Profiling gene alterations in striatonigral neurons associated with incubation of methamphetamine craving by cholera toxin subunit B-based fluorescence-activated cell sorting

• Published in: Frontiers in cellular neuroscience 2025-02, Vol.19, p.1542508
• Main Authors: Altshuler, Rachel D, Burke, Megan A M, Garcia, Kristine T, Class, Kenneth, Cimbro, Raffaello, Li, Xuan
• Format: Article
• Language: English
• Subjects: Abstinence; Behavior; Catheters; Caudate-putamen; Cholera; Cholera toxin; cholera toxin subunit B; Dopamine; Enzymes; Experiments; FDA approval; Flow cytometry; Fluorescence; fluorescence-activated cell sorting; gene expression; Genes; Laboratory animals; Methamphetamine; methamphetamine craving; Molecular modelling; Narcotics; Neostriatum; Neuroscience; Polyethylene; Proteins; Roles; Spiny neurons; striatonigral projection neurons; Surgery; Toxins
• ISSN: 1662-5102; 1662-5102
• DOI: 10.3389/fncel.2025.1542508

In both rats and humans, methamphetamine (Meth) seeking progressively increases during abstinence, a behavioral phenomenon termed "incubation of Meth craving". We previously demonstrated a critical role of dorsal striatum (DS) in this incubation in rats. However, circuit-specific molecular mechanisms in DS underlying this incubation are largely unknown. Here we combined a newly developed fluorescence-activated sorting (FACS) protocol with fluorescence-conjugated cholera toxin subunit B-647 (CTb-647, a retrograde tracer) to examine gene alterations in the direct-pathway (striatonigral) medium spiny neurons (MSNs) associated with incubation of Meth craving. We injected CTb-647 bilaterally into substantia nigra before or after training rats to self-administer Meth or saline (control condition) for 10 days (6 h/d). On abstinence day 1 or day 28, we collected the DS tissue from both groups for subsequent FACS and examined gene expressions in CTb-positive (striatonigral MSNs) and CTb-negative (primarily non-striatonigral MSNs). Finally, we examined gene expressions in DS homogenates, to demonstrate cell-type specificity of gene alterations observed on abstinence day 28. On abstinence day 1, we found mRNA expression of decreased only in CTb-positive (but not CTb-negative) neurons of Meth rats compared with saline rats, while mRNA expression of decreased in all sorted DS neurons. On abstinence day 28, we found increased mRNA expression for , and in all sorted DS neurons, but not DS homogenate. Together, these data demonstrated that incubation of Meth craving was associated with time-dependent, circuit-specific, and cell type-specific gene alterations in DS involved in glutamatergic, GABAergic, opioidergic, and protein degradation signaling.