Figure 3.

Two-photon calcium imaging of Hb9 interneurons during fictive locomotion. A, Experimental setup. AM-ester calcium-sensitive fluorescent dyes were pressure injected into the ventromedial region of an in vitro spinal cord from a neonatal Hb9–GFP/lacZ mouse. Suction recording electrodes were used to record extracellular electrical signals from ipsilateral and contralateral ventral roots (e.g., iL1 or iL2 and cL1 or cL2). Fictive locomotion was evoked by either bath application of NMDA, 5-HT, and DA, or electrical stimulation of the caudal tip of the cord. B, Two-photon projection images show cells near the injection sites that were indiscriminately loaded with Fluo-3 AM and several cells that were expressing GFP. The GFP-labeled interneurons were subsequently identified to be either Hb9 INs or ectopically labeled by the post hoc X-gal stain. C, Typical fluorescent traces show calcium transients in individual GFP-labeled interneurons during fictive locomotion. Electrical stimulation of the caudal tip of the cord elicited fictive locomotor episodes lasting ∼30 s. Extracellular recordings of ventral roots and their corresponding low-pass-filtered traces showed the signature left–right alternation of fictive locomotion. Hb9 INs, cells 1–3, had calcium peaks when ipsilateral ventral root was active (shaded gray), whereas the ectopically labeled cell, cell 4, did not have calcium peaks. Onsets of calcium peaks are indicated (triangles; black if in phase with ipsilateral root activity, red if out of phase). For the same set of cells, calcium transients were also imaged during fictive locomotion evoked by 9 μm NMDA, 12 μm 5-HT, and 18 μm DA. In chemically induced fictive locomotion, Hb9 INs fired in phase with ipsilateral ventral root activity, whereas ectopically labeled cell fired tonically in both ipsilateral and contralateral phases of fictive locomotion.