SEARCH

SEARCH BY CITATION

Keywords:

  • Cocaine;
  • Glutamate receptor;
  • Nucleus accumbens;
  • Prefrontal cortex;
  • Ventral tegmental area;
  • Striatum

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

Abstract : The expression of glutamate receptor/subunit mRNAs was examined 3 weeks after discontinuing 1 week of daily injections of saline or cocaine. The level of mRNA for GluR1-4, NMDAR1, and mGluR5 receptors was measured with in situ hybridization and RT-PCR. In nucleus accumbens, acute cocaine treatment significantly reduced the mRNA level for GluR3, GluR4, and NMDAR1 subunits, whereas repeated cocaine reduced the level for GluR3 mRNA. Acute cocaine treatment also reduced the NMDAR1 mRNA level in dorsolateral striatum and ventral tegmental area. In prefrontal cortex, repeated cocaine treatment significantly increased the level of GluR2 mRNA. The GluR2 mRNA level was not changed by acute or repeated cocaine in any other brain regions examined. Repeated cocaine treatment also significantly increased mGluR5 mRNA levels in nucleus accumbens shell and dorsolateral striatum. Functional properties of the ionotropic glutamate receptors are determined by subunit composition. In addition, metabotropic glutamate receptors can modulate synaptic transmission and the response to stimulation of ionotropic receptors. Thus, the observed changes in levels of AMPA and NMDA receptor subunits and the mGluR5 metabotropic receptor may alter excitatory neurotransmission in the mesocorticolimbic dopamine system, which could play a significant role in the enduring biochemical and behavioral effects of cocaine.

The repeated administration of cocaine produces an abundance of long-lasting neuroadaptations. This includes enduring changes in gene expression, neurotransmission, and behavior (for reviews, see Nestler, 1993 ; White et al., 1995a ; Hyman, 1996 ; Pierce and Kalivas, 1997). Because cocaine binds to the dopamine transporter and the resulting elevation in extracellular dopamine level has been linked to many of the acute effects of cocaine (for review, see White et al., 1995a ; Pierce and Kalivas, 1997), the majority of studies investigating neural plasticity associated with cocaine addiction have focused on changes related to pre- and postsynaptic dopamine transmission. Furthermore, most studies have concentrated on the changes in dopamine transmission in the mesocorticolimbic projection, which arises from dopamine neurons in the ventral tegmental area (VTA) and innervates the nucleus accumbens and prefrontal cortex (Fallon and Moore, 1978). Accordingly, it has been shown that long-term alterations in the releasability of dopamine from accumbens and prefrontal terminals are produced by repeated cocaine administration in rats (for review, see Pierce and Kalivas, 1997). Also, long-term changes in cell transduction initiated by dopamine receptor stimulation have been identified in the nucleus accumbens (for review, see White et al., 1995a ; Hyman, 1996).

In addition to dopamine, more recent studies have investigated involvement of excitatory amino acid neurotransmission in the nucleus accumbens and VTA in the long-term neuroadaptations produced by repeated cocaine. Acute administration of high doses of cocaine elevates extracellular glutamate content in the nucleus accumbens and VTA (Kalivas and Duffy, 1995 ; Smith et al., 1995), and this effect is augmented in rats pretreated with daily cocaine injections (Pierce et al., 1996 ; Reid and Berger, 1996 ; Kalivas and Duffy, 1998). Furthermore, there is a long-term elevation in the behavioral response elicited by microinjection of (±)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) into the nucleus accumbens of rats pretreated with daily cocaine (Pierce et al. 1996). There is also a short-lived reduction in the electrophysiological response produced by iontophoresis of AMPA onto neurons in the nucleus accumbens (White et al., 1995b) and a short-lived increase in the responsiveness to AMPA on dopamine cells in the VTA of rats pretreated with repeated injections of cocaine (Zhang et al., 1997).

There are both ionotropic and metabotropic (mGluR) glutamate receptors (GluRs) in the nucleus accumbens and VTA. The subunits of the ionotropic receptors are classified according to their pharmacological properties and sequence similarity into AMPA, kainate, or N-methyl-d-aspartate (NMDA) receptor families (Hollmann and Heinemann, 1994). In particular abundance in the nucleus accumbens are the GluR1, GluR2, and GluR3 subunits of the AMPA-preferring receptor, as well as the NMDA receptor NMDAR1 and NMDAR2B subunits (Albin et al., 1992 ; Wullner et al., 1994 ; Standaert et al., 1994). In contrast, the GluR4 subunit is in low abundance and localized only to interneurons (Tallaksen-Greene and Albin, 1994 ; Bernard et al., 1997). The VTA neurons also express functional AMPA and NMDA receptors, although information regarding the relative expression level of the individual subunits is not available (for review, see White, 1996). Fitzgerald et al. (1996) reported that repeated cocaine administration did not alter levels of immunoreactive GluR1, GluR2/3, or NMDAR1 protein in the nucleus accumbens, whereas the protein expression of the GluR1 and NMDAR1 subunits was elevated in the VTA. However, these changes were measured only at 1 day after the last daily injection of cocaine, and it is not known if they constitute long-term neuroadaptations. In addition to the ionotropic GluRs, there are eight mGluRs that are coupled to second messenger systems via G proteins and are classified into three groups according to their affector systems and sequence similarity (Conn and Pin, 1997). The group 1 receptor mGluR5 (coupled to membrane phosphoinositide hydrolysis) is especially enriched in the nucleus accumbens (Testa et al., 1995). However, no information exists on the regulation of mGluR expression or function by repeated cocaine administration.

The present study was designed to determine if repeated cocaine administration produces a long-term change in the expression of the ionotropic receptor subunits GluR1, 2, 3, and 4, NMDAR1, or the metabotropic receptor mGluR5 in the nucleus accumbens, VTA, striatum, and prefrontal cortex. The expression of mRNA encoding these proteins was estimated using quantitative RT-PCR and in situ hybridization in the brain of rats pretreated with daily saline or cocaine injections for 1 week and killed 3 weeks after the last daily injection.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

Animal housing

Male Sprague-Dawley rats weighing 250-300 g were obtained from Simonsen Laboratories (Gilroy, CA, U.S.A.). The animals were individually housed with food and water available ad libitum. A 12/12-h light/dark cycle was used with the lights on at 7 : 00 a.m., and all saline or cocaine injections were performed during the light cycle.

Drug treatment regimens

Table 1 shows the treatment regimens used. All animals were handled and injected with saline (0.9% NaCl, sterile, 1 ml/kg) on day 1. Thereafter, subjects were assigned to one of two groups (16 rats each) and were injected daily for 1 week with either saline or cocaine (supplied by the National Institute on Drug Abuse). Three weeks after the last daily injection (day 29) the cocaine and saline treatment groups were divided in half. Half of the animals in each repeated treatment group were injected acutely with cocaine (15 mg/kg, i.p.), and the other half were injected with saline (1 ml/kg, i.p.). The subjects were decapitated 24 h after the acute injection of cocaine or saline, and the brains were processed as described below.

Table 1. Protocol for drug treatment The number after cocaine refers to the daily dose (in mg/kg, i.p.) administered to rats.
  Repeated daily treatment 
Treatment (repeated/acute)Adaptation (day 1)Day 2Day 3-7Day 8Challenge (day 29)
Saline/salineSalineSalineSalineSalineSaline
Saline/cocaineSalineSalineSalineSalineCocaine 15
Cocaine/salineSalineCocaine 15Cocaine 30Cocaine 15Saline
Cocaine/cocaineSalineCocaine 15Cocaine 30Cocaine 15Cocaine 15

Semiquantitative RT-PCR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

The brain was removed and hand-dissected on an ice-cooled plate to isolate the prefrontal cortex, nucleus accumbens, and VTA. The dissected tissue was immediately frozen on dry ice and stored at -80°C.

The PCR protocol was adopted with modification from those of Leonard et al. (1993) and Somogyi et al. (1995). Total tissue RNA was extracted using RNAzol (Life Technologies, Gaithersburg, MD, U.S.A.) according to the manufacturer's protocol, and samples were stored at -80°C. For RT, 500,000 copies of the internal standard RNA (pAW 109 ; Perkin-Elmer, Foster City, CA, U.S.A.) were added to a 5-μg sample of tissue RNA. The first-strand cDNA was synthesized using 250 ng of hexanucleotide random primer (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 200 units of Superscript II reverse transcriptase (Life Technologies) according to the manufacturer's protocol.

PCR primer sequences were taken from the following sources : GluR1-4, Holzwarth et al. (1994) ; NMDAR1, Niedzielski and Wenthold (1995) ; mGluR5, Ghasemzadeh et al. (1996) ; and pAW 109, Somogyi et al. (1995). PCR primer sequences were as follows : GluR1 (345 bp), 5′-ATGCCGTACATCTTTGCC-3′ and 5′-AACAGGAAAACTTGGAGTA-3′ ; GluR2 (434 bp), 5′-GGTGTCTCTTCTAACAGC-3′ and 5′-AGAACAGCTTGCAGTGTTG-3′ ; GluR3 (329 bp), 5′-ATGGGGCAAAGCGTGCTCC-3′ and 5′-AAGGAGGTCAGGGTGTTCAT-3′ ; GluR4 (281 bp), 5′-ATGAGGATTATTTGCAGG-3′ and 5′-ATGGCAAACACCCCTCTAG-3′ ; NMDAR1 (1,012 bp), 5′-ACGGAATGATGGGCGAGC-3′ and 5′-GGCATCCTTGTGTCGCTTGTAG-3′ ; mGluR5 (349 bp), 5′-TCCAATCTGCTCCTCCTACC-3′ and 5′-CAACGATGAAGAACTCTGCG-3′ ; and pAW 109 (149 bp), 5′-CCAGCCATCCTTCGAGATTTCT-3′ and 5′-GTTGTTCCTCCAGTTCTTTCTCACC-3′. All PCR primers were based on unique sequences obtained from GenBank and verified by the NCBI BLAST program (Altschul et al., 1990). PCR primers were synthesized and purified by Custom Primers, Life Technologies.

Ten percent of the synthesized first-strand cDNA was used as a template in PCR experiments. Each PCR tube contained 1× PCR buffer (Perkin-Elmer), 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer), each of the GluR primers at 0.5 μM, each standard primer (pAW 109) at 0.5 μM, 0.2 mM deoxynucleotide triphosphates, and 2.5 mM MgCl2 in a final volume of 50 μl and was covered with a layer of oil. The PCR amplification conditions were 95°C for 4 min for one cycle (initial denaturation step), after which MgCl2 was added (Hot Start PCR), and then the following conditions were used : 94°C for 1.5 min, 55°C for 1.5 min, and 72°C for 1 min for a total of 37 cycles. The samples underwent a final extension time of 10 min at 72°C. To minimize sample-to-sample variation, all samples were reverse-transcribed in one batch using the same chemical ingredients and enzyme. Also, all samples were amplified in one batch using the same PCR master mix. A 15-μl aliquot of the PCR products was analyzed by electrophoresis on a 1.5% agarose gel in 1× Tris-acetate-EDTA (TAE) buffer and subsequently stained with ethidium bromide (1 μg/ml) for 20 min and destained in 1× TAE buffer for 40-60 min. The bands were visualized with UV light and documented with black and white instant Polaroid film 665.

Band intensities were analyzed using computerized laser scanning densitometry (Molecular Dynamics, Sunnyvale, CA, U.S.A.). The target band intensity of each sample was normalized to the internal standard (pAW 109) present in each sample, and the normalized value was used as an indicator of the relative abundance of target mRNA. An RT-PCR calibration experiment was done for each of the GluR subunits and for mGluR5 using 0.5-5 μg of total RNA with 500,000 copies of the pAW 109 RNA added to each RNA tubes. Subsequently, all RNA samples were reverse-transcribed, and cDNAs were used in PCR procedures as described above. PCR experiments for each RNA concentration were done in triplicates. The results indicated that the amplified cDNAs were within the linear range of the amplification curve.

Quantitative in situ hybridization

The procedure for in situ hybridization has been described previously (Testa et al., 1995). Oligodeoxyribonucleotide probes specific for individual mRNAs were made according to published cDNA sequences. The oligodeoxyribonucleotide probes were synthesized and purified by GibcoBRL (Custom Primers, Life Technologies). The sequences of the probes used for the AMPA receptor subunits (GluR1-4) are from Keinanen et al. (1990) : GluR1, 5′-GTCACTGGTTGTCTGGTCTCGTCCCTCTTCAAACTCTTCGCTGTG-3′ ; GluR2, 5′-TTCACTACTTTGTGTTTCTCTTCCATCTTCAAATTCCTCAGTGTG-3′ ; GluR3, 5′-AGGGCTTTGTGGGTCACGAGGTTCTTCATTGTTGTCTTCCAAGTG-3′ ; and GluR4, 5′-CTGGTCACTGGGTCCTTCCTTCCCATCCTCAGGTTCTTCTGTGTG-3′. The GluR1-4 probes recognize both flip and flop splice variants of the mRNA. The NMDAR1 probe was from Standaert et al. (1994) and recognizes all known splice variants, and the sequence is 5′-AAACCAGACGCTGGACTGGTGGGAGTAGGGCGGCACCGTGCGAAG-3′. The probe for mGluR5 was from Testa et al. (1995), with a sequence of 5′-GGAGCGGAAGGAAGAAGATCCATCTACACAGCGTACCAAACCTTC-3′. The sequence of the randome probe is 5′-GATGCGAGCGTACACGACCTTTGCTGACGGACAAGCGTAAAGTCAGGT-3′. All of the probes were 45-50 bases in length with a GC content of 49-65% and were 3′-end-tailed with terminal deoxynucleotidyltransferase using 35S-dATP nucleotide (Du Pont-NEN, Boston, U.S.A.).

Cryostat sections (12 μm) were cut in the coronal plane, mounted on glass slides (Superfrost/Plus ; Fisher, Pittsburgh, PA, U.S.A.), and stored at -70°C. The slides were brought to room temperature under a stream of cool air. For prehybridization, slides were rinsed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (10 min) ; 3× phosphate-buffered saline (5 min) ; 0.1 M triethanolamine (pH 8.0) with 0.25% acetic acid anhydride (10 min) ; phosphate-buffered saline (5 min) ; and graded ethanol solutions (2 min each). Hybridization was performed in a buffer of 50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0), 10% dextran sulfate, 1× Denhardt's solution, 100 mM dithiothreitol, and ~30,000 dpm/μl labeled probe at 37°C overnight. Cold competition controls were hybridized with a 25-fold excess of unlabeled probe added to the hybridization buffer. Posthybridization washes were performed to a maximal stringency of 0.5× standard saline citrate (SSC) at 60°C for 40 min. The sections were exposed to β-Max Hyperfilm (Amersham, Arlington Heights, IL, U.S.A.) for 3 weeks. For quantitative analysis five rats per treatment group were used. Four sections from each rat were used in the hybridization experiments. All sections from all treatment groups were processed for in situ hybridization in a single batch to ensure identical experimental conditions and treatment. Optical density measurements were obtained using an image analysis shareware (NIH Image ver. 1.6) and corrected for background density.

Statistics

All data were evaluated using a two-way ANOVA with chronic and acute treatment as factors. If a significant interaction F score was measured, post hoc comparisons between treatment groups were conducted using a two-tailed unpaired Student's t test. Table 2 lists the probability values ≤0.1 arising from the two-way ANOVAs conducted for each GluR subunit in each brain region.

Table 2. Statistically significant changes in mRNA expression of ionotropic GluR subunits and mGluR5 A dash indicates that the two-way ANOVA revealed no significant effect of acute or repeated treatment or interaction between acute and repeated treatment with cocaine. The probability value for the F score of the two-way ANOVA is shown if p≤ 0.1. When the interaction gave p≤ 0.05, a post hoc Student's t test was conducted to determine which treatment groups differed by p≤ 0.05. Groups with significant differences are indicated in parentheses : ss, repeated saline/acute saline ; sc, repeated saline/acute cocaine ; cs, repeated cocaine/acute saline ; cc, repeated cocaine/acute cocaine.
Receptor subunit/subtype, brain regionAcuteRepeatedInteraction
GluR1   
VTA
NA shell0.077
NA core0.0700.049 (cc vs. cs)
Dorsolateral striatum
Prefrontal cortex
GluR2   
VTA
NA shell
NA core
Dorsolateral striatum
Prefrontal cortex< 0.001
GluR3   
VTA
Nucleus accumbens0.023< 0.001
Prefrontal cortex
GluR4   
VTA0.072
Nucleus accumbens0.0160.0800.013 (ss vs. sc, cs, cc)
Prefrontal cortex0.020 (ss vs. sc)
NMDAR1   
VTA0.050
NA shell
NA core0.027
Dorsolateral striatum0.004
Prefrontal cortex
mGluR5   
VTA
NA shell0.005 (cs vs. ss, cc)
NA core
Dorsolateral striatum0.015 (cs vs. ss, sc, cc)
Prefrontal cortex

Quantification of mRNAs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

In situ hybridization was used to obtain optical density measures of levels of mRNAs encoding GluR1, GluR2, NMDAR1, and mGluR5 in the shell (NAshell) and core (NAcore) of the nucleus accumbens and in the dorsolateral striatum (Fig. 1). The level of mRNA for GluR3 and GluR4 subunits was not sufficient to be accurately quantified using in situ hybridization. Therefore, the amount of transcripts encoding these glutamate receptor subunits was quantified using RT-PCR. In addition, RT-PCR was conducted on tissues from the prefrontal cortex and VTA to obtain relative mRNA levels for the five ionotropic GluR subunits, as well as mGluR5. The conditions for RT and PCR amplification were optimized for quantitative measurements of levels of the mRNA transcripts. A PCR calibration curve was constructed for each GluR subunit/subtype (Fig. 2) to ensure linear amplification of the target transcript under the conditions used in this study.

image

Figure 1. Representative results of in situ hybridization experiments for various probes and the anatomical regions that were used in quantitative in situ hybridization experiments. All sections chosen for the experiments were at the level of plate 11-13 of the rat brain atlas (Paxinos and Watson, 1986). The optical density measured in anterior commissure was used as the background value for NAcore (NAc) and NAshell (NAs), and similarly, corpus callosum was used as background for dorsolateral striatum (STR). Two optical density measurements were obtained from the NAc (indicated by the two circles in the core), and one measurement was made from NAs on each side of the section, and the background subtracted values were averaged. The result of hybridization with a random oligodeoxyribonucleotide probe is also shown, which shows no specific labeling.

Download figure to PowerPoint

image

Figure 2. RT-PCR calibration curves for each GluR subunit/subtype. Data are mean ± SEM (bars) values (n = 3). Total RNA from whole brain tissue was used to synthesize cDNA for these experiments. The RT and PCR amplification conditions were the same used for experimental samples.

Download figure to PowerPoint

GluR1.

A two-way ANOVA of the data indicated that the only significant effect of cocaine on GluR1 mRNA level was in the NAcore, where a significant interaction between acute and repeated treatment was measured (Table 2 and Fig. 3B). A post hoc Student's t test revealed a significant reduction in GluR1 mRNA content by acute cocaine administration in subjects pretreated with repeated cocaine compared with animals pretreated with repeated cocaine and given an acute injection of saline 24 h before they were killed. In addition, post hoc analysis showed a near significant (p = 0.056) increase in GluR1 mRNA level in NAcore in animals pretreated with repeated cocaine compared with subjects pretreated with repeated saline followed by an acute saline injection. The ANOVAs also showed a near significant effect of acute treatment on GluR1 mRNA levels in both the NAcore and NAshell (Table 2).

image

Figure 3. Effect of repeated cocaine on GluR mRNA levels in nucleus accumbens (NA). See Table 1 for description of the treatment groups. A : Results of RT-PCR experiments. For these experiments, NA tissue containing both core and shell compartments was dissected. Data are mean ± SEM (bars) values (n = 7-8 rats per group). B and C : Results of in situ hybridization (ISH) experiments on NAcore and NAshell, respectively. The drug treatment and experimental paradigm were identical to those of the RT-PCR experiments. Data are mean ± SEM (bars) values (n = 5 rats per group) (see Table 1). All data were evaluated using a two-way ANOVA, and results are presented in Table 2. A two-tailed unpaired Student's t test was used for post hoc comparison of individual groups when ANOVA revealed a significant (p≤ 0.05) interaction (see Table 2). *p < 0.05 compared with saline/saline group ; +p < 0.05 compared with cocaine/saline group. NR1, NMDAR1.

Download figure to PowerPoint

GluR2.

The only significant effect of cocaine on GluR2 mRNA levels was in the prefrontal cortex, where repeated cocaine administration significantly elevated the level of GluR2 mRNA (Table 2 and Fig. 4B).

image

Figure 4. Effect of repeated cocaine treatment on GluR mRNA levels in VTA and prefrontal cortex (PFC). The relative mRNA levels were determined using RT-PCR. Data are mean ± SEM (bars) values (n = 6-8 rats per group). Two-way ANOVA was used for statistical analysis, and results are presented in Table 2. A two-tailed unpaired Student's t test was used for post hoc comparison of individual groups. *p < 0.05 compared with saline/saline group. NR1, NMDAR1.

Download figure to PowerPoint

GluR3.

Significant effects of cocaine on GluR3 mRNA were measured in the nucleus accumbens using RT-PCR (Table 2 and Fig. 3A). Both acute and repeated cocaine treatments significantly reduced the GluR3 mRNA level compared with rats receiving repeated saline with an acute saline injection.

GluR4.

Similar to the GluR3 subunit, there was a significant reduction by acute treatment on GluR4 mRNA level in nucleus accumbens and a near significant reduction by repeated treatment (Table 2 and Fig. 3A). There was also a significant interaction between acute and repeated treatment in the nucleus accumbens, and post hoc analysis revealed a significant reduction in GluR4 mRNA level in all treatment groups compared with the subjects given repeated saline treatment and an acute saline injection 24 h before the animals were killed (Fig. 3A). The prefrontal cortex also demonstrated a significant interaction that arose primarily from an elevation in mRNA level by acute cocaine in repeated saline-treated animals and a blunting of this effect in repeated cocaine-treated subjects (Table 2 and Fig. 4B). There was also a near significant effect of repeated treatment on GluR4 mRNA level in the VTA (Table 2).

NMDAR1.

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

Acute treatment was a consistent and statistically significant factor in the regulation of NMDAR1 mRNA in the striatum. NAcore, and VTA (Table 2). In NAcore acute cocaine administration reduced the NMDAR1 mRNA level primarily in the repeated saline-treated subjects (Fig. 3B). However, in striatum and VTA acute cocaine treatment reduced the NMDAR1 mRNA levels in subjects treated with either repeated saline or cocaine (Figs. 4A and 5). There was no effect on NMDAR1 mRNA levels in NAshell and prefrontal cortex by any drug treatment.

image

Figure 5. Effect of repeated cocaine on GluR mRNA levels in dorsolateral striatum. The experimental paradigm was the same as described in the legend to Fig. 2. A two-way ANOVA was used for statistical analysis, and the results are presented in Table 2. A two-tailed unpaired Student's t test was used for post hoc comparison of individual groups. Data are mean ± SEM (bars) values (n = 5 rats per group). *p < 0.05 compared with saline/saline group ; +p < 0.05 compared with cocaine/saline group ; #p < 0.05 compared with saline/cocaine group. NR1, NMDAR1.

Download figure to PowerPoint

mGluR5.

There was a significant interaction between the effect of acute and repeated treatments on mGluR5 mRNA level in both the NAshell and dorsolateral striatum (Table 2). In both the NAshell and striatum the interaction arose from a significant elevation in mRNA levels by repeated cocaine compared with repeated saline treatment and the capacity of an acute cocaine injection to reverse the increase in mRNA level in repeated cocaine-pretreated subjects without reducing the mRNA levels in daily saline-treated rats (Figs. 3C and 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

In this study in situ hybridization and RT-PCR were used to study the effect of acute and repeated cocaine treatments on expression level of mRNA for ionotropic GluR subunits and mGluR5 in prefrontal cortex, nucleus accumbens, VTA, and dorsolateral striatum. Significant changes in the levels of mRNA encoding the ionotropic receptor subunits and mGluR5 were measured that varied with the brain region examined.

Glutamate transmission in nucleus accumbens

Repeated cocaine administration reduces the basal level of extracellular glutamate and enhances the capacity of acute cocaine to elevate glutamate levels in the nucleus accumbens (Pierce et al., 1996 ; Reid and Berger, 1996). Also, repeated cocaine administration augments the behavioral activation elicited by either an AMPA or NMDA agonist microinjected into the nucleus accumbens (Bell and Kalivas, 1996), as well as increasing the level of immunoreactive GluR1 in the nucleus accumbens (Churchill et al., 1997). In agreement with the elevation in content of GluR1 subunit protein produced by repeated cocaine, the post hoc analysis revealed a near significant increase (p = 0.056) in GluR1 mRNA content in NAcore. Repeated cocaine also resulted in a significant or near significant reduction in the level of GluR3 and GluR4 mRNA, respectively, and consistent with previous observations that GluR2 subunit protein levels were unchanged (Churchill et al., 1997), the level of GluR2 mRNA was unaffected by repeated cocaine.

Recently, Lu et al. (1997) have shown that repeated amphetamine administration for 5 days followed by 2 weeks of withdrawal produced a decrease in GluR1 and GluR2 mRNA levels in the nucleus accumbens with no change in level of the GluR3 subunit mRNA. These observations are in contrast to the cocaine effects seen after 3 weeks of withdrawal in our study. It is possible that the neuroadaptation in GluR expression is different after repeated amphetamine exposure than after cocaine treatment, similar to other reported differences in pharmacological and behavioral effects of amphetamine and cocaine (for discussion, see Pierce and Kalivas, 1997).

The proportion of GluR2 subunit making up the AMPA-gated channel is inversely related to calcium conductance (Geiger et al., 1995). Thus, although repeated cocaine may modestly increase GluR1 mRNA expression and decrease that of GluR3 or GluR4, the relative permeability of the AMPA channel to calcium may not be altered. The AMPA receptor subunits exist in two splice forms, named flip and flop (Sommer et al., 1990), which are developmentally regulated (Monyer et al., 1991) and differ in their desensitization kinetics and other pharmacological properties (Lomeli et al., 1994 ; Mosbacher et al., 1994 ; Geiger et al., 1995). In striatum and nucleus accumbens, the expression of the flop variant, which exhibits faster desensitization kinetics, is dominant, with the expression of the GluR1flip and GluR4flip at a very low level and the GluR2 and 3flip variants expressed at a significantly lower level than the flop form (Wullner et al., 1994). Recent studies have shown concordant modulation of the flop and flip forms in schizophrenia and after some pharmacological treatments (Eastwood et al., 1997 ; Lason et al., 1997 ; McCoy et al., 1998). In the present study, we investigated the effect of cocaine treatment on both flip and flop splice variants of AMPA receptor subunits using pan probes/primers that recognize both forms. However, we cannot rule out the possibility that cocaine treatment may have distinct effects on the two splice variants.

Studies using in vitro expression systems and a recent study examining striatal neurons reveal that AMPA receptor channels constructed of GluR1 and GluR2 flip or flop variants desensitize to agonist binding slower than channels that include GluR3 flop or GluR4 flop forms (Lomeli et al., 1994 ; Mosbacher et al., 1994 ; Geiger et al., 1995 ; Gotz et al., 1997). Thus, AMPA channels with a relative increase in GluR1 combined with a decrease in GluR3/4 would be predicted to desensitize more slowly and to mediate greater depolarization to agonist stimulation. This is consistent with the increased behavioral responsiveness to AMPA microinjection into the nucleus accumbens of rats pretreated with daily cocaine injections (Bell and Kalivas, 1996 ; Pierce et al., 1996). Although contrary to the observation that the electrophysiological response to iontophoretic glutamate is reduced in the nucleus accumbens of rats pretreated with daily cocaine (White et al., 1995b), this electrophysiological effect has only been examined 3 days after discontinuing repeated cocaine treatment.

In contrast to the AMPA receptor subunits, repeated cocaine did not produce an alteration in the level of NMDAR1 mRNA in the nucleus accumbens. However, in NAshell repeated cocaine significantly elevated the level of mRNA encoding mGluR5, which may constitute a compensatory response to reduced basal levels of extracellular glutamate seen after repeated cocaine (Pierce et al., 1996). Furthermore, an increase in mGluR5 expression would be predicted to increase the behavioral responsiveness to enhanced glutamate transmission in the nucleus accumbens because mGluR agonist microinjection into the nucleus accumbens elicits an increase in motor activity (Attarian and Amalric, 1997 ; Kearney et al., 1997 ; Kim and Vezina, 1997).

Acute administration of cocaine produces a dose-dependent increase in extracellular glutamate in the nucleus accumbens (Smith et al., 1995 ; Reid and Berger, 1996 ; Pierce et al., 1996). The reduction in NMDAR1 mRNA level produced by acute cocaine administration may constitute a compensatory down-regulation in response to elevated synaptic concentrations of glutamate. Likewise, in the nucleus accumbens a significant or near significant down-regulation in mRNA encoding the GluR1, GluR3, and GluR4 subunits was produced by acute cocaine administration. Acute cocaine produced an overall reduction in many GluR subunit mRNAs, but post hoc analysis revealed differences between the effect of acute cocaine in repeated saline-compared with repeated cocaine-pretreated rats. Thus, although repeated cocaine produced apparent tolerance in the capacity of acute cocaine to alter the levels of mRNA encoding GluR3, GluR4, and NMDAR1, the capacity to affect GluR1 and mGluR5 was augmented.

Glutamate transmission in striatum

Repeated cocaine produced a significant increase in the level of mGluR5 receptor mRNA in the dorsolateral striatum that was similar to the alterations seen in the NAshell. In addition to increasing mGluR5 mRNA content, repeated cocaine rendered the level of mRNA sensitive to reduction by an acute cocaine injection. Similar to the the situation in NAcore, acute administration of cocaine reduced the level of mRNA encoding NMDAR1 in the striatum.

Glutamate transmission in VTA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

Repeated cocaine administration produces a long-term augmentation in the capacity of an acute injection of cocaine to elevate extracellular glutamate content in the VTA (Kalivas and Duffy, 1998). The increased releasability of glutamate is not associated with long-lasting alterations in postsynaptic responsiveness to iontophoretic glutamate or AMPA (Zhang et al., 1997). Accordingly, repeated treatment produced no change in the level of any mRNA except for a near significant increase in GluR4 content. However, similar to the nucleus accumbens and striatum, acute treatment significantly reduced the NMDAR1 mRNA content in the VTA.

Glutamate transmission in prefrontal cortex

The response of the prefrontal cortex to repeated cocaine was distinct from that of other brain regions examined. Repeated treatment produced a significant increase in the level of mRNA encoding GluR2 subunit. As outlined above, the robust increase in GluR2 mRNA content would be expected to decrease calcium conductance through the AMPA channel (Geiger et al., 1995). Unlike other brain regions examined, the prefrontal cortex did not show a reduction in NMDAR1 mRNA level following acute cocaine administration. However, acute cocaine injection elicited a unique increase in the level of GluR4 mRNA. This acute effect of cocaine was present only in animals pretreated with repeated saline and was absent in subjects pretreated with repeated cocaine. A relative increase in the proportion of GluR4 subunit in the AMPA channel will promote desensitization of the channel, resulting in a reduced activation of the receptor (Lomeli et al., 1994 ; Mosbacher et al., 1994 ; Geiger et al., 1995). Repeated amphetamine followed by 2 weeks of withdrawal did not show any changes in GluR1, 2, and 3 mRNA levels in prefrontal cortex (Lu et al., 1997). The discrepancy with our data may arise from differences between amphetamine and cocaine (see above).

General effects of acute and repeated cocaine

Repeated cocaine treatment produced various longterm changes in GluR mRNA levels in regions of the brain associated with the enduring behavioral effects of cocaine. Based on what is known about the physiological effects of altered proportions of receptor subunits on AMPA channel conductance, changes in the nucleus accumbens are likely to promote excitation by decreasing receptor desensitization and increasing current flow. In general, these alterations are consistent with data showing that repeated cocaine increases glutamate transmission in the nucleus accumbens (Bell and Kalivas, 1996 ; Pierce et al., 1996 ; Reid and Berger, 1996). In contrast to the nucleus accumbens, in the prefrontal cortex the predicted changes in receptor subunit balance would be expected to decrease calcium conductance.

Acute cocaine administration elicited changes in the level of GluR mRNAs consistent with a compensatory reduction in postsynaptic responsiveness. Notably, NMDAR1 mRNA content was reduced in striatum, nucleus accumbens, and VTA. However, the changes in AMPA receptor subunits in response to acute cocaine varied depending on the brain regions with reductions in GluR3 and GluR4 levels in the nucleus accumbens, indicating a decrease in the rate of desensitization and an increase in GluR4 content in the prefrontal cortex, portending an increased rate of desensitization.

The physiological relevance of the changes in glutamate subunit/receptor mRNA levels elicited by repeated and acute cocaine awaits further analysis of complementary changes in protein expression and receptor function. However, these data are consistent with other studies showing that repeated cocaine administration produces long-lasting alterations in gene expression, especially in dopamine terminal fields (Hope et al., 1994 ; Moratalla et al., 1996 ; Cha et al., 1997). Long-term behavioral alterations exist associated with cocaine addiction, including the development of paranoia and drug craving (Satel and Edell, 1991 ; Childress et al., 1998), and discovering alterations in gene expression produced by repeated cocaine administration that are of equivalent duration is a primary step toward elucidating the neuroadaptations mediating addiction-related behaviors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Semiquantitative RT-PCR
  5. RESULTS
  6. Quantification of mRNAs
  7. NMDAR1.
  8. DISCUSSION
  9. Glutamate transmission in VTA
  10. Acknowledgements

We thank Kari Johnson for skillful technical assistance. This work was supported in part by the Washington State Alcohol and Drug Abuse Program (to M.B.G.) and U.S. Public Health Service grants DA 03906 and MH40817 and Research Career Development Award DA00158 (to P.W.K.).

  • 1
    Albin R.L., Makowiec R.L., Hollingsworth Z.R., Dure L.S.IV, Penney J.B., Young A.B. (1992) Excitatory amino acid binding sites in the basal ganglia of the rat : a quantitative autoradiographic study.Neuroscience 46,3548.
  • 2
    Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. (1990) Basic local alignment search tool.J. Mol. Biol. 215,403410.DOI: 10.1006/jmbi.1990.9999
  • 3
    Attarian S. & Amalric M. (1997) Microinjection of the metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid into the nucleus accumbens induces dopamine-dependent locomotor activation in the rat. Eur. J. Neurosci. 9,809816.
  • 4
    Bell K. & Kalivas P.W. (1996) Context-specific cross-sensitization between systemic cocaine and intra-accumbens AMPA infusion in the rat.Psychopharmacology (Berl.) 127,377383.
  • 5
    Bernard V., Somogyi P., Bolam J.P. (1997) Cellular, subcellular, and subsynaptic distribution of AMPA-type glutamate receptor subunits in the neostriatum of the rat.J. Neurosci. 17,819933.
  • 6
    Cha X., Pierce R.C., Kalivas P.W., Mackler S.A. (1997) NAC-1, a rat brain mRNA, is increased in the nucleus accumbens three weeks after chronic cocaine self-administration.J. Neurosci. 17,68646871.
  • 7
    Childress A.R., Mozley P.D., McElgin W., Fitzgerald J., Reivich M., O'Brien C.P. (1998) Limbic activation during cue-induced cocaine craving.Am. J. Psychiatry (in press).
  • 8
    Churchill L., Ghasemzadeh M.B., Kalivas P.W. (1997) Glutamate receptor subunits (GluR1 and NMDAR1) increase in the nucleus accumbens of rats 3 weeks after repeated cocaine exposure.Soc. Neurosci. Abstr. 23,260.
  • 9
    Conn P.J. & Pin J. -P. (1997) Pharmacology and functions of metabotropic glutamate receptors.Annu. Rev. Pharmacol. Toxicol. 37,205237.
  • 10
    Eastwood S.L., Burnet P.W., Harrison P.J. (1997) GluR2 glutamate receptor subunit flip and flop isoforms are decreased in the hippocampal formation in schizophrenia : a reverse transcriptase-polymerase chain reaction (RT-PCR) study.Mol. Brain Res. Brain Res. 44,9298.
  • 11
    Fallon J.H. & Moore R.Y. (1978) Catecholamine innervation of basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and striatum.J. Comp. Neurol. 180,545580.
  • 12
    Fitzgerald L.W., Ortiz J., Hamedani A.G., Nestler E.J. (1996) Drug of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area : common adaptations among cross-sensitization agents.J. Neurosci. 16,274282.
  • 13
    Geiger J.R.P., Melcher T., Koh D., Sakmann B., Seeburg P.H., Jonas P., Monyer H. (1995) Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.Neuron 15,193204.
  • 14
    Ghasemzadeh M.B., Sharma S., Surmeier D.J., Eberwine J.H., Chesselet M. -F. (1996) Multiplicity of glutamate receptor subunits in single striatal neurons : an RNA amplification study.Mol. Pharmacol. 49,852859.
  • 15
    Gotz T., Kraushaar U., Geiger J., Lubke J., Berger T., Jonas P. (1997) Functional properties of AMPA and NMDA receptors expressed in identified types of basal ganglia neurons.J. Neurosci. 17,204215.
  • 16
    Hollmann M. & Heinemann S. (1994) Cloned glutamate receptors.Annu. Rev. Neurosci. 17,31108.
  • 17
    Holzwarth J.A., Gibbons S.J., Brorson J.R., Philipson L.H., Miller R.J. (1994) Glutamate receptor agonists stimulate calcium responses in different types of cultured rat cortical glial cells.J. Neurosci. 14,18791891.
  • 18
    Hope B.T., Nye H.E., Kelz M.B., Self D.W., Iadarola M.J., Nakabeppu Y., Duman R.S., Nestler E.J. (1994) Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments.Neuron 13,12351244.
  • 19
    Hyman S.E. (1996) Addiction to cocaine and amphetamine.Neuron 16,901904.
  • 20
    Kalivas P.W. & Duffy P. (1995) D1 receptors modulate glutamate transmission in the ventral tegmental area.J. Neurosci. 15,53795388.
  • 21
    Kalivas P.W. & Duffy P. (1998) Repeated cocaine administration alters extracellular glutamate in the ventral tegmental area.J. Neurochem. 70,14971502.
  • 22
    Kearney J.A.F., Frey K.A., Albin R.L. (1997) Metabotropic glutamate agonist-induced rotation : a pharmacological, FOS immunohistochemical, and [14C]-2-deoxyglucose autoradiographic study. J. Neurosci. 17,44154425.
  • 23
    Keinanen K., Wisden W., Sommer B., Werner P., Herb A., Verdoorn T.A., Sakmann B., Seeburg P.H. (1990) A family of AMPA-selective glutamate receptors.Science 249,556560.
  • 24
    Kim J. & Vezina P. (1997) Activation of metabotropic glutamate receptors in the rat nucleus accumbens increases locomotor activity in a dopamine-dependent manner.J. Pharmacol. Exp. Ther. 283,962968.
  • 25
    Lason W., Turchan J., Przewlocka B., Labuz D., Mika J., Przewlocki R. (1997) Seizure-related changes in the glutamate R2 and R5 receptor genes expression in the rat hippocampal formation.J. Neural Transm. 104,125133.
  • 26
    Leonard M.W., Lim K., Engel J.D. (1993) Expression of the chicken GATA factor family during early erythroid development and differentiation.Development 119,519531.
  • 27
    Lomeli H., Mosbacher J., Melcher T., Hoger T., Geiger J.R.P., Kuner T., Monyer H., Higuchi M., Bach A., Seeburg P.H. (1994) Control of kinetic properties of AMPA receptor channels by nuclear RNA editing.Science 266,17091713.
  • 28
    Lu W., Chen H., Xue C., Wolf M.E. (1997) Repeated amphetamine administration alters the expression of mRNA for AMPA receptor subunits in rat nucleus accumbens and prefrontal cortex.Synapse 26,269280.DOI: 10.1002/(SICI)1098-2396(199707)26:3<269::AID-SYN8>3.0.CO;2-5
  • 29
    McCoy L., Cox C., Richfield E.K. (1998) Antipsychotic drug regulation of AMPA receptor affinity states and GluR1, GluR2 splice variant expression.Synapse 28,195207.
  • 30
    Monyer H., Seeburg P.H., Wisden W. (1991) Glutamate-operated channels—developmentally early and mature forms arise by alternative splicing. Neuron 6,799810.
  • 31
    Moratalla R., Elibol B., Vallejo M., Graybiel A.M. (1996) Network-level changes in expression of inducible Fos-Jun proteins in the striatum during chronic cocaine treatment and withdrawal.Neuron 17,147156.
  • 32
    Mosbacher J., Schoepfer R., Monyer H., Burnashev N., Seeburg P.H., Ruppersberg J.P. (1994) A molecular determinant for submillisecond desensitization in glutamate receptors.Science 266,10591062.
  • 33
    Nestler E.J. (1993) Cellular responses to chronic treatment with drugs of abuse.Crit. Rev. Neurobiol. 7,2339.
  • 34
    Niedzielski A.S. & Wenthold R.J. (1995) Expression of AMPA, kainate, and NMDA receptor subunits in cochlear and vestibular ganglia.J. Neurosci. 15,23382353.
  • 35
    Paxinos G. & Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney.
  • 36
    Pierce R.C. & Kalivas P.W. (1997) A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants.Brain Res. Rev. 25,192216.
  • 37
    Pierce R.C., Bell K., Duffy P., Kalivas P.W. (1996) Repeated cocaine augments excitatory amino acid transmission in the nucleus accumbens only in rats having developed behavioral sensitization.J. Neurosci. 16,15501560.
  • 38
    Reid M.S. & Berger S.P. (1996) Evidence for sensitization of cocaine-induced nucleus accumbens glutamate release.Neuroreport 7,13251329.
  • 39
    Satel S.L. & Edell W.S. (1991) Cocaine-induced paranoia and psychosis proneness.Am. J. Psychiatry 148,17081711.
  • 40
    Smith J.A., Mo Q., Guo H., Kunko P.M., Robinson S.E. (1995) Cocaine increases extraneuronal levels of aspartate and glutamate in the nucleus accumbens.Brain Res. 683,264269.
  • 41
    Sommer B., Keinanen K., Verdoorn T.A., Wisden W., Burnashev N., Herb A., Kohler M., Takagi T., Sakmann B., Seeburg P.H. (1990) Flip and flop : a cell-specific functional switch in glutamate-operated channels of the CNS.Science 249,15801585.
  • 42
    Somogyi R., Wen X., Wu M., Barker J.L. (1995) Developmental kinetics of GAD family mRNAs parallel neurogenesis in the rat spinal cord.J. Neurosci. 15,25752591.
  • 43
    Standaert D.G., Testa C.M., Young A.B., Penney J.B.J r. (1994) Organization of N-methyl-d-aspartate glutamate receptor gene expression in the basal ganglia of the rat. J. Comp. Neurol. 343,116.
  • 44
    Tallaksen-Greene S.J. & Albin R.L. (1994) Localization of AMPA-selective excitatory amino acid receptor subunits in identified populations of striatal neurons.Neuroscience 1,509519.
  • 45
    Testa C.M., Standaert D.G., Landwehrmeyer G.B., Penney J.B.J, Young A.B. (1995) Differential expression of mGluR5 metabotropic glutamate receptor mRNA by rat striatal neurons.J. Comp. Neurol. 354,241252.
  • 46
    White F.J. (1996) Synaptic regulation of mesocorticolimbic dopamine neurons.Annu. Rev. Neurosci. 19,405436.
  • 47
    White F.J., Xiu Y., Henry D.J., Zhang X. -F. (1995a) Neurophysiological alterations in the mesocorticolimbic dopamine system during repeated cocaine administration, inThe Neurobiology of Cocaine (Hammer R. P. Jr., ed), pp. 99120, CRC Press, Boca Raton, Florida.
  • 48
    White F.J., Hu X., Zhang X., Wolf M.E. (1995b) Repeated administration of cocaine or amphetamine alters neuronal responses to glutamate in the mesoaccumbens dopamine system.J. Pharmacol. Exp. Ther. 273,445454.
  • 49
    Wullner U., Standaert D.G., Testa C.M., Landwehrmeyer B., Catania M.V., Penney J.B.J, Young A.B. (1994) Glutamate receptor expression in rat striatum : effect of deafferentation.Brain Res. 674,209219.
  • 50
    Zhang X.F., Hu X.T., White F.J., Wolf M.H. (1997) Increased responsiveness of ventral tegmental area dopamine neurons to glutamate after repeated administration of cocaine or amphetamine is transient and selectively involves AMPA receptors.J. Pharmacol. Exp. Ther. 281,699706.