In vivo labeling and molecular characterization of cocaine memory-specific active neurons using the photo-convertible calcium integrator CaMPARI2

Authors

• A Zhang
• M Moayeri
• SH Leppla
• TH Bugge

Abstract

Background: In abstinent drug users, drug-associated cues can provoke craving and relapse long after cessation of drug use. These maladaptive drug-cue associations are encoded in sparse patterns of strongly activated neurons (ensembles). However previous immediate early gene based labeling approaches lack temporal specificity needed to study short behavioral events (e.g, lever press). To address this gap, we developed procedures to label and characterize active neurons in vivo with sub-second temporal specificity using the green-to-red photo-convertible calcium-based activity marker CaMPARI2, and single-nucleus RNA sequencing (snRNAseq).

Methods: We expressed CaMPARI2 in infra-limbic cortex (IL) of male and female Sprague-Dawley rats, implanted optical fibers for photo-conversion and inserted jugular catheters for cocaine self-administration. Following self-administration training and 21 abstinence days, we used green-to-red CaMPARI2-photoconversion to permanently label IL cocaine-memory ensembles during a 1-min cocaine-seeking test. We collected brains either immediately after test (0-min group) or waited 10 minutes for experience-induced gene expression (10-min group). We isolated red (active) and green (inactive) CaMPARI2-labeled nuclei and performed snRNAseq.

Results: We observed reliable cocaine self-administration during training and robust cue-induced cocaine seeking during the 1-min test. CaMPARI2-snRNAseq revealed distinct clusters of glutamatergic and GABAergic IL neurons that sub-clustered into expected layer and subtypes. Further, IEGs were selectively induced in red neurons from 10-min, but not 0-min group.

Discussion: We will identify unique molecular alterations within IL cocaine-memory ensembles and investigate their distribution across IL cell types. Molecular and cell-type characterization of drug-memory ensembles could identify new targets to selectively weaken persistent drug memories and prevent relapse.

Scientific Focus Area: Neuroscience

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