM, MTEP, or its vehicle (20% w/v 2-hydroxypropyl-β-cyclodextrin) 10 min before methamphetamine (= 10) or saline (= 12) once-daily for 3 days (Days 1–3) and motor activity was quantified as described above. On WD 14, rats were killed via rapid decapitation to collect tissue for biochemical analyses.

Protein cross-linking with Bis[sulfosuccinimidyl]suberate (BS3)

The brains were removed within 45–60 s after decapitation and immediately chilled in ice-cold artificial cerebrospinal fluid (CSF). The mPFC, NAc, and VP were dissected out from the brain (Fig. 1) and chopped into 400 μm slices using a McIlwain tissue chopper (The Vibratome Company, O'Fallon, MO, USA). Methods for receptor cross-linking were based on (Boudreau and Wolf 2005; Boudreau et al. 2007, 2012; Conrad et al. 2008; Nelson et al. 2009; Herrold et al. 2011; Mickiewicz and Napier 2011). Briefly, the slices were transferred to centrifuge tubes containing artificial CSF and 2 mM BS3 (Pierce, Rockford, IL, USA) and incubated for 30 min on a rocker at 4°C. The cross-linking reaction was terminated by the addition of 100 mM glycine for 10 min at 4°C. The slices were pelleted by 2 min of centrifugation (20817.16 g; rotor F45-30-11, Eppendorf, Hauppauge, NY, USA) and the supernatant discarded. The pellets were re-suspended in ice-cold lysis buffer [25 mM HEPES, pH 7.4, 500 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol , 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1x phosphatase inhibitor cocktails I & II (Sigma-Aldrich), 1x protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA), and 0.1% Nonidet P-40]. The samples were sonicated for 5 s, centrifuged for 2 min, aliquotted, and stored at −80°C until analysis. Total protein concentration of lysates was determined according to the Bradford method (Bradford 1976).

image

Figure 1. Illustration of brain regions used for immunoblotting. Sections were redrawn from Paxinos and Watson (1998). The bold outlines correspond to dissection boundaries of the medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and ventral pallidum (VP) tissues assayed in this study. Numbers above each section indicate distance (in mm) from Bregma. ac, anterior commissure.

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Immunoblotting GluA1, GluA2, mGlu5, and STEP

Samples (20 μg) were loaded and electrophoresed on 4–15% gradient Tris-HCl gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes for immunoblotting. Non-specific binding sites were blocked using 1% normal goat serum and 5% non-fat dry milk in TBS-Tween 20 (TBS-T; Sigma), pH 7.4 for 1 h at 22°C. Membranes were incubated in primary antibody for GluA1 (1 : 1000; Millipore, Billerica, MA, USA), GluA2 (1 : 4000; Millipore), mGlu5 (1 : 15 000; Millipore), STEP (1 : 4000; Millipore), and actin (1 : 20 000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C with gentle shaking. Following six washes in TBS-T for 5 min each, membranes were incubated in horseradish peroxidase-conjugated secondary antibody for 1 h at 22°C as follows: for GluA1 and GluA2 immunoblots, goat anti-rabbit (1 : 10 000; Millipore) was used, for mGlu5 immunoblots goat anti-rabbit (1 : 15 000; Jackson ImmunoResearch, West Grove, PA, USA), for actin immunoblots, goat anti-rabbit (1 : 20 000; Jackson), and for STEP immunoblots, rabbit anti-mouse (1 : 4000; Jackson, for STEP). Membranes were again washed in TBS-T (6 × 5 min), immersed in enhanced chemiluminescent substrate (SuperSignal West Pico; Pierce, Rockford, IL, USA) for 5 min, and exposed to HyBlot CL film (Denville Scientific, Metuchen, NJ, USA). The bands were visualized on film. A dense, high molecular weight band of ~ 400–600 kDa represented the surface pool (S) of cross-linked GluA or mGlu5, a band at ~ 106 kDa or ~ 135 kDa represented intracellular (I) GluA or mGlu5, respectively. A band at ~ 61 kDa represented the STEP61 protein isoform. Optical densities of the immunoreactive bands were analyzed using Lab Works (UVP, Upland, CA, USA) or Un-Scan It (Silk Scientific, Orem, UT, USA) software. mGlu5 is highly expressed in the limbic brain regions assayed. To determine the optimal exposure time for mGlu5 immunoblots, a linear curve of optical density and exposure time was generated (data not shown). All mGlu5 data presented were derived from immunoblots at exposure times within this linear curve (and thus, do not reflect saturation). Images were captured using a BioChemi Imaging System (UVP) coupled to a CCD camera or a scanner (Epson Electronics America, Inc., San Jose, CA, USA) coupled to a PC computer. All samples were run at least twice and the data were averaged across runs.

We previously verified that the BS3 cross-linking method was selective for surface proteins by assessing the cytosolic protein extracellular regulated kinase 2 in our samples (Mickiewicz and Napier 2011).

Data summaries and analyses

Behavior

Motor activity data were truncated at 90 min for morphine and 60 min for methamphetamine to capture the peak effect of each drug. For the acute treatment study, a Student's t-test was used to compare motor activity data on day 1 between saline and drug treatment. For the repeated treatment study, development of sensitization was determined using a one-way repeated measures analysis of variance (rm anova) comparing motor activity on days 1–3 within a treatment group, followed by a post hoc Newman–Keuls test for multiple comparisons. To determine expression of sensitization, a Student's t-test was used to compare motor responses to an acute challenge of morphine or methamphetamine. All data are presented as mean ± SEM. Significance was a priori set at α = 0.05.

Immunoblots

The surface/intracellular ratio (S/I) of GluA1, GluA2, and mGlu5 was calculated by dividing the optical densities of the surface band by the intracellular band. Total GluA1, GluA2, and mGlu5 were determined by adding the optical densities of the surface and intracellular bands and dividing by the loading control actin. The portion of GluA1, GluA2, or mGlu5 on the surface or intracellularly located was calculated by dividing the optical density of the surface or intracellular band (respectively) by actin. Results from morphine- and methamphetamine-treated rats were normalized to saline-treated (control) values for each brain region. A Student's t-test was used to compare between saline control and drug treatment groups, with a Bonferroni correction for comparing the same control group to each of the two treatment groups, setting α = 0.025 for significance. All data are presented as mean + SEM (in figures) or mean ± SEM (in tables).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Acute drug administration

Rats treated with a single morphine injection (8 mg/kg sc) demonstrated a significant reduction in horizontal activity (555 ± 126) versus saline-administered rats (1306 ± 50) (t(14) = 4.438, < 0.001), as well as stereotypic activity (339 ± 74) versus saline (822 ± 46) (t(14) = 4.682, < 0.001); no significant differences were obtained for vertical activity or vertical time. A single injection of methamphetamine (1 mg/kg sc) significantly enhanced vertical activity (1924 ± 385) versus saline treatment (113 ± 32) (t(14) = 3.6, < 0.01) and vertical time (651 ± 143) versus saline (70 ± 20) (t(14) = 3.1, p < 0.01); no differences were seen in horizontal activity or stereotypic activity.

Glutamate receptor proteins were evaluated in brain tissue harvested 1 day after a single morphine or methamphetamine treatment. No measured parameter in the VP was altered by the acute treatments (Tables 1 and 2). For the NAc, most of the measured proteins also were not changed (Tables 3 and 4). The exception was the GluA1 S/I ratio, which was significantly decreased for morphine-treated rats compared to saline-treated controls (t(18) = 2.6, = 0.02); total (S+I, t(19) = 0.2, = 0.8), surface (t(19) = 0.1, = 0.9) and intracellular (t(18) = 1.4, = 0.2) GluA1 proteins remained unchanged (Fig. 2). In the mPFC, methamphetamine did not alter any measured parameter (Table 5). This was in contrast to morphine treatment in which the S/I was decreased for GluA1 (t(19) = 2.9, = 0.01), reflecting a non-significant reduction in average surface expression (t(20) = 2.2, = 0.04; α = 0.025, per Bonferroni correction), and a trend for enhancement of intracellular levels (t(20) = 2.0, = 0.06; Fig. 3a). Another morphine-induced change involved a reduction in surface expression of GluA2 (t(18) = 2.5, = 0.02) without an effect on S/I ratio (t(18) = 0.87, = 0.40; Fig. 3b). There was no change in the mPFC between saline- and morphine-treated rats in mGlu5 S/I ratio (t(17) = 1.5, = 0.16), total (S+I, t(17) = 1.3, = 0.21), surface (t(17) = 1.3, = 0.21), or intracellular proteins (t(16) = 1.4, = 0.17; Fig. 3c). Finally, there was no difference between saline- and methamphetamine-treated rats in mPFC tissues with any glutamate receptor protein assayed (Table 5).

Table 1. Ventral pallidum: 1 day after morphine
ProteinComponentSalineMorphineStatistics
GluA1

S/I

Total

100.9 ± 12.2

99.6 ± 5.7

133.6 ± 21.8

111.5 ± 7.0

t(20) = 1.370, p = 0.186

t(20) = 1.328, p = 0.199

GluA2

S/I

Total

100.0 ± 7.1

100.0 ± 4.5

115.0 ± 8.8

96.0 ± 3.9

t(18) = 1.340, p = 0.197

t(18) = 0.649, p = 0.525

mGlu5

S/I

Total

100.0 ± 9.7

95.6 ± 5.14

131.4 ± 22.1

118.2 ± 12.5

t(20) = 1.381, p = 0.182

t(19) = 1.729, p = 0.100

Table 2. Ventral pallidum: 1 day after methamphetamine
ProteinComponentSalineMethamphetamineStatistics
  1. S/I ratio and Total (S+I) protein were assessed for GluA1, GluA2, and mGlu5 in the VP tissues after one day of acute saline (1 mL/kg, sc) or morphine (8 mg/kg, sc) or methamphetamine (1 mg/kg, sc) treatment. There were no differences between saline and morphine treatment groups for any glutamate receptor protein component assayed. Shown are mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics, α = 0.025.

GluA1

S/I

Total

100.9 ± 12.2

99.6 ± 5.7

108.7 ± 16.0

101.9 ± 6.3

t(20) = 0.397, p = 0.696

t(20) = 0.279, p = 0.783

GluA2

S/I

Total

100.0 ± 7.1

100.0 ± 4.5

105.3 ± 11.4

98.3 ± 6.2

t(18) = 0.412, p = 0.685

t(18) = 0.222, p = 0.827

mGlu5

S/I

Total

100.0 ± 9.7

95.6 ± 5.1

97.8 ± 14.6

135.1 ± 18.3

t(19) = 0.129, p = 0.898

t(19) = 2.164, p = 0.043

Table 3. Nucleus accumbens: 1 day after morphine
ProteinComponentSalineMorphineStatistics
GluA2

S/I

Total

97.6 ± 5.2

115.9 ± 7.6

113.7 ± 10.7

112.5 ± 6.6

t(17) = 1.394, p = 0.181

t(18) = 0.335, p = 0.741

mGlu5

S/I

Total

100.0 ± 7.8

98.7 ± 10.2

101.7 ± 16.0

114.8 ± 11.3

t(18) = 0.103, p = 0.919

t(19) = 1.061, p = 0.302

Table 4. Nucleus accumbens: 1 day after methamphetamine
ProteinComponentSalineMethamphetamineStatistics
  1. Shown are the mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics. S/I ratio and Total (S+I) protein were assessed for GluA1, GluA2, and mGlu5 in NAc tissues after 1 day of a single injection of saline (1 mL/kg, sc), morphine (8 mg/kg, sc), or methamphetamine (1 mg/kg, sc). There was a significant decrease in the GluA1 S/I ratio in the NAc of morphine-treated rats *p < 0.025. However, there were no other differences between saline and methamphetamine treatment groups for any glutamate receptor protein component assayed. Shown are mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics, with Bonferroni correction as saline groups were used for morphine and methamphetamine comparisons, α = 0.025.

GluA1

S/I

Total

103.1 ± 10.6

100.3 ± 6.9

142.4 ± 28.3

112.1 ± 6.4

t(18) = 1.405, p = 0.177

t(19) = 1.251, p = 0.226

GluA2

S/I

Total

97.6 ± 5.2

115.9 ± 7.6

97.3 ± 4.5

108.1 ± 12.7

t(18) = 0.047, p = 0.963

t(18) = 0.550, p = 0.589

mGlu5

S/I

Total

100.0 ± 7.8

98.7 ± 10.2

103.5 ± 12.1

106.5 ± 8.8

t(20) = 0.246, p = 0.808

t(20) = 0.577, p = 0.570

Table 5. Medial prefrontal cortex: 1 day after methamphetamine
ProteinComponentSalineMethamphetamineStatistics
  1. S/I ratio and Total (S+I) protein were assessed for GluA1, GluA2, and mGlu5 in the mPFC tissues after 1 day of acute saline (1 mL/kg, sc) or methamphetamine (1 mg/kg, sc) treatment. There was no difference between treatment groups for any glutamate receptor protein assayed. Shown are mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics, α = 0.025 (Bonferroni correction).

GluA1

S/I

Total

100.9 ± 17.2

100.7 ± 3.7

81.5 ± 19.6

92.4 ± 4.9

t(18) = 0.733, p = 0.473

t(19) = 1.376, p = 0.185

GluA2

S/I

Total

100.4 ± 9.7

101.5 ± 6.7

95.4 ± 14.3

88.6 ± 5.8

t(17) = 0.296, p = 0.770

t(17) = 1.285, p = 0.216

mGlu5

S/I

Total

101.5 ± 10.3

100.1 ± 6.7

136.2 ± 27.6

100.2 ± 8.4

t(16) = 1.281, p = 0.218

t(16) = 0.008, p = 0.994

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Figure 2. Decreased expression of GluA1 occurred in the nucleus accumbens (NAc) following acute morphine treatment. Representative immunoblot for GluA1 in NAc tissues harvested 1 day after a single injection of saline (SAL, 1 mL/kg, sc) or morphine (MOR, 8 mg/kg, sc) (left). The GluA1 S/I ratio was decreased in morphine-treated rats, but there was no change in the amount of surface, intracellular, or total protein. Numbers within bars represent sample size. Student's t-test, *< 0.025.

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image

Figure 3. Decreased expression of GluA1 and GluA2 occurred in the medial prefrontal cortex (mPFC) of rats following acute morphine treatment. At left, representative immunoblots for (a) GluA1, (b) GluA2, and (c) mGlu5 in mPFC tissues harvested 1 day after a single injection of saline or morphine. (a) (right) The GluA1 S/I ratio was decreased in morphine-treated rats, but there was no statistically significant change in the amount of surface, intracellular, or total protein. (b) (right) The GluA2 surface component was decreased in morphine-treated rats, but there was no statistically significant change in the GluA2 S/I ratio, intracellular GluA2, or total protein. (c) (right) There was no statistically significant change in the distribution or total protein levels of mGlu5 following morphine treatment. SAL, saline (1 mL/kg, sc); MOR, morphine (8 mg/kg, sc). Numbers within bars represent sample size. Student's t-test, *< 0.025.

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Repeated drug administration and protracted withdrawal

The behavioral responses to repeated morphine and methamphetamine administration are illustrated in Figs 4 and 5, respectively, wherein the development of sensitization was demonstrated by comparing motor activity on days 1, 2, and 3. Our prior studies with automated tallies and observational evaluations demonstrated that horizontal activity and stereotypic activity are reliable indices of morphine-induced behavioral sensitization (McDaid et al. 2006a; Mickiewicz et al. 2009). Similar to these prior studies, horizontal activity (F(2,14) = 7.9, p = 0.01; Fig. 4a) and stereotypic activity (F(2,14) = 6.5, p = 0.01; Fig. 4b) were significantly increased by repeated morphine treatments in this study. Likewise, we previously determined that vertical activity and vertical time are reliable indices of methamphetamine-induced behavioral sensitization (McDaid et al. 2006b). Consistent with these prior reports, vertical activity (F(2,14) = 5.5, p = 0.02; Fig. 5a) and vertical time (F(2,14) = 4.7, p = 0.03; Fig. 5b) were significantly increased in methamphetamine-treated rats in this study. The behavioral sensitization was maintained for at least 14 days for both drugs. A between-treatment group analysis showed that responding to an acute morphine challenge (8 mg/kg sc; given on WD 14) was greater in rats with a morphine history compared to those that received morphine for the first time (horizontal activity, t(13) = 2.8, p = 0.02; stereotypic activity, t(14) = 2.8, p = 0.02; Fig. 4). When challenged with methamphetamine on WD 14, rats with a treatment history of methamphetamine also had higher counts of activity compared to those with a saline treatment history (vertical activity, t(14) = 2.5, p = 0.03; vertical time, t(14) = 2.7, p = 0.02; Fig. 5), indicating that sensitization was maintained for at least 14 days in the methamphetamine-treated animals.

image

Figure 4. Development and expression of behavioral sensitization after repeated morphine. Shown are rats used for behavioral assessments only (squares) as well as those killed on WD 14 for biochemical assessments (circles). Data were collapsed for the 90 min test period after injection. (a) Horizontal Activity and (b) Stereotypic Activity. There was a significant increase in both motor parameters between morphine treatment days 1 and 3 (filled circles, rats used for biochemistry; filled squares, rats used for behavioral verification). There was no statistical difference between days 1, 2, or 3 for saline treatments (open symbols, = 6 or 8). A challenge injection of morphine was administered on WD 14 to rats with a saline (open square, = 7 or 8) or morphine (filled square, = 8) treatment history; rats with a morphine treatment history expressed sensitization. One-way rm anova with post hoc Newman–Keuls, day 1 different from day 3, ##p < 0.01 (biochemistry group), ††p < 0.01 (behavior group); Student's t-test, *p < 0.05.

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image

Figure 5. Development and expression of motor sensitization after repeated methamphetamine. Data were collapsed across the 60 min test period after injection. (a) Vertical Activity and (b) Vertical Time. There was a significant increase in both motor parameters between treatment days 1 and 3 in rats given once-daily injections of methamphetamine (filled diamonds, rats used for biochemistry; filled triangles, rats used for behavioral verification of expression). There was no statistical difference among days 1, 2, or 3 for repeated saline treatments (open symbols, = 6 or 8). A challenge injection of methamphetamine was administered on WD 14 to rats with a saline (open triangle, = 8) or methamphetamine (filled triangle, = 8) treatment history. Rats with a methamphetamine treatment history expressed sensitization. One-way rm anova with post hoc Newman–Keuls, day 1 different from day 3, ##p < 0.01 (biochemistry group), p < 0.05 (behavior group); Student's t-test, *p < 0.05.

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A separate group of animals was subjected to the same treatment protocols for days 1–3, but on WD 14, brain tissue was harvested for biochemical analyses. As in the previous experiments, these drug-treated rats demonstrated increased motor activity on day 3 compared to day 1. Horizontal activity (F(2,16) = 13.7, p < 0.001; Fig. 4a) and stereotypic activity (F(2,18) = 11.4, p < 0.001; Fig. 4b) were increased following repeated injections of morphine (filled circles). Repeated treatment with methamphetamine (filled diamonds) increased vertical activity (F(2,18) = 6.7, p = 0.01; Fig. 5a) and vertical time (F(2,16) = 7.3, p = 0.01; Fig. 5b). The similarity in responding on days 1 and 3 between the rats demonstrating a sensitized motor response on WD 14 and those killed on WD 14 indicated that the biochemical assessments of the harvested brains indeed reflected a sensitized state. Thus, brain tissue harvested following 14 days of withdrawal from repeated morphine or methamphetamine was used to characterize the brain state associated with the expression of behavioral sensitization.

The VP from rats treated with morphine (t(18) = 2.7, = 0.02) or methamphetamine (t(19) = 3.0, = 0.01) showed increases in mGlu5 S/I ratio compared to saline-treated rats (Student's t-test, > 0.025; Fig. 6a and b); no other protein component of mGlu5 measured was changed (Fig. 6). Also, no changes were detected in any other glutamate receptor proteins assessed in the VP (Tables 6 and 7). Alterations in mGlu5 in the VP did not co-occur with those of GluA1 or GluA2 receptors. Furthermore, levels of the STEP61 protein were also unchanged in the VP 14 days after repeated morphine or methamphetamine (Student's t-test, p > 0.025; Tables 6 and 7).

Table 6. Ventral pallidum: 14 days after morphine
ProteinComponentSalineMorphineStatistics
GluA1

S/I

Total

99.1 ± 9.0

99.7 ± 5.6

123.1 ± 23.4

99.4 ± 8.6

t(19) = 1.057, p = 0.304

t(20) = 0.037, p = 0.970

GluA2

S/I

Total

98.9 ± 6.3

101.1 ± 5.7

99.6 ± 11.0

102.0 ± 10.3

t(20) = 0.058, p = 0.954

t(20) = 0.075, p = 0.941

STEP61 100.0 ± 8.394.1 ± 8.2t(20) = 0.503, p = 0.621
Table 7. Ventral pallidum: 14 days after methamphetamine
ProteinComponentSalineMethamphetamineStatistics
  1. Both the S/I ratio and Total (S+I) protein were assessed for GluA1, GluA2, and STEP61 in the VP tissues after 14 days from repeated saline (1 mL/kg, sc, once daily for 3 days) or morphine (8 mg/kg, sc, for 3 days) or methamphetamine (1 mg/kg, sc, for 3 days) treatment. There were no differences between saline and morphine treatment groups for any protein component assayed. Shown are the mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics, α = 0.025 (Bonferroni correction).

GluA1

S/I

Total

99.1 ± 9.0

100.6 ± 5.8

114.5 ± 15.2

109.6 ± 3.6

t(20) = 0.906, p = 0.376

t(20) = 1.258, p = 0.223

GluA2

S/I

Total

98.9 ± 6.3

101.1 ± 5.7

88.8 ± 13.4

130.0 ± 12.6

t(19) = 0.735, p = 0.471

t(20) = 2.262, p = 0.035

STEP61 100.0 ± 8.3107.3 ± 9.6t(19) = 0.573, p = 0.574
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Figure 6. Increased mGlu5 expression occurred in the VP 14 days after morphine or methamphetamine treatment. (a) Morphine treatment. Representative immunoblot for mGlu5 after repeated morphine (left). The mGlu5 S/I ratio was increased in morphine-treated rats, but there was no change in surface, intracellular or total mGlu5 (right). (b) Methamphetamine treatment. Representative immunoblot for mGlu5 after repeated methamphetamine (left). The mGlu5 S/I ratio was increased in the VP of methamphetamine-treated rats compared with saline-treated rats. However, total, surface or intracellular mGlu5 protein components were not altered by treatment history (right). SAL, saline (1 mL/kg, sc); MOR, morphine (8 mg/kg, sc); METH, methamphetamine (1 mg/kg, sc). Numbers within bars represent sample size. Student's t-test, *< 0.025.

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No changes were detected in the glutamate receptor proteins from NAc tissue following 14 days of withdrawal from repeated morphine (Table 8) or methamphetamine (Table 9). Therefore, STEP61 protein levels were not assessed in the NAc.

Table 8. Nucleus accumbens: 14 days after morphine
ProteinComponentSalineMorphineStatistics
GluA1

S/I

Total

99.9 ± 7.5

106.4 ± 7.2

154.2 ± 32.9

111.1 ± 7.3

t(19) = 1.844, p = 0.081

t(20) = 0.447, p = 0.659

GluA2

S/I

Total

99.6 ± 9.0

99.5 ± 4.5

113.6 ± 16.6

91.2 ± 8.8

t(19) = 0.762, p = 0.455

t(19) = 0.855, p = 0.403

mGlu5

S/I

Total

101.3 ± 11.7

100.9 ± 3.6

129.2 ± 10.8

101.5 ± 6.3

t(20) = 1.726, p = 0.099

t(20) = 0.079, p = 0.938

Table 9. Nucleus accumbens: 14 days after methamphetamine
ProteinComponentSalineMethamphetamineStatistics
  1. There were no differences between saline and morphine (8 mg/kg sc, once daily for 3 days) or saline (1 mL/kg sc, once daily for 3 days) and methamphetamine treatment (1 mg/kg sc for 3 days) groups for any glutamate receptor protein component assayed. Shown are the mean ± SEM optical density values as percent average saline control for each treatment group and corresponding Student's t-test statistics, α = 0.025 (Bonferroni correction).

GluA1

S/I

Total

99.9 ± 7.5

106.4 ± 7.2

84.1 ± 10.2

113.7 ± 7.1

t(19) = 1.278, p = 0.217

t(20) = 0.716, p = 0.482

GluA2

S/I

Total

99.6 ± 9.0

99.5 ± 4.5

101.2 ± 13.5

84.9 ± 9.4

t(18) = 0.102, p = 0.920

t(17) = 1.524, p = 0.146

mGlu5

S/I

Total

101.3 ± 11.7

100.9 ± 3.6

118.5 ± 23.7

94.0 ± 5.1

t(19) = 0.706, p = 0.489

t(20) = 1.141, p = 0.267

There was no change in levels of GluA1 (Fig. 7a) or STEP61 (Fig. 7d) between morphine- and saline-treated rats in mPFC tissue collected on day 17. However, the mPFC from morphine-treated rats showed a significant increase in GluA2 S/I ratio (t(18) = 3.0, = 0.01) largely reflecting a trend for the intracellular component to be less that that detected in saline-treated rats (t(19) = 2.1, = 0.04). GluA2 total protein (t(19) = 0.5, p = 0.64) and surface levels (t(19) = 0.8, = 0.46) were unchanged for the mPFC (Fig. 7b). Morphine decreased total mGlu5 (t(19) = 3.1, = 0.01) and surface (t(19) = 2.7, = 0.01) protein levels in the mPFC (Fig. 7c) with a trend for an increase in mGlu5 S/I ratio (t(19) = 2.1, = 0.05; α = 0.025, per Bonferroni correction). However, there was not a significant difference in the mGlu5 intracellular component of the mPFC between morphine- and saline-treated rats (t(19) = 0.48, = 0.64).

image

Figure 7. Expression of GluA2 was increased in the medial prefrontal cortex (mPFC) 14 days after morphine administration. At left, representative immunoblots for (a) GluA1, (b) GluA2, (c) mGlu5, and (d) STEP61 in mPFC tissues harvested 14 days after repeated injections of saline or morphine. (a) (right) There was no change in mPFC GluA1 14 days after repeated morphine treatment. (b) (right) There was a significant increase in the GluA2 S/I ratio in morphine-treated rats compared to saline-treated rats. However, there was no change in surface, intracellular or total protein levels. (c) (right) There was a significant decrease in surface and total protein levels of mGlu5 in morphine-treated rats without a change in the mGlu5 S/I ratio or intracellular component. (d) (right) There was no difference between saline- and morphine-treated groups in the level of STEP61 in the mPFC. SAL, saline (1 mL/kg, sc); MOR, morphine (8 mg/kg, sc). Numbers within bars represent sample size. Student's t-test, *< 0.025.

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Similar to what was observed subsequent to morphine administration, after 14 days withdrawal from repeated methamphetamine, no change in GluA1 protein levels was observed for the mPFC (Fig. 8a), the GluA2 S/I ratio was increased (t(19) = 3.5, = 0.002; Fig. 8b), with reduction trend in the GluA2 intracellular component (t(20) = 2.4, = 0.03; α = 0.025, per Bonferroni correction). There was no difference between treatment groups in total (t(20) = 0.09, = 0.93) or the surface component (t(20) = 0.5, = 0.60) of GluA2 in the mPFC. Unlike what was detected after morphine treatment, there was a decrease in the level of STEP61 protein in the mPFC of methamphetamine- treated rats (t(19) = 2.5, = 0.02; Fig. 8d), with no change in mGlu5 proteins (Fig. 8c).

image

Figure 8. GluA2 expression was increased and STEP61 levels were decreased in the medial prefrontal cortex (mPFC) 14 days after repeated methamphetamine administration. At left, representative immunoblots for (a) GluA1, (b) GluA2, (c) mGlu5, and (d) STEP61 in mPFC tissues harvested 14 days after repeated injections of saline or methamphetamine. (a) (right) There was no difference in GluA1 in the mPFC between rats with a saline or methamphetamine treatment history. (b) (right) The GluA2 S/I ratio was elevated in methamphetamine-treated compared to saline-treated rats 14 days after drug administration. However, GluA2 surface, intracellular and total protein levels remained unchanged. (c) (right) Methamphetamine treatment history had no effects on mGlu5 expression in the mPFC. (d) (right) STEP61 was significantly reduced in the mPFC of rats 14 days after methamphetamine compared to saline. SAL, saline (1 mL/kg, sc); METH, methamphetamine (1 mg/kg, sc). Numbers within bars represent sample size. Student's t-test, *< 0.025.

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Ability of mGlu5 NAM to mitigate behavioral and glutamate receptor adaptations to repeated methamphetamine administration

To ascertain if mGlu5 were necessary for the development of behavioral sensitization, we determined if the behavior was blocked when the mGlu5 NAM, MTEP, was given prior to methamphetamine on days 1–3 (Fig. 9). We observed that MTEP-treated rats did not develop sensitized responding to methamphetamine (vertical activity, F(2,16) = 1.9, p = 0.19; vertical time, F(2,16) = 1.6, p = 0.23), consistent with the critical nature of mGlu5 for this behavior. To determine if mGlu5 signaling was responsible for the changes in GluA2 surface expression and STEP61 seen in the mPFC following repeated methamphetamine, the mPFC was harvested on WD 14 from the MTEP-pre-treated rats. The changes in GluA2 and STEP61 that previously occurred in the mPFC subsequent to 14 days withdrawal from methamphetamine (refer to Fig. 8a and d, respectively) were not present in MTEP-pre-treated rats; that is, neither GluA2 S/I ratio (t(20) = 0.42, p = 0.68; Fig. 9c) nor STEP61 (t(19) = 0.37, p = 0.72; Fig. 9d) were changed by methamphetamine.

image

Figure 9. Repeated treatment with the mGlu5 negative allosteric modulator, MTEP, attenuated methamphetamine-induced behavioral and biochemical changes. Sensitization did not develop in rats pre-treated with MTEP prior to methamphetamine (filled triangles, = 8) for (a) Vertical Activity or (b) Vertical Time. Representative immunoblots for (c) GluA2 and (d) STEP61 in medial prefrontal cortex (mPFC) tissues (left) harvested 14 days after a repeated injections of vehicle + saline or MTEP + methamphetamine. (c) (right) There was no difference in GluA2 expression between VEH/SAL- and MTEP/METH-treated rats. (d) (right) There was no difference in STEP61 levels between VEH/SAL- and MTEP/METH-treated rats. VEH/SAL; vehicle + saline (1 mL/kg, ip/1 mL/kg, sc); MTEP/METH, MTEP + methamphetamine (5 mg/kg, ip/1 mg/kg, sc). Numbers within bars represent sample size. Student's t-test, > 0.025.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Ionotropic (AMPA) and metabotropic (mGlu5) glutamate receptors are critical mediators of drug-induced neuroplasticity. The aims for this study were to ascertain the trafficking profile of these receptors in brain regions involved in addiction following administration of two distinct classes of abused drugs, and to determine if detected changes co-varied with changes in STEP61, a protein that can mediate mGlu5-induced regulation of AMPA receptor trafficking. Motor activity was used as a functional readout verifying that the acute treatments altered behavior (and thus the brain) and the repeated treatments were sufficient to induce a sensitized state that was present at the time when the brains were harvested to assay glutamatergic markers of neuroplasticity. Of the three brain regions (mPFC, NAc, and VP), two drugs (morphine and methamphetamine), and two treatment protocols (acute, and repeated followed by 14 drug free days) studied, the majority of the changes in glutamatergic indices were observed for the mPFC: (i) The GluA2 S/I ratio increased 14 days after repeated morphine or methamphetamine. (ii) Following repeated morphine, total and surface mGlu5 were reduced, but without changes in STEP61 levels; whereas, following methamphetamine, GluA2 S/I ratio was increased, and STEP61 was reduced. Because levels of STEP61 decreased, less of this protein was available to dephosphorylate, and thus, internalize GluA2 (Zhang et al. 2008). Accordingly, the data concur with our original hypothesis that GluA2 and STEP61 levels would be inversely related. These methamphetamine-induced biochemical effects and associated behavioral sensitization were nullified by MTEP pre-treatment indicating that the protracted changes in glutamatergic function in the mPFC, including alteration of GluA2 and STEP61 expression, were functionally dependent on mGlu5.

The finding that mGlu5 are important for the development of behavioral sensitization to methamphetamine is novel. The use of subtype-selective NAMs have validated that mGlu5 are critical for behaviors mediated by opiates and psychomotor stimulants (Herzig and Schmidt 2004; Herrold et al. 2005; Herzig et al. 2005; Miyatake et al. 2005; Kotlinska and Bochenski 2007; Gass et al. 2009; Veeneman et al. 2011). It is known that mGlu5 are necessary for the development of both methamphetamine-induced conditioned place preference (Miyatake et al. 2005) and cocaine-induced behavioral sensitization (Veeneman et al. 2011), yet the role of mGlu5 in the development of methamphetamine-induced sensitization remains unknown. While few recent studies have examined the role of mGlu5 in the acute effects of amphetamine with mixed results, no study to date has determined the importance of this receptor in methamphetamine motor behaviors. While higher doses (9 mg/kg) of the mGlu5 NAM, MPEP, blunt amphetamine-induced hyperactivity in rats (Gormley and Rompre 2011), lower doses facilitate this behavioral effect in mice (Gormley and Rompre 2011; Manago et al. 2012). However, no published studies to date have determined the role of mGlu5 in hyperactivity or behavioral sensitization induced by methamphetamine. Our finding that mGlu5 are necessary for development of sensitization to methamphetamine indicates a necessary role for this receptor in the maladaptive processes associated with repeated exposure to the psychomotor stimulant.

The cross-linked immunoblotting assay allowed for ex vivo assessment of receptor trafficking as proteins located on the membrane surface, intracellular to the membrane, a ratio of surface to intracellular (S/I), and a sum total of surface and the intracellular proteins can be simultaneously determined. Trafficking of receptors, suggested by changes in S/I ratio, can reflect changes in surface or intracellular protein levels. Increases in the S/I ratio of GluA2, which occurred 14 days following repeated morphine or methamphetamine, resulted from non-significant trends toward a decrease in intracellular GluA2. Such findings have been reported by others (Ferrario et al. 2010), and may have reflected degradation of GluA2 protein rather than increased synthesis of new protein.

This study provides a unique comparison of glutamate receptor adaptations following exposure to two distinct drug classes, opiates and psychomotor stimulants that are abused by humans. One striking difference was that AMPAR and mGlu5 were not altered by acute methamphetamine; however, acute morphine significantly reduced the GluA1 S/I ratio in the mPFC and NAc. The morphine-induced effect reflected decreases in surface expression and increases in intracellular GluA1, suggesting that GluA1 was redistributed from the surface to the intracellular compartment. Surface GluA2 decreased following acute morphine, presenting a picture of AMPAR surface down-regulation. Alternatively, acute methamphetamine (1 mg/kg) did not alter any glutamate receptors in any brain region measured. The limited literature on effects of glutamate receptor trafficking following methamphetamine demonstrate that large doses of methamphetamine (30 mg/kg) increase GluA2 in the frontal cortex 24 h following administration (Simoes et al. 2008). This study revealed strikingly different outcomes 14 days after repeated treatments, and several were common for morphine and methamphetamine treatments. First, the GluA2 S/I ratio was increased in the mPFC 14 days after morphine and methamphetamine administration. Second, the mGlu5 S/I ratio was increased in the VP at the same time point. While repeated treatments to both morphine and methamphetamine resulted in up-regulations in glutamate receptors that occurred after 2 weeks in the mPFC and VP, differences did occur between drug classes after acute treatments. Thus, it would be informative to determine if similar findings could be obtained using other drugs of each class such as heroin (another opiate) and cocaine (another psychomotor stimulant).

We are also able to make a valuable comparison between the brain states of induction of behavioral sensitization versus expression of the behavior following extended withdrawal. While the S/I ratio of GluA1 decreased 1 day after acute morphine, extended withdrawal from repeated morphine returned GluA1 levels to baseline in the mPFC. We previously noted that 1 day after repeated morphine, GluA1 S/I levels decrease in the mPFC (Mickiewicz and Napier 2011) suggesting that a decrease in responsiveness to excitatory inputs occurs within the inter-dosing interval employed here, and prior to extended withdrawal (14 days). Others have reported changes in AMPAR distribution in the NAc associated with extended withdrawal from repeated cocaine treatment. Levels of AMPAR subunits GluA1, GluA2/3, and GluA2 increase in the NAc after 2–3 weeks, but not 1 day of withdrawal from repeated cocaine (Boudreau and Wolf 2005; Boudreau et al. 2007). Similarly, synaptosomal mGlu5 remain unchanged 1 day after repeated cocaine administration, but they are up-regulated following long-term (3 weeks) withdrawal in the NAc (Ghasemzadeh et al. 2009a). However, it was reported that AMPAR S/I ratio was unchanged in the NAc of rats that are sensitized to amphetamine (Nelson et al. 2009). Likewise, this study revealed that GluA1 and GluA2 NAc are unaltered after extended withdrawal from methamphetamine-induced sensitization. Thus, AMPARs appear to be unchanged in the NAc following repeated methamphetamine or amphetamine treatment and this contrasts responses induced by cocaine.

Our results indicate that up-regulation of mGlu5 S/I ratio is a common to withdrawal from a sensitizing regimen of morphine and methamphetamine in the VP. Fourteen days after repeated morphine treatment, the mGlu5 S/I ratio increased attributed to a non-significant increase in the surface component of this receptor. The VP is critically involved in the development (Johnson and Napier 2000; Mickiewicz et al. 2009) and maintenance (Dallimore et al. 2006; McDaid et al. 2006a) of opioid-induced behavioral sensitization, and VP neurons of morphine sensitized rats exhibit an increased propensity to enter depolarization block subsequent to local glutamate application (McDaid et al. 2006a) suggestive of excessive up-regulation of VP glutamate receptors. The activation of mGlu5 influence excitability in many ways including the augmentation of ionotropic glutamate N-methyl-d-aspartic acid receptor (NMDAR) function and suppression of after-hyperpolarization (Mannaioni et al. 2001; Ireland and Abraham 2002). It is possible that augmented mGlu5 S/I ratios in the VP following repeated morphine or methamphetamine excessively promote excitability and glutamate-mediated depolarization-induced inactivation. The contribution of mGlu5 may be particularly relevant for opiates as no alterations in the surface expression of GluA1 or GluA2 subsequent to repeated morphine treatment occurred in the VP. Alternative explanations for the observed increase in VP neuronal functionality include post-translational modifications of AMPARs altering channel conductance (Derkach et al. 1999), or changes in the distribution of other AMPAR subunits (e.g., GluA3) or NMDARs Regardless of the underlying mechanisms, the observed outcomes are consistent with the view that VP neurons are important for coding incentive motivational properties of drug cues following amphetamine sensitization (Tindell et al. 2005), and neuronal markers for activity and synaptogenesis in VP neurons are up-regulated following the expression of drug-induced associative learning (Rademacher et al. 2006).

This study explored the mGlu5-dependent signaling mechanism relevant to behavioral sensitization. After extended withdrawal, both surface and total mGlu5 decreased. Less mGlu5 were available for activation in the mPFC of morphine-treated rats. As activation of mGlu5 result in internalization of AMPARs (Snyder et al. 2001) and surface and total levels of mGlu5 are decreased, it follows that, the GluA2 S/I ratio was significantly increased after 14 days of withdrawal from repeated morphine because fewer mGlu5 were available to be activated. Although STEP61 is a key mediator of mGlu5-dependent internalization of GluA2 (Zhang et al. 2008), this protein remained unchanged 14 days after repeated morphine. After extended withdrawal from morphine, the mPFC is adapting to reduce neuronal calcium levels by favoring AMPAR subunit composition for the calcium-impermeable GluA2 (Liu and Zukin 2007), and decreasing surface and total mGlu5 which would reduce calcium release from intracellular stores (Sladeczek et al. 1985). Similarly, in this study, GluA2 S/I ratio was increased 14 days after repeated methamphetamine, because of a decrease in intracellular GluA2. No alterations occurred in mGlu5 levels in the mPFC. As STEP61 levels were significantly decreased, it appears that less phosphatase was available to internalize GluA2-containing AMPARs and levels increased on the membrane surface relative to the intracellular compartment. This effect was mediated through activation of mGlu5, as a pre-treatment of MTEP prior to methamphetamine administration precluded any changes in mPFC GluA2 or STEP61 levels at 14 days of withdrawal. Thus, the increased GluA2 S/I ratio and decreased STEP61 protein likely reflected activation of mGlu5 through methamphetamine-induced increases in glutamate transmitter in the mPFC (Shoblock et al. 2003; Qi et al. 2009).

In summary, this study addressed several knowledge gaps: Ionotropic and metabotropic glutamate receptor adaptations induced by two drugs of separate classes were characterized. Overlaps for the two drugs included the up-regulation of mGlu5 surface expression in the VP and GluA2 surface expression in the mPFC after long-term withdrawal suggested common glutamatergic mechanisms between the neuronal adaptations that underlie sensitization. Finally, up-regulation of GluA2 S/I ratio and the down-regulation of STEP61 in the mPFC was shown to be mGlu5-dependent. The findings underscore the unique profiles for glutamatergic maladaptations in various brain regions involved in addiction between acute and chronic treatment protocols for opiates and psychomotor stimulants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

The authors have no conflict of interest to declare. This research was supported by US Public Health Service grants DA015760 (T.C.N.), DA023306 (A.A.H. and T.C.N.), and DA019783 (A.L.M. and T.C.N.).

Reference

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference
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