Numerous neurodegenerative and psychiatric disorders are associated with deficits in executive functions such as working memory and cognitive flexibility. Progress in developing effective treatments for disorders may benefit from targeting these cognitive impairments, the success of which is predicated on the development of animal models with validated behavioural assays. Zebrafish offer a promising model for studying complex brain disorders, but tasks assessing executive function are lacking. The Free-movement pattern (FMP) Y-maze combines aspects of the common Y-maze assay, which exploits the inherent motivation of an organism to explore an unknown environment, with analysis based on a series of sequential two-choice discriminations. We validate the task as a measure of working memory and executive function by comparing task performance parameters in adult zebrafish treated with a range of glutamatergic, cholinergic and dopaminergic drugs known to impair working memory and cognitive flexibility. We demonstrate the cross-species validity of the task by assessing performance parameters in adapted versions of the task for mice and Drosophila, and finally a virtual version in humans, and identify remarkable commonalities between vertebrate species' navigation of the maze. Together, our results demonstrate that the FMP Y-maze is a sensitive assay for assessing working memory and cognitive flexibility across species from invertebrates to humans, providing a simple and widely applicable behavioural assay with exceptional translational relevance.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

When exposed to ethanol, Drosophila melanogaster display a variety of addiction-like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma-aminobutyric acid receptor B (GABA B ) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose-dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABA B drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABA B receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (AUD). KEYWORDS addiction behaviours, alcohol use disorder, CGP 54626, Drosophila melanogaster, GABA B receptor, SKF 97541

Simple mazes have provided numerous tasks for assessing working memory. The discrete nature of choices in the T-maze has provided a robust protocol with sensitivity to cognitive deficits, whilst the continuous Y-maze reduces manual handling and pre-trial training. We have combined these attributes to develop a new behavioural task for assessing working memory, the Free-movement pattern (FMP) Y-maze. Using sequentially recorded left and right turns we demonstrate that zebrafish and mice use a single dominant strategy predominantly consisting of alternations between left and right choices trial-to-trial. We further tested this protocol with Drosophila and discovered an alternative invertebrate search strategy. Finally, a virtual human FMP Y-maze confirmed a common strategy among all tested vertebrate species, validating the translational power of the task for human research. The FMP Y-maze combines robust investigation of working memory and high translational power, generating a simple task with far-reaching impact.

Mutations in mitochondrial complex I cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The mechanisms underlying complex I deficiency pathogenesis remain poorly understood, and as a result there are currently no available treatments. To better understand the causes of neuronal dysfunction in complex I deficiency, we modelled complex I deficiency in Drosophila by knocking down the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Remarkably, we find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1 in neurons, which couples NADH oxidation to transfer of electrons into the respiratory chain, reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Metabolomic analysis shows that NDI1 expression also reconfigures neuronal metabolism and implicates increased GABA levels as a contributor to the neurological manifestations of complex I deficiency. Together, these data indicate that NDI1 abrogates UPR signalling and reprogrammes metabolism to alleviate neuronal dysfunction caused by neuronal complex I deficiency.