Elsevier

Biological Psychiatry

Volume 51, Issue 8, 15 April 2002, Pages 612-620
Biological Psychiatry

Original article
Reduction of cocaine place preference in mice lacking the protein phosphatase 1 inhibitors DARPP 32 or Inhibitor 1

https://doi.org/10.1016/S0006-3223(01)01318-XGet rights and content

Abstract

Background: Modulation of protein phosphorylation by dopamine is thought to play an important role in drug reward. Protein phosphatase-1 (PP-1) is known to mediate some of the changes in neuronal signaling that occur following activation of the dopaminergic system.

Methods: Two endogenous inhibitors of PP-1 are dopamine and cyclic 3′, 5′ adenosine monophosphate-regulated phosphoprotein (DARPP-32) and Inhibitor-1 (I-1). Knockout mice lacking one or both of these PP-1 inhibitors were tested for responses to cocaine using in vivo amperometry and conditioned place preference.

Results: Presynaptic dopaminergic function appears to be unaffected by these mutations because stimulation-evoked changes in extracellular dopamine levels were unchanged between wild type mice and mice lacking one or both of these PP-1 inhibitors. In contrast, conditioned place preference to cocaine is reduced in mice lacking DARPP-32, I-1, or both phosphoproteins. This does not appear to be due to a learning deficit because mice lacking both DARPP-32 and I-1 show normal passive avoidance learning.

Conclusions: These data imply that increased PP-1 function as a result of deficits in DARPP-32 or I-1 is sufficient to decrease the rewarding properties of cocaine. Furthermore, the mechanism for this altered cocaine place preference does not involve alteration of dopamine release or reuptake.

Introduction

Psychostimulants are thought to exert their rewarding effects through distinct cellular mechanisms that lead to increased dopaminergic transmission Di Chiara 2000, Di Chiara and Imperato 1988, Kuhar et al 1991, Nestler 1997, Wise and Rompre 1989. In the striatum, dopamine (DA) produces its biological effects through activation of D1- and D2-like receptors and their downstream effector molecules. Protein phosphatase-1 (PP-1), a serine/threonine phosphatase distributed throughout the brain including the striatum, can modulate the phosphorylation state and activity of a variety of effector molecules that may be critical for the behavioral actions of drugs of abuse (Greengard et al 1999). Dopamine and cyclic 3′, 5′ adenosine monophosphate-regulated phosphoprotein, 32kDa (DARPP-32) is a potent inhibitor of PP-1 that is localized to striatal medium spiny neurons Ouimet et al 1984, Ouimet et al 1988 and regulated by phosphorylation by several protein kinases (Greengard et al 1999). Upon D1-like receptor activation, DARPP-32 is phosphorylated by PKA at threonine-34 and activated (Nishi et al 1997). In contrast, following activation of D2-like receptors, DARPP-32 is dephosphorylated through activation of calcineurin (PP2B) and inhibition of PKA King et al 1984, Nishi et al 1999b. Hence, the activity of DARPP-32 can be regulated by dopaminergic signaling through either D1 or D2 receptors. Inhibitor-1 (I-1) is another PP-1 regulatory protein that is present in the basal ganglia, but unlike DARPP-32 it is not restricted to dopaminoceptive neurons. Inhibitor-1 is also activated by phosphorylation and is colocalized with DARPP-32 in dopaminoceptive neurons, although it is present at lower levels (Hemmings et al 1992). Inhibition of PP-1 by DARPP-32 or I-1 results in changes in the phosphorylation state and physiologic activity of ion channels, receptors, and transcription factors, resulting in altered responses to DA (Fienberg and Greengard 2000).

Given the lack of specific agonists and antagonists for DARPP-32 or I-1, mice lacking the genes encoding these molecules provide a model for in vivo study of the functional role of these proteins in responses to drugs of abuse. Previous studies have described the generation and characterization of mice lacking DARPP-32 (Fienberg et al 1998) or I-1 (Allen et al 2000) and have demonstrated biochemically that PP-1 regulation is altered in these mouse lines. DARPP-32 knockout mice previously have been shown to be deficient in some responses to cocaine. At the behavioral level, chronic—intermittent exposure to 10 mg/kg cocaine results in a lower rate of locomotor sensitization that is overcome at 20 mg/kg cocaine Fienberg et al 1998, Hiroi et al 1999. Intracellular and extracellular recordings in the nucleus accumbens of DARPP-32 knockout mice have shown that medium spiny neurons from mutant animals respond less to D-1—like receptor agonists and glutamate (Fienberg et al 1998). In addition, downstream targets of PP-1 are also affected by the absence of the gene encoding DARPP-32; phosphorylation of the NR1 subunit of the NMDA-type glutamate receptor is reduced, as is the activity of the Na+, K+, ATPase, and the induction of chronic fos-related antigens by psychostimulants Fienberg et al 1998, Nishi et al 1999a. The role of I-1 in dopaminergic signaling has not yet been elucidated; however, studies in brain slices indicate that mice lacking I-1 show deficient long-term potentiation (LTP) at perforant path-dentate granule cell synapses, but not at Shaffer collateral-CA1 pyramidal cell synapses. Despite this defect in perforant path LTP, I-1 knockout mice do not appear to have any spatial learning deficits (Allen et al 2000).

In our study, we used the place preference paradigm to determine whether responses to cocaine are affected by the absence of DARPP-32, I-1, or both genes. We found that mice lacking either DARPP-32 or I-1 showed attenuated place preference to cocaine and that mice lacking both genes were more profoundly impaired in their responses to cocaine. Moreover, using in vivo voltammetry, we show that this altered cocaine place preference does not involve alteration of dopamine release or reuptake.

Section snippets

Place preference and cocaine-induced locomotor activity

A clear plastic cage 25.4 cm high × 50 cm long × 25.4 cm wide was used to construct the place preference apparatus, which consisted of a 25-cm square compartment with smooth white plastic flooring and white walls and a second 25-cm square compartment with black-mesh flooring and black-and-white striped (3-cm-wide) walls. A divider separated the two compartments during training sessions and a 10-cm aluminum platform was placed under the divider to discourage animals from lingering in the doorway

Place preference

Injection of saline had no effect on place preference in mice of any of the genotypes tested (Figure 1A). No difference was seen in baseline preference in any group (not shown). The lowest dose of cocaine that could condition a place preference in C57Bl/6 mice under our conditions was 5 mg/kg Zachariou et al 1999, Zachariou et al 2001. At that dose, wild type mice showed a 245 ± 61 sec place preference (Figure 1B). This effect of cocaine was attenuated in animals lacking DARPP-32 (101 ± 34

Discussion

We have shown that mutation of either I-1 or DARPP-32 decreases conditioned place preference to cocaine without affecting cocaine-induced locomotion. Conditioned place preference is a behavioral paradigm that can evaluate the rewarding properties of a drug when the animal is in a drug-free state (Mucha et al 1982). We have previously shown that the threshold dose for cocaine place preference can be established using this paradigm in C57Bl/6 mice or in lines of knockout mice backcrossed onto a

Acknowledgements

This work was funded by Grant Nos. DA00436 and DA08227 (M.R.P.) and DA10044 (PBA, PG) from the National Institutes of Health, and by CNRS UMR 5541, Université Bordeaux 2 and La Région Aquitaine (FG) The authors would like to thank Barbara Caldarone for help with statistical analysis of behavioral data.

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