Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators

Abstract

Neural circuits are non-linear dynamical systems that transform information based on the pattern of input, current state and functional connectivity. To understand how a given stimulus is processed, one would ideally record neural activity across the entire brain of a behaving animal, at cellular or even subcellular resolution, in addition to characterizing anatomical connectivity. Given their transparency and relatively small size, larval zebrafish provide a powerful system for brain-wide monitoring of neural activity. Genetically encoded calcium indicators have been used for this purpose, but cannot directly report hyperpolarization or sub-threshold activity. Voltage indicators, in contrast, have this capability. Here, we test whether two different genetically encoded voltage reporters, ASAP1 and Bongwoori, can be expressed and report activity in the zebrafish brain, using widefield, two-photon and light sheet microscopy. We were unable to express ASAP1 in neurons. Bongwoori, in contrast expressed well, and because of its membrane localization, allowed visualization of axon trajectories in 3D. Bongwoori displayed stimulus-evoked changes in fluorescence, which could be detected in single trials. However, under high laser illumination, puncta on neural membranes underwent spontaneous fluctuations in intensity, suggesting that the probe is susceptible to blinking artefacts. These data indicate that larval zebrafish can be used to image electrical activity in the brain of an intact vertebrate at high resolution, although care is needed in imaging and analysis. Recording activity across the whole brain will benefit from further developments in imaging hardware and indicators.

An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics

“…While calcium indicators have become the standard for imaging neuronal activity, genetically encoded voltage indicators (GEVIs) are a promising technology [45,46], despite important constraints on the technique[12]. In principle, the toolbox could be extended without difficulty to allow the analysis of imaging data using GEVIs.…”

Calcium imaging and dynamic causal modelling reveal brain-wide changes in effective connectivity and synaptic dynamics during epileptic seizures

“…chemoconvulsant exposure) can be evaluated in vivo [2–4]. Zebrafish in particular have been of recent interest for epilepsy research because they (i) are a vertebrate organism, (ii) allow the introduction of genetic mutations [5] and large-scale drug screening [6,7], and (iii) allow recording of neuronal function at high resolution across distributed brain networks [8,9]. There are now several studies of epileptic seizures in zebrafish [10–13] and recent imaging studies have captured network-wide changes in zebrafish brain activity during seizures [14,15].…”

Improving a genetically encoded voltage indicator by modifying the cytoplasmic charge composition

“…Bongwoori is another GEVI that is an ArcLight derivative exhibiting improved speed and a shifted voltage-response to more positive potentials which enabled the optical resolution of action potentials firing at 60 Hz 4 . While an in vivo signal could be observed when Bongwoori was expressed in zebrafish embryos, the signal size was small complicating the detection of neuronal activity 5 . In this report, we describe an improved version of Bongwoori yielding optical signals as high as 20% ΔF/F during the firing of action potentials.…”