Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation

Abstract

The zebrafish (Danio rerio) has become one of the major animal models for in vivo examination of sensory and neuronal computation. Similar to Xenopus tadpoles neural activity in the optic tectum, the major region controlling visually guided behavior, can be examined in zebrafish larvae by optical imaging. Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy. This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons, which is a critical issue when using membrane-permeant dyes. This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae. Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals. We established a procedure to circumvent these problems, enabling in vivo calcium imaging in the optic tectum of adult zebrafish. Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy, because dye labeling remains restricted to tens of neurons or less. Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion. These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique. Overall, our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available. This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits.

Imaging Neuronal Activity in the Optic Tectum of Late Stage Larval Zebrafish

“…Second, calcium reporters have been targeted to the nucleus, using a UAS:gal4 system or a different pan-neuronal and/or specific promoter [ 12 , 14 , 16 ]. Finally, calcium dyes injected into the brain have also successfully been used to image adult zebrafish [ 13 , 44 ]. Each of these approaches has proven useful, within their own limitations.…”

“…An alternative approach of delivering oxygen is used in adult fish, and involves intubation with a small tube supplying water directly to the gills through the mouth. This approach has been used for adult zebrafish [ 13 ], and has recently been successfully applied to juvenile zebrafish (1 month post-fertilisation, [ 46 ]).…”

“…A disadvantage of the commonly used pan-neuronal huc promoter is that the expression of neuronal activity reporters (e.g., GCaMPs) decreases after 6–7 dpf [ 12 ]. To overcome this issue, one can either use GCaMP3-H2B fusion to target the calcium reporter expression to the nucleus [ 12 ] or use synthetic calcium indicators injected in the brain area of interest [ 13 ]. While both approaches have proven useful they have limitations.…”

Spatial Resolution of an Eye Containing a Grouped Retina: Ganglion Cell Morphology and Tectal Physiology in the Weakly Electric Fish Gnathonemus petersii

“…In short, the recorded data were randomly assigned to the different stimulus conditions and ellipses were again fitted to each of 10,000 surrogate datasets. As a measure of error probability we then determined the probability to find OSI values higher or equal to the actually measured value (Kassing et al, ).…”

Direction-selective units in goldfish retina and tectum opticum — review and new aspects

“…DAMJANOVIĆ were absent. DS neurons of the tectal origin were identi¯ed also in the zebra¯sh in a number of electrophysiological studies (Grama & Engert, 2012;Gabriel et al, 2012;Kassing et al, 2013). As a rule, cell bodies of the zebra¯sh DS neurons recorded with the calcium imaging techniques were located in the deep tectal layers (the periventricular zone).…”