Ic capture

Video recording: On Day 21, mice were placed in a cage with raised ceiling to capture any jumping behavior. Mice were video recorded for 20 min (ImagingSource; Model DFK22AUC03) after 5 mg/kg Naloxone and recorded using open-source software I.C Capture from The Imaging Source. Recorded videos were analyzed using VLC media player. Mice were assessed for withdrawal expressed as Wet Dog Shakes, Teeth Chattering, Paw Shakes, Jumps, and Diarrhea. The global withdrawal score was calculated using the formula: Global withdrawal score = jumps*0.8 + wet dog shakes*1 + diarrhea*1.5 + paw shakes*0.35 + teeth chattering*1.5 + body tremor*1.5. All analyses of video recordings were conducted with a scorer blinded to treatment conditions. A correlational analyses for jumping behavior during withdrawal was performed between two different scorers.
LMA chambers: Locomotor activity was assessed in soundproof, light-controlled locomotor activity chambers from Omnitech Electronics. On Day 22, mice were placed in the chamber immediately after receiving 5 mg/kg naloxone and their activity was recorded for 15 min. Movement time was automatically measured in the box based on horizontal and vertical light beam breaks.

Images were captured every 6 s with an Imaging Source DMK 23GP031 camera (2592 × 1944 pixels, The Imaging Source, USA) equipped with a Fujinon lens (HF12.5SA-1, 1:1.4/12.5 mm, Fujifilm Corp., Japan) with a Hoya 49mm R72 Infrared Filter. We used IC Capture (The Imaging Source) to acquire time-lapse images through a gigabit Ethernet connection. All experiments were carried out under dark-field illumination using infrared LED strips (Ledlightsworld LTD, 850 nm wavelength) positioned below the LarvaLodge.

Single zebrafish larvae were mounted on microscope slides and imaged on a Nikon SMZ1500 stereomicroscope. Still images of the retinae were taken using a DMK21AF04 camera and captured using IC Capture (The Imaging Source, Germany). In Image J, the zebrafish retina was encircled using the polygon tool and the area calculated.