Abstract
Functional neuroimaging, using genetically-encoded Ca2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.
Highlights
COPYRIGHT AND REUSE Open Research Exeter makes this work available in accordance with publisher policies
GCaMPs have been employed in a range of model organisms[3]; amongst these, larval zebrafish offer an unparalleled combination of higher vertebrate relevance, optical transparency, and a small brain size[4] allowing simultaneous spatial coverage of multiple brain regions within a fully intact connectome
Using four exemplar drugs known to induce neural network hyperactivity by different molecular mechanisms in both mammals and zebrafish, we utilise a combination of light sheet microscopy (LSM) and a transgenic zebrafish[11] to quantify functional brain responses in 4 dimensions
Summary
COPYRIGHT AND REUSE Open Research Exeter makes this work available in accordance with publisher policies. Functional neuroimaging, using genetically-encoded Ca2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. We use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. We present a computational image analysis framework that enables 3D anatomical registration against a standardised larval zebrafish brain, providing quantitative functional data for defined regions across the whole larval brain Using this methodology we have quantified several measures of neural activity in multiple brain regions, revealing differential spatiotemporal patterns associated with differing molecular mechanisms of drug action. The use of in vivo extracellular local field potential recording from the optic tectum of comparable animals provides additional information on the temporal profile characteristics of drug-induced neural activity to aid interpretation of the GCaMP imaging data
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