Photophobia

The LDB test allow to monitor photophobic behavior which is common behavior seen in migraine patients during attacks (43 (link)). Rats were individually tested in the LDB test on day 4 post-surgery. Rats in their cages were placed in the testing room around 9:30 a.m. for acclimatization, and testing were held between 10 and 12 a.m.
The setup consisted of two plexiglas boxes of identical size (50 × 50 cm and 34 cm in height). The light box had no lid and a light intensity of ~1,000 lux. This specific light intensity was chosen to stimulate a lighter environment, that would discourage rats that experience photophobia, while not discouraging healthy rats to explore. The second box consisted of darker walls and a top to make it pitch-dark, with plexiglass permeable to infra-red (IR) light, making video-recordings possible. A darker environment is believed to be more suitable for rats experiencing photophobia. There was an opening of 10 × 10 cm between the two boxes to facilitate transition between the boxes for the rats.
Rats were taken individually out of their cages and placed in the center of the light box with their heads facing away from the dark box. Rats were recorded and tracked using the ANY-MAZE (Stoetling, USA) software and an IR lamp to monitor their behavior. Time spent in the light zone (seconds) was chosen as parameter for analysis of photophobic behavior. Distance traveled per minute spent in the dark box was a parameter chosen for the reduced physical activity migraineurs experience during migraine attacks. The individual rats were monitored for 10 min. When testing was completed for each rat, both boxes were cleaned with 20% ethanol to remove scent of previous rats to avoid discrepancies and distractions between tests. The investigator was blinded to the group designation.

For a recorded afferent fiber, its mechanosensitive receptive field in the hindpaw glabrous skin was first searched using a glass rod. Poking with the glass rod at the mechanosensitive receptive field of the recorded afferent fiber would result in the detection of APs by the recording electrode. In the present study, all data were collected from mechanosensitive receptive sites, i.e., mechanoreceptors in the hindpaw glabrous skin. Once a mechanoreceptor was identified, mechanical stimulation was applied to the same receptive field with a force-calibrated mechanical indenter (300C-I, Aurora Scientific Inc., Ontario, Canada) to determine mechanical thresholds. The tip size of the indenter was 0.8 mm in diameter. The indenter was connected to a Digidata 1550B Digitizer to allow generating ramp-and-hold mechanical stimulation using the pClamp 11 software. Prior to the application of mechanical stimulation, the tip of the indenter was lowered to the surface of the receptive field with a 10-mN force and then the 10-mN force was canceled to 0 so that the tip of the indenter was just in contact with the receptive field surface. Under the force control module, ramp-and-hold mechanical stimuli were applied to the mechanoreceptor of the glabrous skin. The step force commanders were calibrated by applying indenter at finger tips, paw pads and other areas of plantar skin, and the actual forces after the calibration were used in experiments. The ramp-and-hold force steps were at 0, 5, 30, and 80 mN. The duration of the ramp (dynamic phase) was 10 ms, and the duration of the holding (static phase) was 0.98 s. The minimal force at which AP impulses was elicited was defined as intender mechanical threshold of the mechanoreceptors. In a different set of experiments mechanical stimulation was applied using von Frey filaments to vertically poke the glabrous skin. The von Frey mechanical thresholds were determined by mechanical stimulation with von Frey filaments (0.08 ~ 6 g) onto mechanoreceptors.
To determine whether a mechanoreceptor was from Nav1.8^ChR2-positive or Nav1.8^ChR2-negative afferent fibers, the same mechanosensitive receptive field was stimulated by a blue LED light (Thorlab; M455L4, 455 nm) to test light sensitivity. A mechanoreceptor was from Nav1.8^ChR2-positive afferent fibers if light stimulation evoked impulses. Otherwise, the mechanoreceptor was from light-insensitive or Nav1.8^ChR2-negative afferent fibers. The blue Light was applied through a 40 × objective to a mechanoreceptor with a 1-s light stimulation pulse at the intensity of 50 mW. Afferent impulses evoked by mechanical and light stimulation were recorded using the pressure-clamped single-fiber recordings, and signals were amplified by the Multiclamp 700B amplifier and sampled at 25 kHz with band path filter between 0.1 Hz and 3 kHz on AC recording mode.