Biology subjects

Fontana, B. D.

Publications and source records attributed to Fontana, B. D..

8 recordsLinked to original sources

bioRxiv2026

Behavioral test batteries induce transient, domain-specific effects while preserving global phenotypic structure in zebrafish

Behavioral test batteries are increasingly used to characterize multiple functional domains in zebrafish, yet the potential impact of test sequence on behavioral outcomes remains poorly defined. Here, we systematically evaluated whether test order influences behavioral responses in a three-assay battery comprising the novel tank test (NTT), mirror-induced aggression (MIA), and social preference (SP) test. Adult zebrafish (Danio rerio) were exposed to all possible permutations of the three assays in a fully counterbalanced design, allowing assessment of order effects across locomotor, anxiety-like, aggression-related, and social behaviors. Test order produced modest and parameter-specific effects, primarily affecting locomotor activity in the NTT and social proximity in the SP assay. Time-course analysis revealed within-test behavioral dynamics, with limited evidence that test order modulates early adaptation or late engagement with the testing environment but does not alter overall temporal response profiles. Sex-dependent effects were assay-specific and most pronounced in the NTT, with no consistent sex differences observed in MIA or SP. To evaluate the global structure of behavioral variation, Principal Component Analysis (PCA) was performed across assays. Despite localized effects of test order, no clear multivariate separation between test sequences was observed, indicating that sequential testing does not produce distinct baseline phenotypes. Together, these findings support the robustness and reproducibility of multidomain behavioral batteries while highlighting the importance of standardized test-order reporting to improve cross-study comparability.

animal behavior and cognition↗

bioRxiv2025

Genetic disruption of leucine rich repeat transmembrane protein 4 like 1 induces a pro-social behavioural phenotype in zebrafish

BackgroundSocial behaviour encompasses the wide range of interactions that occur between members of the same species. In humans, disruptions in social behaviour are characteristic of many neuropsychiatric disorders, where both genetic risk factors and synaptic dysfunctions can contribute to the phenotype. Among the genes implicated in synaptic regulation, the synaptic adhesion protein leucine-rich repeat transmembrane protein 4 (LRRTM4) has been identified as a key player in maintaining synaptic function and neuronal circuit integrity. Despite its established role in the nervous system, the potential involvement of LRRTM4 in modulating social behaviour and its contribution to social deficits has yet to be explored. MethodsIn the current study, we used zebrafish to study how genetic deletion of lrrtm4l1, a zebrafish orthologue of LRRTM4, affects sociality. For this, the social behaviour of homozygous lrrtm4l1 knockout (KO) zebrafish was analysed in multiple behavioural assays and the brain transcriptome of mutant animals was investigated by RNAseq. ResultsKO zebrafish displayed a pro-social phenotype in multiple behavioural assays. Groups of lrrtm4l1 KO zebrafish formed more cohesive shoals and KO individuals spent more time in the vicinity of conspecifics during a social interaction test. They were also less aggressive and in contrast to wild-type zebrafish did not differentiate in their interactions with known and unknown groups of fish. Neurotranscriptomic analysis revealed 560 differentially expressed genes including changes in glutamatergic neurotransmitter signalling, tryptophan- kynurenine metabolism and synaptic plasticity. ConclusionThese findings suggest that lrrtm4l1 is an important regulator of social behaviour in zebrafish. In a translational perspective, LRRTM4 is a promising potential therapeutic target that warrants further investigation in the framework of neuropsychiatric conditions characterized by major social impairments.

neuroscience↗

bioRxiv2025

Individual differences in fear memory expression engage distinct functional brain networks

Fearful stimuli elicit a mix of active (e.g., evasion) and passive (e.g., freezing) behaviors in a wide range of species, including zebrafish (Danio rerio). How these different responses are encoded in the brain, and the extent to which individuals vary in their fear responses, is not clear. To investigate this, we first developed a contextual fear conditioning paradigm in adult zebrafish where fish associate a specific tank with an aversive pheromone, conspecific alarm substance (CAS). We collected data from over 300 fish across four different strains (AB, TU, TL, and WIK) and both sexes finding that, as expected, fish exhibit a mix of active and passive responses during CAS exposure and during memory expression. We also found that behavior fell into four distinct groups: non-reactive, evaders, evading freezers, and freezers, with the evading freezer and freezer groups most clearly associated with memory formation. Background strain and sex also influenced how fish respond to CAS, with males more likely to increase evasive behaviors in response to CAS and the TU strain more likely to be non-reactive during recall. Finally, using whole-brain activity mapping, we identified the brain regions associated with active and passive fear responses during memory expression and how the functional brain networks of evading freezers and freezers differed. Freezing behavior was associated with widespread neural activity that was particularly strong in the cerebellum, reticular formation, and parts of the telencephalon associated with olfactory processing. Evasive behavior was associated with an increase in the activity of visual threat detection and a decrease in the activity of brain regions related to foraging and navigation. Network analysis revealed that animals with high freezing and low evasion (i.e., freezers) had strong interactions between the pallium and cerebellum as well as strong interconnectivity between visual (pretectal) and thalamic analog (preglomerular) nuclei. Animals that mix freezing with evasive behaviors (i.e., evading freezers) had higher subpallial connectivity to regions involved in autonomic function and stress responses such as the hypothalamus and preoptic areas.

neuroscience↗

bioRxiv2024

Environmental Enrichment Reduces adgrl3.1-Related Anxiety and Attention Deficits but Not Impulsivity

Environmental factors play a role in the development and severity of neuropsychiatric disorders. Externalizing disorders (EDs) are linked to genetic and environmental factors and frequently co-occur with internalizing disorders (ID), such as anxiety. Individuals experiencing both ED/ID are often among the most likely to seek healthcare services, as this co-occurrence is associated with more severe symptomatology and greater functional impairment. Here, we investigate the role of environmental enrichment (EE) on a gene associated with impulsivity and attention deficits in zebrafish, adgrl3.1. Zebrafish were reared in either standard or enriched environments (from 15 days-post fertilization), and attention, impulsivity, and anxiety-related phenotypes were assessed at adult stages (4 months-post fertilization) using the open field test and 5-choice serial reaction time task. EE mitigated anxiety-related behaviors in adgrl3.1 knockouts, normalizing locomotor patterns and decreasing thigmotaxis. Although attention deficits were reduced in adgrl3.1-/- fish reared in EE, impulsive behaviors were not. Together, these findings suggest that while EE can alleviate some externalizing and internalizing phenotypes in zebrafish, impulsivity remains resistant to environmental modification. This study suggests that impulsivity is a stable trait in adgrl3.1-/- fish, but highlights the importance of EE in mitigating some externalizing and internalizing symptoms. Summary statementEnvironmental enrichment reduces attention deficits and anxiety-like behavior, but not impulsivity in adgr3.1 knockout zebrafish, highlighting the interaction between genetic and environmental factors in shaping externalizing and internalizing phenotypes.

animal behavior and cognition↗

bioRxiv2024

Social Isolation Intensifies adgrl3.1-Related Externalizing and Internalizing Behaviors in Zebrafish

Externalizing disorders (EDs) are characterized by outward-directed behaviors such as aggression and hyperactivity. They are influenced by gene-environment interactions, yet our understanding of the genetic predispositions and environmental contexts that give rise to them is incomplete. Additionally, people with EDs often exhibit comorbid internalizing symptoms, which can complicate the clinical presentation and treatment strategies. Following on from our previous studies, we examined genes x environment interaction as a risk factor for EDs by looking at internalizing and externalizing behaviors after social isolation. Specifically, we subjected adgrl3.1 knockout zebrafish -- characterized by hyperactivity and impulsivity -- to a 2-week social isolation protocol. We subsequently assessed the impact on anxiety-like behavior, abnormal repetitive behaviors, working memory, and social interactions. Genotype-specific additive effects emerged, with socially isolated adgrl3.1 knockout fish exhibiting intensified comorbid phenotypes, including increased anxiety, abnormal repetitive behaviors, reduced working memory, and altered shoaling, when compared to WT fish. The findings demonstrate that genetic predispositions interact with environmental stressors, such as social isolation, to exacerbate both externalizing and internalizing symptoms. This underlines the necessity for comprehensive diagnostic and intervention strategies.

animal behavior and cognition↗

bioRxiv2023

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment

Externalising disorders (ED) are a cause of concern for public health, and their high heritability make genetic risk factors a priority for research. Adhesion G Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown impacts of this gene on several core ED-related behaviors. For example, adgrl3.1-/- zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1-/- zebrafish and found them to be highly impulsive, show boldness in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1-/- vs wild type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

neuroscience↗

bioRxiv2020

Effects of D1 agonist on working memory and cognitive flexibility in a zebrafish model of aging

Healthy aging is associated with a decline in memory and executive function, which have both been linked with aberrant dopaminergic signalling. We examined the relationship between cognitive performance and dopamine function of young and aging zebrafish (Danio rerio). We revealed age-related decreases in working memory and cognitive flexibility in the Free-Movement Pattern (FMP) Y-maze. An increase in drd5 gene expression in aging adults coincided with a decrease in cognitive performance. Treatment with a D1/D5 receptor agonist (SKF-38393, 35 M) 30 minutes prior to behavioural assessment resulted in improved working memory in aging zebrafish, but no effect in younger adults. However, an overdosing effect caused by agonist treatment resulted in downregulation of dat expression in 6-month old, treated zebrafish. The translational relevance of these findings was tested in humans by analysing exploratory behaviour in young-adult, 18-35-year olds, and aged adults, 70+ year olds, in a virtual FMP Y-maze. Our findings revealed similar age-related decline in working memory. Thus, strongly supporting zebrafish as a translational model of aging and cognitive decline.

animal behavior and cognition↗

bioRxiv2020

A Test of Memory: The Fish, The Mouse, The Fly And The Human

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.

animal behavior and cognition↗