The authors would like to thank Anni Lee and Maureen Byrne for their help in establishing the extended conditioned place preference 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
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	<li>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.</li>
	<li>Place a mouse cage with beddi...

<ol>
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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...

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 environment and the rewarding properties of cocaine. After several pairings of cocaine with a particular chamber, animals are allowed to freely explore the cocaine-paired and non-cocaine-paired environments and if they prefer the drug-paired compartment, they are said to have acquired a cocaine-induced place preference. Moreover, following an extinction training period, this paradigm can be used to study context-specific reinstatement of cocaine-seeking behavior.
Compared to other behavioral models of addictive-like behavior, such as psychomotor sensitization, which is used to study long-term cocaine-induced behavioral and molecular plasticity 2,3 and self-administration (SA), that is thought to more accurately mimic addictive-like behavior found in humans, the CPP paradigm is a simple procedure to study cocaine contextual learning 4. CPP protocols can be conveniently extended to include extinction and reinstatement phases, similarly to SA, which allow for investigation of the mechanisms underlying drug craving and relapse 5-7 and which are believed to recapitulate aspects of what occurs in human drug-seeking behavior and drug- and cue-induced relapse 8-10.
One mechanism that underlies the cocaine-induced behavioral plasticity that is characteristic of each of the distinct phases of CPP, including acquisition, extinction, and reinstatement, is activation of unique signatures of gene expression within different brain regions. To directly test which genes within reward-relevant brain regions mediate cocaine-induced behavioral changes, it is useful to be able to manipulate them selectively in a regionally-specific manner. One way of achieving this is to microinject recombinant adenoassociated viral (rAAV) vectors that express Cre recombinase using stereotaxic surgery, into distinct brain areas of mice that have target genes flanked by LoxP sites (floxed mice). This method allows for very precise temporal and regional control over when and where genes are ablated, in both dividing and non-dividing neurons, without inducing immune responses 11-13. This level of control represents an important advantage over traditional Cre-LoxP technology of breeding floxed mice with mice expressing Cre recombinase under the control of an endogenous gene promoter, in that the timing and distribution of gene deletion can be more tightly regulated. Additionally, viral vector-mediated gene knockout circumvents potential developmental compensatory effects that may occur using traditional knockout strategies.
In addition to cocaine CPP, which is described here, microinjection of rAAV-Cre into the brain of floxed mice to assess the role of specific genes can be applied ubiquitously to behavioral paradigms that involve separate phases, including self-administration and psychomotor sensitization. For example, our laboratory has utilized the rAAV-Cre technology to study the role of Cav1.2 L-type Ca2+ channels in cocaine psychomotor sensitization 14. Specifically, rAAV-Cre was microinjected into the nucleus accumbens (NAc) of mice with the gene encoding Cav1.2 floxed, to demonstrate that Cav1.2 acting in this region mediates the expression phase of psychomotor sensitization 14,15. However, the rAAV-Cre strategy cannot be used if mice with a floxed gene of interest do not exist, as was our experience when assessing the role of Cav1.3 L-type Ca2+ channels in psychomotor sensitization. Hence, a limitation of using rAAV-Cre presents itself if conditional mice do not exist for a particular gene of interest. However, rAAVs that express siRNA can be used to knockdown target genes, as we have done to examine the role of Cav1.3 channels 14,15.
Microinjecting rAAV-Cre into discrete brain regions of floxed mice and then testing them in the CPP paradigm allows for investigation into the specific genes that mediate the various phases of addictive-like behavior and where they are acting. Use of this paradigm has aided in our understanding of how repeated cocaine administration in essence hijacks the brains reward circuitry causing maladaptive changes in molecular signal transduction pathways and gene expression that lead to the addicted state 1,16,17.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Conditioned place preference activity chambers</td>
			<td>Med Associates, Inc., St. Albans, VT, USA</td>
			<td>MED-CPP-MS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment system for mouse</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>model 900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringes</td>
			<td>Hamilton Company, Reno, Nevada, USA</td>
			<td>7634-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-Cre-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotaxic microinjection of viral vectors expressing cre recombinase to study the role of target genes in cocaine conditioned place preference

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.

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). ...

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

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.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

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18.5K 조회수.  Weill Cornell Graduate School of Biomedical Sciences. 이 문서에서는 마우스의 뇌에 바이러스 벡터를 microinject하고 수집, 멸종 위기 및 회복 단계를 포함하는 에어컨 장소 환경 패러다임에서 테스트하는 방법에 대해 설명합니다.