Abstract
Visualization and tracking of the facial whiskers is critical to many studies of rodent behavior. High-speed videography is the most robust methodology for characterizing whisker kinematics, but whisker visualization is challenging due to the low contrast of the whisker against its background. Recently, we showed that fluorescent dye(s) can be applied to enhance visualization and tracking of whisker(s) (Rigosa et al., 2017), and this protocol provides additional details on the technique.
Keywords: Whisker, Tracking, Fluorescence, Dye, Tactile perception, Barrel cortex
Background
Over the last 10 years, neuroscientists have begun to focus on sensorimotor processing in behaving rodents. A critical step is to be able to quantify the signals that enter the nervous system from the outside world. For whisker-mediated touch, visualization and tracking of the facial whiskers are required in order to characterize the sensory input. Though many approaches have been employed, only high-speed videography has proven adequate for measuring whisker motion and deformation during interaction with an object. However, whisker visualization and tracking is challenging for multiple reasons, primary among them the low contrast of the whisker against its background. This protocol details a technique for increasing contrast by rendering whiskers fluorescent.
Materials and Reagents
Equipment
Procedure
Notes
The whisker staining (protocol ‘Dye application’) is executed on the sedated animal (protocol ‘Animal sedation’). The sedation should last between 2.5/3 h and between two sedation there should be a resting period of at least 24 h, and in any case no more than twice a week. The dye is subject to photo-bleaching. Though in Rigosa et al. (2017) a time constant of about 14 h was reported (i.e., ca. 25,000 trials of 2 sec each), this result depends on the light density and the amount of dye that was absorbed by the whisker sample. If necessary, the investigator can re-stain the same whisker, as this would not alter its mechanical properties. If staining multiple whiskers, the investigator can obtain the same result by staining one whisker at a time across different sessions.
Acknowledgments
The authors declare no competing financial interests. We acknowledge the financial support of the Human Frontier Science Program (http://www.Hfsp.org; project RG0015/2013), the European Research Council Advanced grants CONCEPT (http://erc.europa.eu; project 294498) and MicroMotility (project 340685), and Italian MIUR grant HANDBOT (http://hubmiur.pubblica.istruzione.it/web/ricerca/home; project GA 280778). GN gratefully acknowledges support by SISSA through the excellence program NOFYSAS 2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
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