Figure 8. Build-up AMPAR-mediated EPSCs can be evoked by ChR2 or electrical evoked transmitter release and may be due to somatic synapses.

(A) Slow build-up EPSC evoked by presynaptic electrical stimulation in UBC. The build-up of inward current is sufficient to cause a burst of spikes beginning at the 5th stimulus. No 4-AP in bath. These currents are distinct from the usual slow EPSC seen in ON UBCs that occurs at the stimulus offset. (B) Another UBC with a similar slow EPSC evoked by 50 Hz train of presynaptic electrical stimulation. The current was blocked by AMPAR antagonist GYKI53655 (50 μM), revealing a small IPSC presumably mediated by mGluR2 known to be present in UBCs. (C) Slow EPSC evoked by ChR2 stimulation of primary vestibular afferent. This UBC was filled with biocytin and recovered post hoc. Super-resolution imaging revealed the mossy fiber made contact with the soma of the UBC and not the brush. (D) IPSCs evoked by ChR2 stimulation of primary vestibular afferent. The fast transient IPSC was blocked by strychnine (0.5 μM) and SR-95531 (5 μM) and the delayed slower IPSC was blocked by LY341495 (1 μM). Biocytin cell fill revealed that this UBC received input to the soma and to the shaft of its dendritic brush. This mGluR2 mediated IPSC was seen in 4 UBCs that received primary afferent input. The slow IPSC amplitude for primary afferent receiving UBCs was 5.43 ± 2.89 pA (mean ± SD), n = 4. (E) This UBC had a response that appeared to be a combination of dendritic and somatic synapses due to the fast EPSC at stimulus onset and buildup of inward current during the train, respectively. Below- Recovery of the cell and the input mossy fiber revealed that this was indeed possible, because the primary vestibular afferent made contact with both the soma and the dendritic brush. This is the cell shown in Figure 7A. Right- Boxed region expanded- Shank1 antibody staining (white) was seen on the periphery of the soma directly opposed to the primary afferent. (F) Slow EPSC evoked by 50 Hz train of either low intensity electrical synaptic stimulation (0.4 V, black) or ChR2 stimulation (blue). The similarity between responses suggests that the same input is being stimulated and the transmitter release is comparable between stimulation methods. Below- the same UBC with two levels of electrical synaptic stimulation. The observation that a stronger electrical stimulation evokes a typical ON UBC response suggests that this cell may have received multiple inputs.