Optimizing Chemogenetic Ablation of Dopaminergic Neurons and Establishing Single-Cell Brain Dissociation Protocols for ScRNA-seq in Adult Zebrafish

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by the loss of midbrain dopaminergic neurons, producing motor and non-motor symptoms. Adult zebrafish (Danio rerio) offer unique advantages for studying both degeneration and regeneration, yet existing chemogenetic PD models are mostly larval and rely on immersion-based prodrug delivery, which yields incomplete or biased ablation. This thesis aimed to establish an adult zebrafish PD model by combining nitroreductase (NTR)-activated prodrugs, metronidazole (MTZ), nifurpirinol (NFP), and ronidazole (RON) with cerebroventricular microinjection (CVMI) for brain-wide delivery. Dopaminergic neuron survival was quantified in the olfactory bulb, telencephalon, and diencephalon using tyrosine hydroxylase (TH) and cyan fluorescent protein (CFP), the latter expressed under regulatory elements of the dopamine transporter (DAT) promoter. Behavioural assays assessed locomotor and olfactory function at 1 and 7 days post-treatment. No significant reductions in dopaminergic neuron counts or behavioural impairments were observed compared with vehicle controls. This outcome likely reflects multiple constraints: the low solubility and limited bioavailability of prodrugs, the modest catalytic efficiency of first-generation NTR, and possible rapid neuronal regeneration in adults. Technical factors such as autofluorescence and reliance on manual counts may also have masked subtle changes. In parallel, this work piloted adult brain dissociation for single-cell RNA sequencing (scRNA-seq). While single-cell suspensions were obtained, viability assessment by microscopy proved insufficient, highlighting the need for quantitative approaches such as flow cytometry. Overall, this thesis identifies key methodological limitations and outlines strategies, including NTR 2.0 transgenics, refined prodrug delivery, and optimized cell dissociation, that will enable robust adult zebrafish PD models for regenerative studies.