Fig. 2 | Scientific Reports

Fig. 2

From: Electrophysiologically calibrated optogenetic stimulation of dentate granule cells mitigates dendritic spine loss in denervated organotypic entorhino-hippocampal slice cultures

Fig. 2

Optogenetic activation of ChR2-expressing dentate GCs in OTCs depends on light intensity and cellular ChR2-expression levels. (a) Patch-clamp recordings of single ChR2-tdTomato expressing GCs were performed while cells were stimulated with either blue (450 nm) or green (505 nm) light. Photostimulation was carried out with high power LED modules consisting of 3 LEDs mounted onto a custom-made fixture and covered with a collimator. (b, c) Current-clamp recordings were performed while GCs were stimulated with 450 nm or 505 nm light pulses (10 ms) at different intensities. Sample traces of stimulations with 505 nm LEDs: While some cells frequently exhibited subthreshold depolarization in response to light pulses at ~ 0.5 mW/mm² LED intensity (b), others reliably fired APs in response to light pulses at all levels of intensity (c). (d, e) Confocal image stacks of patched GCs filled with Alexa 488 visualize ChR2-tdTomato expression of the recorded neurons. (f) Analysis of the minimal light intensity required to prompt spiking revealed a proportion of neurons activated by relatively low light intensities (i.e. <2 mW/mm2; n = 14) while other neurons needed higher light intensities to spike (n = 8) or did not spike (open circles; n = 5). (g) An examination of ChR2 expression in individual GCs showed that cells responding to low light intensities exhibited a relatively high ChR2-related fluorescence density (gray line depicts mean: 19,906 A.U.; n = 11) compared with GCs that required higher light intensities or did not respond at all (mean: 5,483 A.U.; n = 5). (h) The majority of GCs that were stimulated with 505 nm LEDs fired APs in response to light intensities below 2 mW/mm2 (n = 6) while one neuron spiked at ~ 4.0 mW/mm2. Three GCs showed no response. (i) A ChR2 expression analysis showed that GCs that fired APs following stimulation with a low light intensity had higher expression levels (mean: 13,025 A.U.; n = 4) compared with the neuron that only responded to high light intensity (9,472 A.U.; n = 1). A.U.: arbitrary units. n represents number of cells.

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