Optical stimulation of the olfactory bulb

Optical stimulation of the OB has been described in detail in³⁶. In brief, M/T cell spike activity was recorded extracellularly in anesthetized mice while optogenetically activating the surrounding glomeruli in the exposed dorsal surface of the OB. Precise spatial control of optical stimulation was achieved by projecting two-dimensional light patterns onto the dorsal surface of the OB using digital micromirror technology (duration 100 ms) coupled to an optical imaging system. This resulted in an image where each projected pixel corresponded to ~11 µm. Optical stimulation was controlled with the MATLAB psychophysical toolbox.

To avoid possible bias due to respiration phase^37,38, optical stimulation was randomly applied with respect to respiration cycles, with typically 20 repetitions of each spot stimulation. On average 98.34 ± 52.99 spots were stimulated for each recorded neuron.

The light intensity was set to obtain excitatory responses³⁹ and ranged from 10–20 mW/mm² as measured by a photometer (Thorlab PM100D). The expression of Chr2 in the OMP-Chr2 mice varies which result in different light power required to elicit a clear response near the recording electrode. Spot sizes ranged from 55 to150 µm (111 ± 27.3 µm, mean ± s.d.). We scanned the full extent of the exposed dorsal OB divided into an N by M non-overlapping spots. N and M were determined by the size of the craniotomy. The result did not change when we only used experiments with the same spot sizes (e.g. 110 µm).

Finally, due to the spatial organization of the axons of passage (i.e., from rostral to caudal)⁴⁰ activation of axons of passage is more common when stimulating the anterior parts of the OB. To avoid this, in a few neurons in which there was a clear response from the anterior OB parts we tended to focus our light stimulation on the more posterior parts of the OB and ignored the anterior parts.