Morphine regulates astrocyte transcriptional dynamics in the ventral tegmental area by stimulation of glucocorticoid signaling

Reviewer #1 (Public review):

Studies investigating global gene expression changes induced by a single morphine administration have previously been conducted in several rodent brain regions. In this work, the authors focused on the ventral tegmental area (VTA), a key structure of the reward system that has not been extensively characterized in this context. To examine genome-wide transcriptional responses, they employed single-nucleus RNA sequencing (snRNA seq), a method well-suited for profiling gene expression in VTA cells, which are otherwise difficult to isolate.

The effects of morphine on gene expression in VTA cells were assessed in naive animals, in rats exposed to chronic inflammatory pain induced by local CFA injection into the paw, and in animals subjected to both conditions. The study revealed widespread transcriptional changes following morphine administration, whereas inflammation alone produced only limited alterations-an outcome that may reflect the sensitivity or resolution of the sequencing approach used.

Further in vitro experiments conducted in multiple astrocyte models demonstrated that the increase in Fkbp5 expression observed in the VTA is unlikely to result from opioid receptor activation. Instead, the data indicate that this effect is mediated by glucocorticoid receptor stimulation. These findings suggest that the elevated Fkbp5 expression in the rat VTA represents a secondary response rather than a direct consequence of morphine exposure. Comparable transcriptional changes, as well as similar mechanistic interpretations, have been reported in previous studies examining the nucleus accumbens (NAc), reinforcing the view that glucocorticoid-dependent regulation of Fkbp5 may be a broader feature of opioid related neuroadaptations.

The present paper showed largely similar morphine-induced gene changes in both male and female VTA samples. On the other hand, several studies indicate that males and females exhibit differences in dopaminergic activation and distinct gene expression profiles in response to opioids in the reward system. Preclinical studies have found marked sex differences in Fkbp5 expression in the dorsal striatum. This issue should be better addressed both experimentally and theoretically.

Reviewer #2 (Public review):

Summary:

This study addresses an important gap in our understanding of how pain‑related neuroadaptations interact with opioid exposure at the cellular and molecular levels, particularly in terms of cell‑type-specific responses within reward‑related brain regions. By applying single‑nucleus RNA sequencing, the authors generate a comprehensive atlas of transcriptional changes in the rat VTA associated with chronic inflammatory pain and acute morphine administration.

Strengths:

Overall, the study is important, and the experiments are carefully designed and executed. The manuscript is logically structured and well written. The sample size is appropriate: nuclei were collected from 14 male and 14 female Sprague‑Dawley rats, with 6-8 animals per experimental group. The inclusion of both sexes further strengthens the study by enhancing the generalizability of the findings.

To increase translational relevance, the authors also employ a human‑derived astrocyte culture model, which helps bridge findings from rodent tissue to human‑related cellular mechanisms.

Weaknesses:

A limitation is that the study examines only a single time point after morphine administration. However, this is balanced by the use of state‑of‑the‑art , and inherently expensive, molecular tools that allow deep transcriptional profiling.

One area requiring clarification is compliance with methodological standards. The manuscript does not specify whether ARRIVE guidelines were followed, whether a power analysis was performed to justify the number of animals used, or how randomization and blinding procedures were implemented.

Reviewer #3 (Public review):

Summary:

This work examined the transcriptional response to pain induction by CFA and/or morphine treatment in rat VTA at the level of single cells. This builds on prior work using bulk-tissue RNA-seq to evaluate response to SNI pain and/or oxycodone treatment. Here, authors find few lasting gene expression changes with CFA, but a robust transcriptional response to acute morphine, particularly in non-neuronal cells, where an increase in Fkbp5 stood out. The authors validated corticosterone-induced elevations in Fkbp5 in rat glial cell culture and human astrocyte cell culture, which are blocked by the GR antagonist mifepristone and inhibition of Nr3c1, but are not independently induced by the µOR agonist DAMGO.

Strengths:

The authors started with somewhat surprising transcriptional observations and followed the science appropriately to investigate the functional relevance of one particular finding. This work is well-powered and uses state-of-the-art snRNA-seq and CRISPR-based manipulations in both rat glia and human astrocyte cell preparations to determine the functional relevance of Fkbp5-regulated transcriptional activity.

Weaknesses:

(1) It was somewhat surprising that the CFA-Morphine group was not taken at a time point when the morphine treatment was found to be behaviorally effective.

(2) The final conclusion that Nr3c1 repression reduces the response to cort is not novel or surprising, even if it is within human astrocyte culture (which is cool).

(3) This work falls short of bringing the research full circle by applying their Nr3c1-CRISPRi approach in vivo to alter behavioral response to morphine and/or pain.