Regional synapse gain and loss accompany memory formation in larval zebrafish

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

The Cyanotoxin 2,4-DAB Reduces Viability and Causes Behavioral and Molecular Dysfunctions Associated with Neurodegeneration in Larval Zebrafish

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB’s effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish

In early limb embryogenesis, synovial joints acquire specific shapes which determine joint motion and function. The process by which the opposing cartilaginous joint surfaces are moulded into reciprocal and interlocking shapes, called joint morphogenesis, is one of the least understood aspect of joint formation and the cell-level dynamics underlying it are yet to be unravelled. In this research, we quantified key cellular dynamics involved in growth and morphogenesis of the zebrafish jaw joint and synthesised them in a predictive computational simulation of joint development. Cells in larval zebrafish jaw joints labelled with cartilage markers were tracked over a forty-eight hour time window using confocal imaging. Changes in distance and angle between adjacent cell centroids resulting from cell rearrangement, volume expansion and extracellular matrix (ECM) deposition were measured and used to calculate the rate and direction of local tissue deformations. We observed spatially and temporally heterogeneous growth patterns with marked anisotropy over the developmental period assessed. There was notably elevated growth at the level of the retroarticular process of the Meckel's cartilage, a feature known to undergo pronounced shape changes during zebrafish development. Analysis of cell dynamics indicated a dominant role for cell volume expansion in growth, with minor influences from ECM volume increases and cell intercalation. Cell proliferation in the joint was minimal over the timeframe of interest. Synthesising the dynamic cell data into a finite element model of jaw joint development resulted in accurate shape predictions. Our biofidelic computational simulation demonstrated that zebrafish jaw joint growth can be reasonably approximated based on cell positional information over time, where cell positional information derives mainly from cell orientation and cell volume expansion. By modifying the input parameters of the simulation, we were able to assess the relative contributions of heterogeneous growth rates and of growth orientation. The use of uniform rather than heterogeneous growth rates only minorly impacted the shape predictions whereas isotropic growth fields resulted in altered shape predictions. The simulation results suggest that growth anisotropy is the dominant influence on joint growth and morphogenesis. This study addresses the gap of the cellular processes underlying joint morphogenesis, with implications for understanding the aetiology of developmental joint disorders such as developmental dysplasia of the hip and arthrogryposis.

A lexical approach for identifying behavioural action sequences

Animals display characteristic behavioural patterns when performing a task, such as the spiraling of a soaring bird or the surge-and-cast of a male moth searching for a female. Identifying such recurring sequences occurring rarely in noisy behavioural data is key to understanding the behavioural response to a distributed stimulus in unrestrained animals. Existing models seek to describe the dynamics of behaviour or segment individual locomotor episodes rather than to identify the rare and transient sequences of locomotor episodes that make up the behavioural response. To fill this gap, we develop a lexical, hierarchical model of behaviour. We designed an unsupervised algorithm called “BASS” to efficiently identify and segment recurring behavioural action sequences transiently occurring in long behavioural recordings. When applied to navigating larval zebrafish, BASS extracts a dictionary of remarkably long, non-Markovian sequences consisting of repeats and mixtures of slow forward and turn bouts. Applied to a novel chemotaxis assay, BASS uncovers chemotactic strategies deployed by zebrafish to avoid aversive cues consisting of sequences of fast large-angle turns and burst swims. In a simulated dataset of soaring gliders climbing thermals, BASS finds the spiraling patterns characteristic of soaring behaviour. In both cases, BASS succeeds in identifying rare action sequences in the behaviour deployed by freely moving animals. BASS can be easily incorporated into the pipelines of existing behavioural analyses across diverse species, and even more broadly used as a generic algorithm for pattern recognition in low-dimensional sequential data.

Lentinan Impairs the Early Development of Zebrafish Embryos, Possibly by Disrupting Glucose and Lipid Metabolism

LNT is the major biologically active substance extracted from Lentinus edodes (L. edodes). Although functional and pharmacological studies have demonstrated that LNT has multiple benefits for animals and humans, the safety assessment is far from sufficient. To evaluate the potential safety risk, larval zebrafish were continuously exposed to varying concentrations of LNT for 120 h. The 96 h LC50 of LNT was determined to be 1228 μg/mL, and morphological defects including short body length, reduced eye and swim bladder sizes and yolk sac edema were observed. In addition, LNT exposure significantly reduced the blood flow velocity and locomotor activity of larval zebrafish. The biochemical parameters were also affected, showing reduced glucose, triglyceride and cholesterol levels in zebrafish larvae after being exposed to LNT. Correspondingly, the genes involved in glucose and lipid metabolism were disrupted. In conclusion, the present study demonstrates the adverse potential of high concentrations of LNT on the development of zebrafish larvae in the early life stage.

The geometry of decision-making in individuals and collectives

Choosing among spatially distributed options is a central challenge for animals, from deciding among alternative potential food sources or refuges to choosing with whom to associate. Using an integrated theoretical and experimental approach (employing immersive virtual reality), we consider the interplay between movement and vectorial integration during decision-making regarding two, or more, options in space. In computational models of this process, we reveal the occurrence of spontaneous and abrupt “critical” transitions (associated with specific geometrical relationships) whereby organisms spontaneously switch from averaging vectorial information among, to suddenly excluding one among, the remaining options. This bifurcation process repeats until only one option—the one ultimately selected—remains. Thus, we predict that the brain repeatedly breaks multichoice decisions into a series of binary decisions in space–time. Experiments with fruit flies, desert locusts, and larval zebrafish reveal that they exhibit these same bifurcations, demonstrating that across taxa and ecological contexts, there exist fundamental geometric principles that are essential to explain how, and why, animals move the way they do.

Environmental and Molecular Modulation of Motor Individuality in Larval Zebrafish

Innate behavioral biases such as human handedness are a ubiquitous form of inter-individual variation that are not strictly hardwired into the genome and are influenced by diverse internal and external cues. Yet, genetic and environmental factors modulating behavioral variation remain poorly understood, especially in vertebrates. To identify genetic and environmental factors that influence behavioral variation, we take advantage of larval zebrafish light-search behavior. During light-search, individuals preferentially turn in leftward or rightward loops, in which directional bias is sustained and non-heritable. Our previous work has shown that bias is maintained by a habenula-rostral PT circuit and genes associated with Notch signaling. Here we use a medium-throughput recording strategy and unbiased analysis to show that significant individual to individual variation exists in wildtype larval zebrafish turning preference. We classify stable left, right, and unbiased turning types, with most individuals exhibiting a directional preference. We show unbiased behavior is not due to a loss of photo-responsiveness but reduced persistence in same-direction turning. Raising larvae at elevated temperature selectively reduces the leftward turning type and impacts rostral PT neurons, specifically. Exposure to conspecifics, variable salinity, environmental enrichment, and physical disturbance does not significantly impact inter-individual turning bias. Pharmacological manipulation of Notch signaling disrupts habenula development and turn bias individuality in a dose dependent manner, establishing a direct role of Notch signaling. Last, a mutant allele of a known Notch pathway affecter gene, gsx2, disrupts turn bias individuality, implicating that brain regions independent of the previously established habenula-rostral PT likely contribute to inter-individual variation. These results establish that larval zebrafish is a powerful vertebrate model for inter-individual variation with established neural targets showing sensitivity to specific environmental and gene signaling disruptions. Our results provide new insight into how variation is generated in the vertebrate nervous system.