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In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Published: February 10th, 2021



1Cellular and Molecular Neurobiology, Zoological Institute, Technische Universität Braunschweig, 2Institute of Pathophysiology, University Medical Center of the Johannes Gutenberg University, 3Cell Physiology, Zoological institute, Technische Universität Braunschweig

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.

Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Over the recent decades, the zebrafish (Danio rerio) has evolved as one of the most popular vertebrate model organisms for embryonic and larval developmental studies. The large fecundity of zebrafish females coupled with the rapid ex utero development of the embryo and its transparency during early embryonic developmental stages are just a few key factors that make zebrafish a powerful model organism to adress developmental questions1. Advances in molecular genetic technologies combined with high resolution in vivo imaging studies allowed for addressing cell biological mechanisms underlying developmental processes

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All animal work described here is in accordance with legal regulations (EU-Directive 2010/63). Maintenance and handling of fish have been approved by local authorities and by the animal welfare representative of the Technische Universität Braunschweig.

1. Preparation of artificial cerebro spinal fluid (ACSF), low melting agarose and sharp glass needles

  1. Prepare the ACSF by dissolving the listed chemicals at following concentrations in distilled water. 134 mM NaCl (58.44 g/mol),.......

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Figure 3A,C show a 14 dpf larva of the transgenic line Tg[-7.5Ca8:GFP]bz12[15] with the skull still intact. The pigment cells in the overlaying skin are distributed all over the head and are interfering with the fluorescence signal in the region of interest (here, cerebellum). With the larva in this condition, it is not possible to obtain high resolution images of the brain. F.......

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The presented method provides an alternative approach to brain isolation or the treatment of zebrafish larvae with pharmaceuticals inhibiting pigmentation for recording high resolution images of neurons in their in vivo environment. The quality of images recorded with this method is comparable to images from explanted brains, yet under natural conditions.

Furthermore, a loss in intensity of fluorescence is avoided, because there is no need for treatment with fixatives

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We especially thank Timo Fritsch for excellent animal care and Hermann Döring, Mohamed Elsaey, Sol Pose-Méndez, Jakob von Trotha, Komali Valishetti and Barbara Winter for their helpful support. We are also grateful to all the other members of the Köster lab for their feedback. The project was funded in part by the German Research Foundation (DFG, KO1949/7-2) project 241961032 (to RWK) and the Bundesministerium für Bildung und Forschung (BMBF; Era-Net NEURON II CIPRESS project 01EW1520 to JCM) is acknowledged.


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Name Company Catalog Number Comments
Calcium chloride Roth A119.1
Confocal Laser scanning microscope Leica TCS SP8
d-Glucose Sigma G8270-1KG
d-Tubocurare Sigma-Aldrich T2379-100MG
Glass Capillary type 1 WPI 1B150F-4
Glass Capillary type 2 Harvard Apparatus GC100F-10
Glass Coverslip deltalab D102424
HEPES Roth 9105.4
Hoechst 33342 Invitrogen (Thermo Fischer) H3570
Imaging chamber Ibidi 81156
Potassium chloride Normapur 26764298
LM-Agarose Condalab 8050.55
Magnesium chloride (Hexahydrate) Roth A537.4
Microscope Camera Leica DFC9000 GTC
Needle-Puller type 1 NARISHIGE Model PC-10
Needle-Puller type 2 Sutter Instruments Model P-2000
Pasteur-Pipettes 3ml A.Hartenstein 20170718
Sodium chloride Roth P029.2
Sodium hydroxide Normapur 28244262
Tricain Sigma-Aldrich E10521-50G
Waterbath Phoenix Instrument WB-12
35 mm petri dish Sarstedt 833900
90 mm petri dish Sarstedt 821473001

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