DOI: 10.5061/dryad.qrfj6q5nx
title: Drugs of abuse hijack a mesolimbic pathway that processes homeostatic need

description: Drugs of abuse are thought to promote addiction in part by
 "hijacking" brain reward systems, but the underlying mechanisms
 remain undefined. Using whole-brain FOS mapping and in vivo single-neuron
 calcium imaging, we found that drugs of abuse augment dopaminoceptive
 ensemble activity in the nucleus accumbens (NAc) and disorganize
 overlapping ensemble responses to natural rewards in a cell-type-specific
 manner. Combining FOS-Seq, CRISPR-perturbation, and snRNAseq, we
 identified Rheb as a molecular substrate that regulates cell-type-specific
 signal transduction in NAc while enabling drugs to suppress natural reward
 consumption. Mapping NAc-projecting regions activated by drugs of abuse
 revealed input-specific effects on natural reward consumption. These
 findings characterize the dynamic molecular and circuit basis of a common
 reward pathway, wherein drugs of abuse interfere with the fulfillment of
 innate needs.
description: # Drugs of abuse hijack a mesolimbic pathway that processes homeostatic
 need
 [https://doi.org/10.5061/dryad.qrfj6q5nx](https://doi.org/10.5061/dryad.qrfj6q5nx) A detailed description of methodology and analysis that were followed in order to generate and process all included files can be found in the associated publication "Materials and Methods" section ([https://pubmed.ncbi.nlm.nih.gov/38669575/]()). #### **1. Dataset: Fos-Seq** * **Description:** This dataset contains the results of a computational correlation analysis between brain-wide FOS expression (measured via whole-brain clearing) and the Allen Institute’s *in situ* hybridization (ISH) gene expression atlas. * **Methodology:** Custom code was used to correlate spatial FOS patterns across experimental conditions with the expression patterns of specific genes to identify genetic markers associated with neuronal activation. #### **2. Dataset: snRNA-seq** * **Description:** Single-nucleus RNA sequencing (snRNA-seq) data from the Nucleus Accumbens (NAc). * **Sample Info:** Samples were collected from dissected NAc tissue where virally delivered guide RNAs (gRNAs) were expressed. #### **3. Dataset: FOS_expression** * **Description:** Z-scored datasets of brain-wide FOS expression levels. * **Methodology:** Samples were processed using whole-brain clearing (LifeCanvas Technologies) and stained with FOS-specific antibodies to map neuronal activity. Z-score transformation was applied along the sample axis per batch in order to correct for batch effects. #### **4. Dataset: FOS_expression_by_sample** * **Description:** Z-scored datasets of brain-wide FOS expression levels from individual samples. * **Methodology:** Samples with repeated exposure to cocaine or morphine were processed using whole-brain clearing (LifeCanvas Technologies) and stained with FOS-specific antibodies to map neuronal activity. Samples with acute exposure to cocaine and morphine or withdrawal from repeated exposure were processed using a section-based approach and stained with FOS-specific antibodies to map neuronal activity. Z-score transformation was applied along the sample axis per batch in order to correct for batch effects. ## Description of the data and file structure ##### **1. Fos-Seq Dataset** **File/Folder:** `Fos-Seq.zip` ##### **Sub-folder: PearsonR** Contains 6 CSV files. These files represent the output of the correlation analysis across different experimental conditions. * **Variables:** * `Gene`: Official gene symbol. * `Pearson_R`: Computed Pearson correlation coefficient representing the spatial relationship between FOS and gene expression. * `P-value`: Statistical significance of the correlation. ##### **Sub-folder: Fos-Seq (Implementation Files)** These 6 files are required to run the custom analysis code provided in the Jupyter Notebook. * `allGeneStructureInfo_allgenes_summary_struct.csv`: Dataset derived from the Allen Institute ISH datasets; contains gene expression values mapped to specific brain structures. * `brain_region_dict.pkl`: Python dictionary mapping brain region names to their specific Structure IDs. * `cfos_brain_acronym.pkl`: Pickled list of brain acronyms used in the whole-brain FOS mapping datasets. * `gene_list_across_regions.pkl`: Pickled data containing specific gene sets categorized by brain region. * `structure_tree_2017.csv`: Hierarchical tree structure for brain regions (2017 Allen Brain Atlas version). * `Fos-Seq_function_Dryad.ipynb`: Jupyter Notebook containing the Python code to implement the Fos-Seq analysis. ##### **2. snRNA-seq Dataset** **File/Folder:** `snRNA-seq.zip` **Description:** This dataset consists of single-nucleus RNA sequencing (snRNA-seq) data from dissected Nucleus Accumbens (NAc) samples. The experiment utilized virally delivered guide RNAs (gRNAs) to identify cell-type-specific transcriptomic responses. ##### **Sub-folder: HH2NKDRX3** * **Description:** This folder contains the **raw sequencing datasets** (e.g., FASTQ or sparse matrix files) required for single-cell sequencing analysis and alignment, as well as a 230731_A00815_0649_BHH2NKDRX3.md5 file containing hashes for all FASTQ files to verify data integrity. These files serve as the primary data for processing through pipelines such as Cell Ranger. ##### **Metadata File: feature_reference.csv** * **Description:** A reference file used during the sequencing analysis to map and recover specific guide RNA (gRNA) sequences from the raw data. ##### **3. FOS_expression Dataset** **File/Folder:** `FOS_expression.zip` ##### **Included Files:** * `acute_cocaine_morphine_zscore.csv`: Z-scored FOS expression data comparing responses to a single (acute) dose of cocaine and morphine. * `chronic_cocaine_morphine_zscore.csv`: Z-scored FOS expression data following repeated exposure to cocaine and morphine. * `withdrawal_cocaine_morphine_zscore.csv`: Z-scored FOS expression data during spontaneous withdrawal from repeated cocaine and morphine exposure. ##### **Key Variables & Interpretation:** * **`Specificity`** **(Categorical Key)**: This variable identifies the drug-specific nature of the FOS response: * **0**: No significant response relative to the saline group. * **1**: Cocaine-specific response. * **2**: Morphine-specific response. * **3**: Shared response (significant for both cocaine and morphine). ##### **4. FOS_expression_by_sample Dataset** **File/Folder:** `FOS_expression_by_sample.zip` ##### **Included Files:** * `acute_withdrawal_cocaine_morphine_saline.xlsx`: Z-scored FOS expression data comparing responses to a single (acute) dose of cocaine and morphine, or withdrawal from repeated exposure to cocaine and morphine per sample. * `chronic_cocaine_morphine_saline.xlsx`: Z-scored FOS expression data following repeated exposure to cocaine and morphine per sample. ##### **Key Variables & Interpretation:** * **`Row 1 `(Categorical Key)**: each sample * **`Column A `(Categorical Key)**: each brain region ## Sharing/Access information For additional details or specific inquiries regarding this dataset, please contact the authors by email. ## Code/Software Please refer to the file **`Fos-Seq_function_Dryad.ipynb`** located within the Fos-Seq.zip dataset for the custom Python code used to perform the analysis and generate the correlations.