If the amplitude had a negative value with respect to the baseline, that site was added to the abduction map. In the case of bidirectional movement profiles where both the positive and negative components satisfied the amplitude criteria, the corresponding site was included in both the abduction and adduction maps and counted as overlap between maps. For each map, the center of gravity was calculated along with the mean amplitude and latency for the nine pixels closest to the center point. Maps with mean amplitude of <0.1 mm at the center were excluded from further analysis.

Separation between Mab and Mad was defined as the distance between the centers of gravity for each map. After completing two to five INCB024360 research buy motor maps, mice were raised into a sitting posture with their forelimbs hanging freely. Stimulus sites were placed as close to the centers of the abduction and adduction representations as possible without targeting major blood vessels, since these absorb light strongly (Ayling et al., 2009).

Fifty-one frames were captured at a rate of 100 Hz beginning 10 ms prior to laser stimulus onset, and paw trajectories were generated from the raw image sequences using the plugin “MTrack2” for ImageJ. Ten to 20 repetitions were then averaged for each trial, and speed and angle profiles were calculated based on this average trajectory. ChR2 transgenic mice were implanted with optical fibers (Thorlabs Apoptosis Compound Library datasheet BFH48-200) extending to the cortical surface and terminating in a ferrule connector (Precision Fiber Products) fixed to the skull with dental acrylic and bone screws. Two fibers were implanted, targeted to the mean coordinates of the Mab and Mad map centers. These locations were stimulated alternately (5 mW 5 ms pulses at 100 Hz for 500 ms) using a 473 nm laser (IKECOOL IKE-473-100-OP) connected via an optical commutator (Doric). Stimulus evoked behavior was recorded by a CCD camera

(Dalsa 1M60) and frame grabber (EPIX). Limb trajectories were analyzed in the same manner as the anesthetized data, except that paw position was tracked using the plugin “Manual Tracking” for ImageJ. Glass pipets (tip width 10–20 μm) containing a 0.25 mm bare silver wire were filled with 1% fast green PDK4 in 3 M sodium chloride. A micromanipulator (Sutter) was used to advance the pipet to a depth of 700 μm. Stimulation sites were matched with those targeted by laser stimulation in the same animals. Trains of 200 μs 100 μA pulses at 200 Hz with 10–500 ms durations were generated by an AM systems stimulator and a WPI stimulus isolator. For motor mapping experiments involving virally transduced mice, 1–2 μl of adeno-associated virus (serotype 2/1 CAG-ChR2-GFP) was injected through a burr hole into the sensorimotor cortex of ChR2-negative mice 2 mm lateral of bregma at a depth of 500 μm using a 5 μl Hamilton syringe with a 33 gauge needle and a syringe pump (WPI). Mice recovered for 2–4 weeks before being used in experiments.