, 1998; Fujisawa et al., 2008), making the separation of direct and synaptically

mediated effects difficult in recurrent networks. Third, even very low stimulus intensities can recruit distant neurons through direct axonal stimulation (Histed et al., 2009), preventing the possibility of high spatial resolution stimulation. Although the use of the optogenetic tools discussed here can largely eliminate most of these shortcomings, a number of precautions should be taken. First, although the passive structure of axons makes them relatively harder to activate with ChR2 than soma–dendrite regions (Johnston selleckchem & Wu, 1995), ChR2 expression can potentially be high enough in axons for them to be directly excited by light stimuli (Petreanu et al., 2007 andPetreanu et al., 2009). Therefore neurons can still be recruited via antidromic axon stimulation by brief large-amplitude light pulses. Second, brief light pulses also tend to synchronously activate ChR2-expressing neurons, with the associated issues mentioned above. The problem of synchrony-induced spike superimposition can be avoided through the use of low-frequency sine wave stimuli.

The 5-Hz sinusoid stimulation used here, close to the see more natural theta oscillation frequency of the hippocampal networks, eliminated the induction of population spikes and did not alter the spike waveforms. As a result, light-activated pyramidal neurons could be readily identified following spike sorting by routine clustering methods. In addition, the use of sine wave stimuli should lower the chance of indirect synaptic activation of pyramidal cells because of the nonsynchronized discharges they generate compared to short pulses. In our experiments, the chance of indirect synaptic activation was low because of the sparsity of recurrent collaterals between CA1 principal neurons (Amaral & Witter, 1989). Finally, we speculate that slow stimulus

waveforms should further reduce the chances of axonal stimulation at light levels sufficient to activate somata. Indeed, as selleck chemicals the somata have higher low-pass filtering properties than axons, the impact of light-induced potentials should be relatively low in somata when using high-frequency stimuli, but not for low-frequency stimuli. Silencing of neuronal populations is particularly advantageous for the dissection of network components. For the identification of neuron types, light suppression of NpHR-expressing neurons (Han & Boyden, 2007; Zhang et al., 2007b) should be the preferred method as it avoids the synchrony-induced spike superimposition problem and makes the separation of direct and synaptically mediated effects straightforward. Yellow light pulses robustly silenced PV-containing interneurons in our experiments.