To distinguish whether directional selectivity

in the presence of inhibitory blockers arose pre- or postsynaptically, the properties of ganglion cell light-evoked synaptic inputs were analyzed using whole-cell voltage-clamp techniques. In these experiments, after measuring spikes in cell-attached mode in the presence of blockers (Figure 6A), the same cell was patched with an electrode containing intracellular solution. After learn more break-in, the DSGC was dialyzed with QX314 and Cs+ and repeatedly injected with brief depolarizing pulses (−60–0 mV) until Na+ currents and a large fraction of voltage-gated K+ currents were blocked. Under these conditions, moving spots elicited large inward currents in both the null and preferred directions (VHOLD = −60 mV; Figure 6B). When the cell was held ∼0 mV, the inhibitory inputs that are usually associated with stimulating these cells (Figures 3A and S3) were not apparent, confirming that they were effectively blocked with the cocktail of antagonists (also see Figure S4). At +40 mV, light evoked outward currents. Importantly, the

temporal characteristics of currents measured at −60 and +40 mV were similar (Figure S5), indicating that they were not contaminated by voltage-dependent conductances, and thus provided a reliable readout of bipolar cell output. Reversal of the excitatory currents also indicated that gap junctions did not significantly contribute to the synaptic responses (Ackert et al., Akt inhibitor 2009). Under conditions in which inhibitory receptors and active postsynaptic conductances were blocked, preferred and null-direction stimuli evoked excitatory currents that were similar in size. The amplitude of the peak currents was not significantly different

whether measured at −60 mV (preferred: −228 ± 30 pA and −136 ± new 24 pA, for ON and OFF, respectively; null: −206 ± 30 pA and −131 ± 18 pA for ON and OFF, respectively; p > 0.6; n = 6) or +40 mV (preferred: 353 ± 64 pA and 214 ± 44 pA, for ON and OFF, respectively; null: 373 ± 67 pA and 216 ± 40 pA for ON and OFF, respectively; p > 0.6; n = 6; Figure 6C). Similarly, the total charge of the response was similar in magnitude in the null and preferred directions, indicating that moving spots stimulated an equal number of inputs in both directions (−60 mV ON, −113 ± 18 nC for preferred compared to −126 ± 21 nC for null; −60 mV OFF, −54 ± 13 nC for preferred compared to −66 ± 16 nC for null; +40 mV ON, 227 ± 52 nC for preferred compared to 265 ± 54 nC for null; +40 mV OFF, 124 ± 26 nC for preferred compared to 141 ± 29 nC for null; p > 0.5; n = 6; Figure 6D). The symmetry in input strength contrasts with the asymmetry in the spiking responses and suggests that nonlinearities within the ganglion cell must contribute to direction discrimination.