# Integrative single-cell spatial transcriptomic, anatomical, and functional profiling of brain-wide ensembles engaged by opioid relapse

> **NIH NIH R01** · UNIVERSITY OF WASHINGTON · 2024 · $790,368

## Abstract

PROJECT SUMMARY
Opioid abuse is devastating communities across the United States and is responsible for untold suffering. The
synthetic opioid fentanyl, whether due to prescription or illicit use, is involved in nearly half of reported opioid-
related deaths. Unlike heroin and morphine, fentanyl is commonly administered through non-intravenous routes,
but we have a limited mechanistic understanding of fentanyl use and abuse vulnerability. A leading hypothesis
for the transition from use to abuse, in vulnerable individuals, is the recruitment of drug-induced gene expression
changes in certain brain circuits and cells following repeated drug exposure. However, identifying such circuit-
and cell-type specific populations, in combination with their underlying genetic and functional adaptations, is
often limited by the resolution, throughput, and data registration of current assays. The overall goal of this project
is to (i) generate machine--guided behavioral characterization of oral and intravenous fentanyl self-administration
and reinstatement in male and female mice that captures individual risk vulnerability, and (ii) perform brain-wide
mapping by integrating new enabling technological platforms including single-cell spatial transcriptomics (Pixel-
seq), whole brain cell type specific circuit connectivity, and functional distributed brain-wide neural activity
recordings (Neuropixel 2.0), recently developed by PIs in our team. To realize the potential for the molecular and
functional brain mapping and data registration, we have three Specific Aims: 1) perform single-cell spatial
transcriptomic profiling, with circuit-specificity, in mice with varying degrees of fentanyl-seeking behavior; 2)
perform single-cell whole-brain activity mapping, with cell-type and circuit-specificity, in mice with varying
degrees of fentanyl-seeking behavior; 3) perform brain-wide distributed large-scale electrophysiological
recordings during oral fentanyl-seeking and integrate spatially resolved transcriptomic data with recording data.
In the first Aim, Pixel-seq will be used to generate cell atlases, analyze drug- and behavior-associated spatially
conserved gene expression, and map neuronal connectivity by coupling with retrograde viral tracing. In the
second Aim, we will perform immediate early gene-based whole-brain activity mapping, contextualized by cell
type-specific afferent connectivity, after fentanyl self-administration and reinstatement. In the third Aim, we will
first perform Neuropixel2.0 electrophysiological recording of the brain-wide distributed regions of interest, and
then integrate and align Neuropixel2.0 and Pixel-seq data in a new assay called NeuroPixel-seq (NP-seq). The
proposed project is innovative as our integrative approaches will, for the first time, generate spatial multimodal
data of unprecedented depth and resolution within the context of opioid relapse risk. It is significant because our
data will provide a much-needed accessible oral fe...

## Key facts

- **NIH application ID:** 10931509
- **Project number:** 5R01DA059374-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Sam Golden
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $790,368
- **Award type:** 5
- **Project period:** 2023-09-30 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10931509

## Citation

> US National Institutes of Health, RePORTER application 10931509, Integrative single-cell spatial transcriptomic, anatomical, and functional profiling of brain-wide ensembles engaged by opioid relapse (5R01DA059374-02). Retrieved via AI Analytics 2026-06-08 from https://api.ai-analytics.org/grant/nih/10931509. Licensed CC0.

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