एक व्यवहार के लिए हल्की तीव्रता में परिवर्तन के लिए Zebrafish की जवाबदेही उपाय परख
Summary
हम दृश्य – मोटर रिस्पांस विकसित करने के लिए प्रकाश वेतन वृद्धि और decrements के जवाब में लार्वा zebrafish की मोटर उत्पादन quantitate. हम भी कोई optokinetic प्रतिक्रिया म्यूटेंट (NRC), जो पूरी तरह से अंधा होने लगा रहे थे जब एक और दृष्टि परख, optokinetic पलटा के द्वारा परीक्षण सहित zebrafish दृष्टि म्यूटेंट, जांच की.
Abstract
The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.
Protocol
Discussion
प्रयोगात्मक प्रक्रियाओं हम फिल्म में दिखाने WT मछली के सभी प्रतिनिधि हैं. हालांकि, इन प्रयोगों के उत्परिवर्ती मछली के रूप में अच्छी तरह से (प्रतिनिधि परिणाम अनुभाग देखें) पर हो analogously किया जा सकता है. हम प्लेट WT और ?…
Acknowledgements
यह काम स्वास्थ्य EY0081 और 5T32UY07145 अनुदान के राष्ट्रीय संस्थान द्वारा और शूरवीरों टमप्लर नेत्र फाउंडेशन द्वारा समर्थित किया गया. जेसन Rihel ब्रिस्टल Squibb लाइफ साइंसेज रिसर्च फाउंडेशन के एक साथी है.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Microplate devices | Tool | Whatman | 7701-1651 | |
| Transfer pipetes | Tool | VWR | 202205 | |
| Fish water | Reagent | refer to reference #4 | ||
| Recording chambers (Zebrabox) | Tool | Viewpoint Lifesciences | ||
| Videotrack Software | Tool | Viewpoint Lifesciences |
Referências
- Allwardt, A. B., Lall, B. A., Brockerhoff, S. E. Synapse formation is arrested in retinal photoreceptors of the zebrafish nrc mutant. J Neurosci. 21, 2330-2330 (2001).
- Emran, F., Rihel, J., Adolph, A. R. OFF ganglion cells cannot drive the optokinetic reflex in zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 104, 19126-19126 (2007).
- Prober, D. A., Rihel, J., Onah, A. A. Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J Neurosci. 26, 13400-13400 (2006).
- Westerfield, M. . The zebrafish book: a guide for the laboratory use of zebrafish. , (2000).
