, 2012). At higher stimulation frequencies the response became increasingly sinusoidal and decreased in amplitude. There has long been evidence that ChR-2-infected neurons have difficulty following stimulation patterns at >40 Hz kinase inhibitors of signaling pathways (Yizhar et al., 2011). A decrease in LFP response amplitude might therefore be assumed at frequencies

>40 Hz as a result of less reliable spike generation: fewer neurons are following the stimulus and generating action potentials, so the signal conducted to the hippocampus – manifested in the hippocampal post-synaptic LFP – is reduced. However, the stimulation frequencies we explored are within this experimentally determined acceptable window. We hypothesize instead that the pattern of decreasing amplitude with increasing stimulation frequency is instead a consequence of the photocycle of ChR2. ChR2 is believed to possess a four-stage photocycle consisting of two open states with different ion conductances, and two closed states (Berndt et al., 2010). The first open state, which is triggered by sudden light intensity changes, results in the non-specific conduction of several ionic

species. The second open state, which occurs with prolonged illumination, follows the first open state and is associated with a decrease in the total conductance, in part due to increased selectivity for H+ ions, as well as the accumulation of channels in non-conducting states. The waveform response properties we observed may then be a result of similar accumulation of ChR2 channels in these non-conducting states, whereas low-frequency stimulation is able to more maximally activate a recycled and conductive population

of light-sensitive ion channels. This hypothesis also provides an explanation for the observation that longer pulse widths tended to alter the time-to-peak responses with different intensities. With short pulse widths the primary conductive mediator would be the first, fast open state. With longer pulse widths Carfilzomib the second, slower conducting open state could come into play, delaying the time-to-peak with a later contribution to the response waveform. Computer modeling of these dynamics could provide more quantitative hypotheses that would better reveal the influence of stimulation parameters on these responses, as well as greater insight into the ChR2 channel. The large influence of stimulation parameters on the response waveform in these characterization experiments suggest that care must be taken in experimental design. Intensity will influence the volume of neural tissue activated, as has been modeled (Adamantidis et al., 2007), but the frequency and pulse width of the stimulation may also influence its impact.