Characterising the functional homology of central nervous system drug targets in larval zebrafish (Danio rerio)

Pharmaceuticals are increasingly detected in aquatic environments at concentrations ranging from ng/L to mg/L, yet their potential impacts on non-target organisms remain poorly understood. Central nervous system (CNS)-targeting drugs are of particular concern because they are designed to act at low concentrations on highly conserved molecular targets. While sequence-level conservation of these targets between humans and fish is well established, it remains unclear whether this translates into functional conservation in terms of physiological and neurobiological responses. Addressing this gap is critical for improving environmental risk assessment frameworks, which currently rely largely on apical toxicity endpoints and consider pharmacological mode of action to a lesser degree. This thesis investigates whether conservation of CNS drug targets in fish predicts functional effects, using larval zebrafish (Danio rerio) as a model system. The study focuses on nine widely prescribed and environmentally relevant compounds: three anticonvulsants (carbamazepine, lamotrigine, gabapentin), three antidepressants (venlafaxine, escitalopram, trazodone), and three antipsychotics (aripiprazole, clozapine, risperidone). Chapter 1 reviews the occurrence, mechanisms of action, and environmental relevance of CNS-active pharmaceuticals, identifying key knowledge gaps in linking molecular conservation to ecological outcomes. Major neurotransmitter systems, including serotonergic, dopaminergic, glutamatergic, GABAergic, cholinergic, and adrenergic pathways, are shown to be highly conserved between humans and fish. Chapter 2 examines the evolutionary conservation of CNS drug targets across ray-finned fish using bioinformatic analyses of 67 species from the Ensembl database. High levels of sequence and binding site conservation were identified across multiple receptor families and ion channels, including dopaminergic, serotonergic, and histaminergic receptors, as well as voltage-gated Na⁺ and Ca²⁺ channels. However, gene duplication and the presence of 3 paralogues introduce complexity in predicting functional equivalence, highlighting limitations of sequence-based approaches alone. Chapter 3 evaluates uptake and developmental toxicity of the selected compounds using a modified OECD fish embryo toxicity assay in larval zebrafish. Compound-specific differences in bioconcentration and toxicity were observed, and bioconcentration factors (BCFs) were determined for 8 of the 9 compounds. The uptake of gabapentin was below the instruments limit of detection. Sub-lethal endpoints, including touch responsiveness, were sensitive to exposure. This chapter also established no observable effect concentrations (NOECs), which informed exposure levels for subsequent functional analyses. Chapter 4 investigates the neurophysiological effects of chronic exposure using whole-brain Ca²⁺ imaging. Distinct, drug-specific alterations in neural activity were observed, with region specific responses across multiple regions. Most compounds produced a unique pattern of neural activation and suppression, although risperidone showed no measurable effect under the conditions tested. Interestingly. gabapentin demonstrated significant impact on neural activity patterns, despite showing no measurable uptake in chapter 2. Functional connectivity analysis demonstrated that trazodone exposure alters functional connectivity providing direct evidence of pharmacologically relevant effects on the fish CNS. Chapter 5 assesses molecular responses using quantitative polymerase chain reaction (qPCR). Most gene expression changes were not statistically significant; however, increased expression of htr2cl1 was observed following exposure to a human therapeutically relevant concentration of escitalopram. Additionally, differential expression of the paralogues drd2a and drd2b was identified following exposure to aripiprazole, with no differences observed in paralogue expression within controls. These molecular responses did not consistently align with neural activity patterns or predicted target conservation. Together, these findings demonstrate that while CNS drug targets are highly conserved at the molecular level and most compounds were taken up by larvae, conservation and uptake 4 do not reliably predict functional outcomes. Instead, biological responses are influenced by a combination of target conservation, bioavailability, and system-level neural dynamics. This work provides a mechanistic framework linking molecular conservation to functional effects in aquatic organisms and highlights the importance of integrating pharmacological knowledge into environmental risk assessment. By combining bioinformatics, toxicology, neuroimaging, and molecular biology, this thesis advances understanding of how neuroactive pharmaceuticals impact non-target species and underscores the need to consider evolutionary and functional context when assessing environmental risk.<p></p>

Authors dc:creator

• Siobhân Alexandra Monaghan (23946420)
• Siobhan Monaghan (21040757)

• Uncategorised value

Statement dc:rights

• All rights reserved
• Open Access after 2027-05-21

Identifier

10779/exe.32346996.v1

OAI identifier oai:identifier

oai:figshare.com:article/32346996