This experiment was designed to examine the responses of different cell types to photostimulation in the same functional system in an optogenetic model. A Cre-mediated genetic approach was utilized to generate mice in which channelrhodopsin-2 (ChR2), fused with a fluorescent reporter, is selectively expressed by GABAergic neurons. We focused on GABAergic cells in the mouse cerebellum, where such cells are abundant (Han et al., 2014).
Slices (typically sagittal, 200 µm in thickness) were prepared from the cerebellum of ~1 month-old mice of either sex. (Several experiments were also carried out in mice at postnatal day 6.) Whole-cell patch recordings were performed under visual control. In some experiments, neurobiotin or biocytin was included in the internal solution to label the recorded cells for later morphological identification. TTL signals were used to trigger 473 nm blue laser light pulses, which were delivered to the tissue via a 200 μm optical fiber. The tip of the optical fiber was submerged in the recording chamber ACSF at a tip distance of about 1-1.5 mm from the recording site. The power level was ~1 mW/mm2 and the duration of the TTL signals, 1-500 ms, was adjusted based on the responses. The inward currents (pA) or depolarization (mV) elicited by the light pulses were measured in voltage- or current-clamp mode, respectively. Data acquisitions and analysis was carried out using the P Clamp 9. Labeled cells were visualized with the streptavidin-conjugated fluorescent dye for confocal image analysis and cell-type identification.
As expected from it being the principal GABAergic cell-type in the cerebellum, Purkinje cells (PCs) had robust responses to photostimulation. Under current clamp, light pulse as brief as 1 ms consistently evoked > 10 mV of membrane depolarization and generated spikes with 13.15±3.1 mV amplitudes (n=47). Under voltage-clamp, a 1 ms light pulse evoked inward currents ranging 200-350 pA. When the duration of the light pulse was increased to 50-100 ms, the inward current became stable, reaching a maximum of 910±119 pA (n=47). Surprisingly, however, responses of all other GABAergic cell types in the cerebellar cortex were significantly smaller: basket cells 33±16.4 pA (n=8); stellate cells 27±11.4 pA; (n=); Golgi cells 110±34.8 pA (n=4). We also recorded from 18 smaller cells in the deep cerebellar nuclei region, likely local GABAergic interneurons, showing 72 ± 15.6 pA inward currents and 1.5 ± 0.55 mV depolarization in response to photostimulation. Among these cells, 3 responded with spikes in some of the tests.
These results indicate that responses to optical stimulation by all non-principal GABAergic cell types in our Ai27 and Ai32 mouse model were much weaker than that those of PCs. The mechanisms for such differences deserve further investigation.