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.

It consists of two major subdivisions, the medial and lateral habenula, and receives input from several regions of the forebrain (Mok & Mogenson, 1974; Herkenham & Nauta, 1977; Turner et al., 2016). The lateral habenula has become a focus

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Equations: 0 Total number of words: 5191 Number of words in abstract: 177 2 Abstract Habenula neurons are constantly active. The level of activity affects mood and behaviour, with increased activity in the lateral habenula reflecting exposure to punishment and a switch to passive coping and depression. Here, we identify GABAergic neurons that could influence activity in the lateral habenula of larval zebrafish. GAD65/67 immunohistochemistry and imaging of gad1b:DsRed transgenic fish suggest the presence of GABAergic terminals in the lateral habenula. Retrograde tracing with the lipophilic dye DiD indicates that this GABAergic innervation derives from the thalamus and a nucleus lateral to the posterior tuberculum, the putative M2 nucleus. Two-photon calcium imaging indicates that blue light causes excitation of thalamic GABAergic neurons and concomitant inhibition of a subpopulation neurons in the lateral habenula. Whole-cell recording confirmed that blue-light induced hyperpolarization of lateral habenula neurons. No response to blue light was detected in the putative M2 neurons. These observations suggest that GABAergic input from the thalamus mediates inhibition of the zebrafish lateral habenula, possibly contributing to the beneficial effects of blue light. Stimuli acting via the putative preglomerular complex remain to be defined.