# Microglia and Opioid Withdrawal: Mechanisms of Negative Reinforcement

> **NIH NIH R01** · SEATTLE INST FOR BIOMEDICAL/CLINICAL RES · 2022 · $408,546

## Abstract

The current epidemic of opioid overdoses has been propelled by both illicit and prescribed narcotic pain
medications. Extensive opioid use and repeated abstinence increases the likelihood of severe withdrawal and
perpetuates the vulnerability to relapse via means of negative reinforcement. The negative emotional valence
of withdrawal can last long after the initial, dramatic physical signs, involving a protracted negative emotional
state, drug craving, and a high likelihood of relapse. These combined symptoms are commonly referred to as
being “dope sick”. Addicted individuals often prefer to continue drugs rather than face withdrawal. Being
“dope-sick” has many attributes of a severe inflammatory state and this led us to investigate the involvement of
microglia, the innate immune cells that reside in the brain, in opioid tolerance and withdrawal, and this was
supported by a CEBRA R21 grant (R21-DA044757).
That CEBRA R21 grant resulted in our findings of dramatic changes in ribosome-bound mRNAs—the
“translatome”—in microglia using RNA sequencing of RiboTag purified microglial RNAs. Those results
provided us with the leads that form that basis for this proposal. Many of the changes related to cyclic AMP
signaling and its downstream targets, and experimental chemogenetic stimulation of Gi/o signaling was found
to actually worsen opioid withdrawal. With the understanding that glia are partners in plasticity, we suspect that
the relapsing/remitting nature of opioid dependence serves to prime and condition microglia, shifting the impact
from tempering withdrawal during initial stages to exacerbating withdrawal and opioid seeking after multiple
cycles of tolerance and withdrawal. Thus, investigations into the role of glia in withdrawal may provide new
therapeutic avenues. We propose three Aims using fentanyl and a recently developed transgenic mouse that
allows conditional and microglia-specific Cre and TdTomato expression without disrupting microglia function. In
Aim 1 we will analyze the trajectory of the changing microglial translatome after one vs. five cycles of opioid
dependence and spontaneous withdrawal. In Aim 2 we will examine the physical and behavioral
consequences of one vs. five cycles of opioid dependence and withdrawal, to explore the idea that intermittent
cycles of dependence and withdrawal exacerbate the negative consequences of withdrawal. We will then
investigate the hypothesis that the purinergic receptors P2Y12 and P2X7 are involved in microglial responses
during initial and delayed phases of opioid withdrawal. In Aim 3 we will use an in vitro brain slice model with 2-
photon confocal imaging of microglia. We will study the microstructure and motility of microglia using time-
lapse microscopy. We will measure real-time changes in cyclic AMP using a FRET-based biosensor and
calcium dynamics with GCaMP6. These three Aims integrate the temporal, behavioral, and molecular
consequences of microglial engagement during opioid dependence ...

## Key facts

- **NIH application ID:** 10458741
- **Project number:** 5R01DA052618-02
- **Recipient organization:** SEATTLE INST FOR BIOMEDICAL/CLINICAL RES
- **Principal Investigator:** John F Neumaier
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $408,546
- **Award type:** 5
- **Project period:** 2021-08-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458741

## Citation

> US National Institutes of Health, RePORTER application 10458741, Microglia and Opioid Withdrawal: Mechanisms of Negative Reinforcement (5R01DA052618-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10458741. Licensed CC0.

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