Increased AGS3 expression during withdrawal
Immunoblots were obtained in tissue collected from the brain regions that are commonly associated with the effects of abused drugs (Everitt and Wolf, 2002
). Increased content of AGS3 was observed in the PFC and the core region of the nucleus accumbens following 3 weeks of withdrawal from repeated cocaine, but not in the shell of the accumbens, ventral tegmental area or striatum (). By 8 weeks of withdrawal the levels of AGS3 remained elevated in the accumbens core, and while significantly elevated above control in the PFC, the levels were reduced compared to 3 weeks of withdrawal (). AGS3 expression was not different between repeated cocaine and saline treatment groups at earlier withdrawal time points (one and 7 days), or after acute cocaine treatment in any brain region examined ( and ). Finally, animals were trained to self-administer cocaine for two weeks, and after 3 weeks of withdrawal the level of AGS3 in the PFC was elevated compared with yoked saline control subjects ().
Figure 1 AGS3 was upregulated during withdrawal from repeated cocaine administration. (A and B) After 3 and 8 weeks of withdrawal, AGS3 was upregulated in the prefrontal cortex (PFC) and nucleus accumbens core (NAcore), but not in the shell of the nucleus accumbens (more ...)
Mesocorticolimbic AGS3 expression following withdrawal from repeated cocaine.
Mesocorticolimbic AGS3 expression following withdrawal from acute cocaine.
Elevating AGS3 caused a cocaine sensitization-like phenotype
Giα inhibition by AGS3 can be mimicked by a consensus peptide derived from the four GPR domains contained within AGS3, and a single point mutation in the GPR domain (mGPR) abolishes Giα binding (Bernard et al., 2001
; Peterson et al., 2002
). The GPR peptides were rendered cell permeable by fusing the protein transduction domain from HIV-Tat to the amino terminus of the GPR consensus peptide (Schwarze et al., 1999
). Like the GPR domain of AGS3, Tat-GPR peptides inhibited [35
S]GTPγS binding to purified Giα, while Tat-mGPR was without effect (). Also, high affinity agonist binding to α2A/D
adrenergic receptors in DDT-MF2 cell membrane extracts was inhibited by Tat-GPR, but not by Tat-mGPR (). Thus, Tat-GPR effectively mimicked the ability of the AGS3 GPR domain to inhibit Giα. It should be noted that in the brain LGN, Rap1Gap, RGS12 and RGS14 contain at least one GPR domain each (Blumer and Lanier, 2003
), and increasing intracellular content of the 4 GPR domains contained in AGS3 by Tat-GPR could also be mimicking the action of theses proteins.
Figure 2 Tat-GPR prevents Giα signaling. (A) [35S]GTPγS binding to 100 nM purified Giα was blocked by increasing concentrations of Tat-GPR (100 μM) while Tat-mGPR (100 μM) was ineffective. (B) High affinity (4 nM [3H]-UK14304) (more ...)
A Tat-fluorescein conjugate was microinjected into the PFC, and in vivo transduction of cells was examined 30 min following microinjection. Transduction of both neurons and glia was found at the site of microinjection (). Nissl staining of adjacent sections revealed an abundance of healthy appearing neurons and no apparent neurotoxicity outside the mechanical damage resulting from cannula implantation (). Immunoblotting for Tat-GPR at different times after microinjection into the PFC revealed maximum Tat-GPR content at 30 min that gradually diminished to undetectable levels by 120 min ().
Figure 3 Tat-peptides were efficiently transduced and exhibited no apparent toxicity. (A) Epifluorescent micrograph illustrates Tat-fluorescein containing cells at the tip of the microinjection cannulae 30 minutes after microinjection. Cannulae placements are (more ...)
The behavioral effect of transducing PFC cells with Tat-GPR was examined in drug naïve animals using photobeam breaks to estimate locomotor activity. Thirty min after microinjecting Tat-peptide, cocaine (15 mg/kg, ip) was administered and the motor stimulant response was markedly enhanced between 10 and 60 min after cocaine injection in Tat-GPR pretreated animals compared to subjects microinjected with Tat-mGPR (). In contrast there was no difference in behavioral activation produced by injecting saline between the Tat-GPR and Tat-mGPR pretreated groups (). Both distance traveled (estimate of locomotion, ) and number of stereotypies (repeated breaking of the same photocell beam to estimate stereotyped behavior, ) were augmented by Tat-GPR, indicating that the increase in motor activity probably did not involve switching between competing behaviors, but rather that inhibition of Giα by Tat-GPR imparts increased sensitivity to the motor stimulant properties of cocaine. In the experiment shown in , subjects were microinjected with Tat-GPR or Tat-mGPR at one week intervals in a random design such that each animal received all four treatments. Thus, in accord with the apparent short half-life of the Tat-GPR fusion peptide (), the capacity of Tat-GPR pretreatment to produce a sensitization-like augmentation in the behavioral stimulant effect of cocaine was reversible.
Figure 4 Tat-GPR in the PFC produces a sensitization-like behavioral and neurochemical phenotype. (A and B) Microinjection of Tat-GPR into the PFC produced a sensitized motor response to cocaine (15 mg/kg, ip) in drug-naïve subjects. Animals were habituated (more ...)
In addition to motor stimulation, the capacity of Tat-GPR to potentiate cocaine-primed reinstatement of drug-seeking was examined (). The reinstatement of drug-seeking behavior is a widely employed animal model of relapse (Shalev et al., 2002
). Animals were trained to self-administer intravenous cocaine, and following extinction training were administered an injection of cocaine at a dose (5 mg/kg, ip) that produces submaximal reinstatement of drug-seeking (Baker et al., 2003
). Two separate reinstatement trials were conducted and animals were microinjected with either Tat-GPR or Tat-mGPR in counterbalanced order 30 min prior to injecting cocaine ( shows protocol). The number of active lever presses elicited by cocaine was significantly augmented in the group pretreated with Tat-GPR ().
Animals that develop behavioral sensitization or have been trained to self-administer cocaine demonstrate an elevation in extracellular glutamate in the core compartment of the nucleus accumbens following a subsequent injection of cocaine and/or exposure to an environment that was paired with cocaine administration, and the enhanced glutamate release arises primarily from prefrontal cortical glutamatergic afferents (Hotsenpiller et al., 2001
; Li et al., 1999a
; McFarland et al., 2003
; Pierce et al., 1996
; Pierce et al., 1998
). Corresponding with Tat-GPR inducing a behavioral sensitization-like phenotype, cocaine produced an increase in extracellular glutamate in the accumbens core of drug-naïve animals following Tat-GPR pretreatment of the PFC (). Similar to previous reports in drug-naïve animals (McFarland et al., 2003
; Smith et al., 1995
), extracellular glutamate in the accumbens core was not altered by an acute injection of cocaine (15 mg/kg, ip) in animals pretreated with Tat-mGPR ().
Antisense oligonucleotides reversibly lower levels of AGS3
An antisense oligonucleotide strategy was used during withdrawal to reversibly inhibit the increase in AGS3 in the PFC. Two antisense constructs, one that flanked the AGS3 initiation codon (AS-1) or one directed within the AGS3 open reading frame (AS-2), were continuously infused bilaterally into the PFC using subcutaneously implanted osmotic minipumps. To determine a concentration of AS that would restore the cocaine withdrawal-elevated AGS3 content to approximate control levels (e.g. ~40% reduction, see ), AS-1 or AS-2 was continuously infused into one side of the PFC, while a control, scrambled oligonucleotide sequence was infused into the opposite hemisphere for one or 2 weeks. An antisense infusion rate of 504 pmol/24 hr was found to reduce AGS3 expression by ~35–60% (). Additional immunoblotting revealed that two weeks of AS-1 infusion did not affect expression of proteins in the PFC that are functionally related to signaling though Giα including Giα1,3 (111 ± 9.5%; n = 8; mean optical density ± sem change from scrambled oligonucleotide) or Gβ (110 ± 11%; n = 8). In some animals, the osmotic minipumps were removed after two weeks to determine if the reduction in AGS3 by AS-1 was reversible. Two weeks after discontinuing oligonucleotide infusion, AGS3 content was equivalent between the AS-1 and scrambled control group (). Moreover, animals were administered daily saline or cocaine for 7 days, and one week later implanted with bilateral minipumps to deliver AS-1 or scrambled oligonucleotide into the PFC for 2 weeks. Two weeks following pump removal the level of AGS3 in the PFC of both oligonucleotide groups was elevated in the animals treated with chronic cocaine compared with chronic saline (), indicating that cocaine-induced elevations in AGS3 were restored after discontinuing AS-1 infusion.
Figure 5 Regulating AGS3 expression with antisense oligonucleotides. (A) AGS3 was downregulated following either 1 or 2 weeks continuous infusion of antisense into the PFC and returned 2 weeks after antisense infusion was terminated. (B) Animals were injected (more ...)
Neurotoxicity is produced by relatively high concentrations of phosphorothioate substituted oligonucleotides (Braasch and Corey, 2002
). Due to using low concentrations and having only the 5 terminal bases phophorothiate substituted, after two weeks of infusion no evidence of neurotoxicity was found outside of the mechanical disruption produced by implantation of the infusion cannula (). Nissl staining revealed an abundance of healthy appearing cells (), and staining for glial proliferation with glial fibrillary acidic protein (GFAP) showed equivalent staining between the three oligonucleotide infusion groups ().
Figure 6 Lack of neurotoxicity after 2 weeks of oligonucleotide infusion into the PFC. (A, D, and G) Cannulae placements were near the prelimbic/infralimbic border of the PFC (Paxinos and Watson, 1998). bar = 1 mm, cc – corpus callosum. (B, E, and H) Visual (more ...)
Elevated AGS3 contributes to reduced Giα signaling produced by repeated cocaine
The effect of increased AGS3 expression on G protein signaling after 3 weeks withdrawal from repeated exposure to cocaine was assayed via agonist-induced guanosine 5′-[γ-35
S]GTPγS) binding in ex vivo
PFC membranes with ligands selective for Giα-coupled group II metabotropic glutamate receptors (mGluR2/3) and D2/3 dopamine receptors, or Gsα-coupled D1 dopamine receptors. At one week after the last daily cocaine or saline injection, either scrambled oligonucleotide or AS-1 was infused into the PFC for 2 weeks. Following infusion of scrambled oligonucleotide, [35
S]GTPγS binding induced by stimulating mGluR2/3 or dopamine D2/3 receptors with 2R,4R-4-aminopyrrolidine-2,4-dicaroxylate (APDC) or quinpirole, respectively, was significantly decreased in cocaine withdrawn versus control subjects (e.g. compare Sc + Coc with Sc + Sal in ). Agonist-induced [35
S]GTPγS binding was restored in rats withdrawn from repeated cocaine to levels equivalent to control by infusing AS-1 (e.g. no significant difference existed between AS + Coc, Sc + Sal and AS + Sal groups). In contrast, AS-1 in repeated saline animals did not significantly alter the level of quinpirole stimulated [35
S]GTPγS binding (this treatment group was not examined with APDC treatment ). Consistent with the selective binding of AGS3 to Giα (Bernard et al., 2001
S]GTPγS binding induced by stimulating the Gsα-coupled dopamine D1 receptors was equivalent in all groups (). The restoration of quinpirole and APDC induced [35
S]GTPγS binding by AS-1 was not associated with alterations in PFC content of D2 (AS-1 = 96.8 ± 6.2, n
= 8, data normalized to percent change from Sc) or mGluR2/3 (monomer, AS-1 = 100.1 ± 1.3, n
= 8; dimer, AS-1 = 92.4, n
= 8) ().
Figure 7 Elevation of AGS3 parallels decreased signaling through Giα coupled receptors. (A) 3 weeks withdrawal from one week of daily cocaine administration decreased (−)-quinpirole stimulated Giα signaling at D2/3 receptors in the PFC. (more ...)
Decreasing AGS3 reversibly inhibits behavioral sensitization
Repeated exposure to cocaine produces an enduring augmentation (sensitization) in the motor stimulant effects of a subsequent cocaine injection (Everitt and Wolf, 2002
). Some lesion studies (Pierce et al., 1998
; Tzschentke and Schmidt, 1999
), but not all (Li et al., 1999b
), demonstrate that the glutamatergic PFC projection to the core of the nucleus accumbens is necessary for the expression of behavioral sensitization following repeated cocaine administration. Experiments were conducted to determine if the upregulation of AGS3 in the PFC during withdrawal from repeated treatment with cocaine contributes to the expression of cocaine-sensitized motor behavior.
Rats were repeatedly administered intraperitoneal cocaine for one week and following one week of withdrawal osmotic minipumps were implanted to deliver AS-1 or scrambled oligonucleotide into the PFC (). After two weeks of oligonucleotide infusion, all animals were administered cocaine (15 mg/kg, ip) to test for the presence of a sensitized motor response. The pumps were removed and two weeks later all animals were again tested for the presence of behavioral sensitization to cocaine. The first injection of cocaine (i.e. on day 1 before oligonucleotide infusion) elicited an equivalent motor response in both groups (). Following three weeks of withdrawal from daily cocaine and two weeks of oligonucleotide infusion, the motor response to cocaine in the scrambled group was significantly augmented compared to day 1 (). In contrast, infusion of AS-1 inhibited the expression of a sensitized motor response (day 28; ). Two weeks following pump removal, both oligonucleotide groups showed significant and equivalent behavioral sensitization in response to an injection of cocaine (). The sensitized response in the scrambled group was equivalent between days 28 and 42, while the response on day 42 was significantly increased over day 28 in the AS-1 group. Thus, in accord with the protein expression studies (), the inhibition of sensitized motor activity by AS-1 was reversible.
Figure 8 Reduction of AGS3 in the PFC by antisense oligonucleotide inhibits locomotor sensitization. (A) Timeline summarizing the behavioral protocol. (B) The cumulative motor response to the cocaine at days 1, 28 and 42. (C) The motor response to cocaine injection (more ...)
In contrast to the marked effect of AS-1 on the expression of the sensitized motor response to cocaine, two weeks of AS-1 infusion did not affect the motor stimulant response to an acute injection of cocaine (15 mg/kg, ip) in drug-naïve rats (). Similarly, the exploratory response to novelty (habituation to the photocell chamber) or the locomotor response to an acute injection of or saline was not affected by AS-1 infusion ().
Decreasing AGS3 reversibly inhibits cocaine reinstatement of drug-seeking
Pharmacological inactivation of the PFC prevents the expression of drug-seeking behavior induced by a cocaine injection or an environmental stressor (Capriles et al., 2003
; McFarland and Kalivas, 2001
). Also, the release of glutamate from the PFC into the accumbens core is required for cocaine-induced reinstatement of drug-seeking (McFarland et al., 2003
). Based on the critical role of the PFC in drug-seeking behavior, experiments were conducted to evaluate the hypothesis that the up-regulation of AGS3 in the PFC during withdrawal from cocaine self-administration mediates the reinstatement of cocaine-primed drug-seeking.
Rats were trained to self-administer cocaine by associating a lever press with a cocaine infusion. Lever pressing for cocaine was extinguished by replacing cocaine with saline, and during extinction training, AS-1, AS-2, or scrambled oligonucleotides were bilaterally infused into the PFC for 2 weeks. No difference between oligonucleotide treatment groups was measured in the number of days required to achieve the extinction criterion (see methods). After 14 days of oligonucleotide infusion, rats were administered a single injection of cocaine (10 mg/kg, ip) to reinstate drug-seeking (lever pressing for saline). Following this reinstatement trial, oligonucleotide infusion was terminated by removing the osmotic minipumps and the propensity to reinstate drug-seeking was reassessed two weeks later ().
Figure 9 Prefrontal cortical AGS3 regulates cocaine-seeking behavior. (A) Timeline outlining the experimental protocol. (B) Oligonucleotide infusion did not impair food seeking. (C) Bilaterally reducing AGS3 blocked the reinstatement of drug-seeking behavior while (more ...)
Drug-seeking was elicted by a cocaine injection on day 28 in the group infused with scrambled oligonucleotide (). In contrast, drug-seeking was prevented by infusion of either of the two antisense oligonucoeotides (). However, a second cocaine injection administered to the same animals two weeks after discontinuing oligonucleotide infusion (day 42) elicited reliable reinstatement to a cocaine injection in all oligonucleotide treatment groups (). The reduction in AGS3 by AS-1 did not affect food-induced reinstatement in rats trained to self-administer food pellets instead of cocaine, indicating that AS-1 did not produce motor or learning deficits that prevented responding to the cocaine-priming injection ().
The site of oligonucleotide infusion () or Tat-GPR microinjection () was primarily in the ventral prelimbic and dorsal infralimbic cortex (Paxinos and Watson, 1998
). The dialysis probes were implanted into the core of the nucleus accumbens with <15% of the active membrane dorsal or ventral to the accumbens core, in the striatum or ventral limb of the accumbens shell, respectively (e.g. see Figure 4 in McFarland et al., 2003
, for similar probe placements).