Bridging in vivo and in vitro approaches to understand the physiological and behavioural effects of substances of abuse including poly-substance use and novel synthetic substances

Novel synthetic and poly-substance misuse pose major public health concerns due to their rapidsynthesis, widespread availability, unknown toxicity, and high lethality. Rapid and reliablehazard assessment methods are therefore urgently needed to determine the toxicity of thesesubstances as soon as they are identified, however current approaches are limited by time andethical constraints. This thesis evaluates the 4 days post-fertilisation (dpf) larval zebrafish(Danio rerio) model as a rapid, high-throughput, and ethical tool bridging in vitro and in vivoapproaches. The light/dark behavioural assay, which is the most used for investigating drug-induced effects, lacked experimental consistency and was initially validated throughexperimental optimisation and development of a robust normalisation approach to improvereproducibility. Model predictive validity was then confirmed with fentanyl, diacetylmorphine(heroin), and remifentanil. Fentanyl bioavailability was assessed using liquid chromatographywith tandem mass spectrometry (LC-MS/MS). Behavioural effects were reversed withnaloxone, confirming opioid receptor activation. In vivo whole-brain Ca2+-transient imagingidentified significantly affected brain regions with fentanyl (e.g., anterior commissure) and lowconcentration hyperactivity was reversed with the dopamine D2 receptor antagonist, sulpiride,implicating dopaminergic mechanisms. Functional connectivity brain analyses revealeddisrupted sensory-motor pathways between the torus semicircularis and eminentia granularis.Furthermore, the model demonstrated suitability for assessing the behaviour of g-aminobutyricacid (GABA)A positive allosteric modulator’s (PAM) and N-methyl-D-aspartate (NMDA)receptor antagonists. Finally, co-administration of fentanyl and ethanol produced a novelhyperactive phenotype, replicable in adult zebrafish, involving multiple neurotransmittersystems with significant neuronal dysregulation identified. Taken together, these findingsdemonstrate the utility of the model as a first-tier rapid hazard assessment tool of syntheticsubstances and poly-substance interactions, supporting their integration into toxicologicalresearch. However, caution for translational extrapolation to human risk evaluations remain.