Adult zebrafish (Danio rerio) were maintained and bred under standard conditions at 26.5°C. Embryos and larvae of a double-transgenic line (elavl3:GCaMP5 x vglut:DsRed)45 (link),46 (link) in nacre background were raised at 28.5°C in standard E3 medium47 .
Imaging experiments were performed as described previously48 (link),49 (link). In brief, larvae 4 - 5 days post fertilization were contained in a small drop of aerated E3 without methylene blue or N-phenylthiourea. Larvae were then paralyzed by addition of 20 μl of fresh mivacurium chloride (Mivacron, GlaxoSmithKline, Munich, Germany)50 (link) and embedded in 2% low-melting agarose (type VII; Sigma, St Louis, MO, USA) in a perfusion chamber that was inclined by 30° to improve dorsal optical access to the OBs. Agarose covering the noses was carefully removed. A constant stream of E3 (2 ml/min) was delivered through a tube in front of the nose and removed by continuous suction. Throughout the experiment it was ensured that larvae showed normal heartbeat. Larvae that were not fixed for EM recovered from paralysis after a few hours and continued to develop without obvious defects. All animal procedures were performed in accordance with official animal care guidelines and approved by the Veterinary Department of the Canton of Basel-Stadt (Switzerland). The sex of zebrafish larvae is not yet determined at the age used in this study.
Imaging experiments were performed as described previously48 (link),49 (link). In brief, larvae 4 - 5 days post fertilization were contained in a small drop of aerated E3 without methylene blue or N-phenylthiourea. Larvae were then paralyzed by addition of 20 μl of fresh mivacurium chloride (Mivacron, GlaxoSmithKline, Munich, Germany)50 (link) and embedded in 2% low-melting agarose (type VII; Sigma, St Louis, MO, USA) in a perfusion chamber that was inclined by 30° to improve dorsal optical access to the OBs. Agarose covering the noses was carefully removed. A constant stream of E3 (2 ml/min) was delivered through a tube in front of the nose and removed by continuous suction. Throughout the experiment it was ensured that larvae showed normal heartbeat. Larvae that were not fixed for EM recovered from paralysis after a few hours and continued to develop without obvious defects. All animal procedures were performed in accordance with official animal care guidelines and approved by the Veterinary Department of the Canton of Basel-Stadt (Switzerland). The sex of zebrafish larvae is not yet determined at the age used in this study.
