Graduation Year

2026

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Neuroscience

Reader 1

Lucy Vulchanova

Reader 2

Sandra Watson

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Terms of Use for work posted in Scholarship@Claremont.

Abstract

Alcohol use disorder (AUD) is the U.S.’s predominant substance use disorder, affecting about 20 million people annually, and has high comorbidity with chronic pain (CP) due to overlapping neural circuitry. Pain signaling is partly modulated in the brainstem, which has neuronal populations that project to reward-processing regions implicated in AUD. Published works support the hypotheses that the dorsal raphe nucleus (DRN) in the brainstem has altered neuronal activity under chronic alcohol exposure (CAE) and that CP is mediated by an excitatory pathway originating in the DRN. This study used RNAscope fluorescent in situ hybridization (FISH) to identify glutamatergic subpopulations active in the DRN post-CAE and CP. Male and female mice underwent voluntary-intermittent CAE or water treatment for 9 weeks. In week 6, mice received a sham or sciatic nerve crush injury to induce neuropathic pain. Weekly von Frey testing measured tactile sensitivity and showed delayed recovery from persistent pain in alcohol-crush mice post-injury. Fresh frozen brain tissue was collected and neuronal activation was measured by cFos expression. Vesicular glutamate transporters, VGLUT2 or VGLUT3, marked two distinct glutamatergic neuronal populations in the DRN. Protocol optimization enhanced slide-tissue adherence and autofluorescence to improve mRNA puncta quantification. Qualitative improvements involved increasing tissue fixation, lowering protease digestion, and decreasing fluorophore incubation time. Images were taken on a Keyence BZ-X800 microscope to collect 40x objective z-stacks and analyzed using CellProfiler for a tailored image-processing pipeline. Ongoing FISH quantitative analysis is expected to show CAE-mice have increased glutamatergic activation compared to water treatment mice.

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