Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Summary

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Abstract

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

Introduction

Cocaine is a highly reinforcing psychostimulant. Following repeated exposure, several molecular and cellular adaptations occur in reward-relevant brain circuitry that are believed to result in compulsive drug-seeking behavior, prompting high rates of relapse which pose a serious clinical problem ¹. Cocaine exerts these long-lasting behavioral effects by regulating gene expression. To study the adaptations that arise from chronic cocaine use, preclinical rodent models have been used extensively. One such model is the conditioned place preference (CPP) paradigm. This model involves the development of a learned association between a previously neutral environm....

Protocol

All procedures are conducted in accordance with the Weill Cornell Medical College Institutional Animal Care and Use Committee rules.

1. Preparation and Setup for Stereotaxic Delivery of Viral Vectors

1. If an instrument, sterile swab, or hand wearing sterile glove is contaminated by coming into contact with a non-sterile surface, discard or re-sterilize the instrument using a hot bead sterilizer, discard the swab or change to new sterile gloves.
2. Place a mouse cage with bedding on a heat block to warm for post-operative recovery.
3. Set up electric razor for shaving head and ethanol and iodine to sterilize the scalp....

Results

CPP

After performing CPP on microinjected mice, one should verify that the cohort of control-injected (rAAV-GFP) mice have normally acquired preference for the drug-paired chamber (Figure 1A, 1B). Mice are considered to have acquired preference for a particular chamber when cocaine preference (time spent in the cocaine-paired chamber minus time spent in the saline-paired chamber) is significantly higher compared to baseline preference score (Figure 1B, A vs. B). .......

Discussion

Regionally- and temporally-specific gene ablation via stereotaxic microinjection of viral vectors combined with CPP that includes extinction and reinstatement phases allows for investigation of the specific contributions of genes to three distinct phases of addictive-like behavior. While conditional knockout that is achieved utilizing the traditional Cre-LoxP system provides for spatio-temporally restricted gene ablation, stereotaxically microinjecting Cre-recombinase into discrete brain areas of floxed mice allows for e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Conditioned place preference activity chambers	Med Associates, Inc., St. Albans, VT, USA	MED-CPP-MS
Stereotaxic alignment system for mouse	David Kopf Instruments, Tujunga, CA, USA	model 900
Hamilton syringes	Hamilton Company, Reno, Nevada, USA	7634-01
rAAV2-Cre-GFP	Vector BioLabs, Philadelphia, PA, USA	7016
rAAV2-GFP	Vector BioLabs, Philadelphia, PA, USA	7004