We described a procedure for the fabrication of optoelectronic probes (optrodes), tools that combine the advantages of optogenetics and silicon probes, enabling both fine scale stimulation and large-scale recording of neurons in behaving animals. A key advantage of these devices is the enhanced spatial precision of stimulation that is achieved by delivering light close to the recording sites of the probe. Additional cell-type specificity is achieved through genetic targeting of the light-activated current sources. Our experimental findings illustrate these capabilities.
Microstimulation is an important tool for investigating the contribution of small groups of neurons to the network patterns (Salzman et al., 1990
; Cohen and Newsome, 2004
; Butovas et al., 2006
; Butovas and Schwarz, 2003
; Seidemann et al., 2002
). For this purpose, electrical stimulation has some limitations. First, it generates local electrical artifacts that are typically larger than the extracellular spike signals, requiring complex methods to extract the neuronal waveforms (Olsson et al., 2005
). Second, it activates neurons in a highly synchronous manner, preventing the reliable isolation of individual neuron by clustering methods for large-scale recordings. The induced synchronous discharge is also more effective at synaptically activating other neurons (Csicsvari et al., 1998
; Fujisawa et al., 2007), 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 ability to perform stimulations of high spatial resolution. 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 and Wu, 1995
), ChR2 expression can potentially be high enough in axons for them to be directly excited by light stimuli (Petreanu et al., 2007
). 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 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 non-synchronized discharges they generate compared to short pulses. In our experiments, the chance of indirect synaptic activation was low because of the sparse recurrent collateral between CA1 principal neurons (Amaral and 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, since the somata have higher low-pass filtering properties than axons, the impact of light-induced potentials should be relatively decreased in somata when using fast frequency stimuli, but not for slow frequency stimuli.
Silencing of neuronal populations is particularly advantageous for the dissection of network components. For the identification of neuron types, light suppression of halorhodopsin-expressing neurons (Han and Boyden, 2007
; Zhang et al., 2007
) should be the preferred method since 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. Although synaptically-mediated disinhibitory effects of pyramidal cells and other putative interneurons were evident in several experiments, suppression of activity for the entire duration of stimulation is expected to occur only in halorhodopsin-expressing neurons.