Optrode recordings can be used for more than validating ChR2 activation. Awake electrophysiology has historically been limited by a lack of reliable methods for identifying specific cell types in extracellular recordings. By combining optogenetics with awake recordings, specific cell types can be identified by testing their responsiveness to light. Once identified, the activity of these subtypes can be tracked to determine their behavioral correlates [44
Several issues need to be addressed when identifying cell types based on ChR2 expression. The first concerns stimulation parameters for identifying ChR2-expressing neurons. It is possible to use brief (<10msec), high powered (>10mW) laser pulses to drive single action potentials in neurons [63
]. However, illuminations like this can cause large populations of ChR2-expressing neurons to fire at once, which can increase synchronous multi-unit activity on a recording electrode, making spike sorting difficult [44
]. This multi-unit activity can also infiltrate the recording of a well isolated unit and spuriously lead a researcher to conclude that the isolated unit expresses ChR2. Finally, high powered laser pulses can cause photoelectric artifacts on recording electrodes [44
]. In the striatum, we have found that longer (1s), low power (~0.1 to 3.0mW) laser pulses are sufficient to drive spiking in ChR2 expressing neurons, while avoiding both large increases in multi-unit activity and photoelectric effects. Useful stimulation parameters are dependent on the recorded brain structure, and different stimulation parameters have been used to mitigate these issues in the hippocampus [44
After determining useful stimulation parameters, identifying ChR2-expressing neurons can still be difficult due to the high interconnectivity among neurons in most brain regions. For example, a non-ChR2 expressing neuron could appear to be light-responsive if it receives excitatory input from a neighboring ChR2-expressing neuron. In brain regions that are interconnected by excitatory synapses exhibiting short-term depression of glutamate release, this issue can be addressed with trains of moderate frequency laser pulses. Due to the rapid kinetics of ChR2, neurons that express ChR2 reliably respond to each light pulse in a train (at rates <40Hz). In contrast, synaptically activated neurons respond unreliably after the first few pulses, presumably as the readily releasable pool of glutamate is depleted [64
This specific confound is less likely in inhibitory structures. For example, the striatum does not contain any excitatory neurons, so increases in spiking that follow the laser pulse cannot be due to local excitatory drive. However, inhibitory structures like the striatum often exhibit lateral inhibition which can overpower light-activated currents in ChR2-expressing neurons. In these cases, a lower laser power can reveal ChR2-mediated responses. For example, some striatal neurons respond most strongly to low power laser illumination, because higher laser powers either depolarize neurons too much (inactivating sodium channels and driving neurons into a depolarization block) or drive more lateral inhibition from other ChR2-expressing cells (). Other striatal neurons respond better to higher laser powers (), likely because they express lower levels of ChR2 or do not receive adequate illumination at lower laser power. In such cases, it can be useful to illuminate with a series of pulses of incrementing laser powers to determine which neurons express ChR2. As a final note, while it is possible to identify ChR2 expressing neurons with these techniques, it is difficult to conclude anything about the cell type of neurons that do not respond to the light. Non-responsive neurons may not express high enough levels of ChR2 to drive spiking, may not be effectively illuminated, or could be synaptically inhibited.
Optogenetic identification of specific cell types in vivo
Theoretically, it should also be possible to identify and track more than one cell type simultaneously. This would involve targeting multiple optogenetic proteins to different cell types, and using the unique characteristics of these proteins to identify each cell type. For example, spectrally-distinct channelrhodopsin variants could be targeted to two cell populations, which can be selectively stimulated with different wavelengths of light [19
]. The unique kinetics of different optogenetic proteins can also be used to identify different cell types. We have expressed ChR2 in one neuron type in the striatum and a step-function variant of ChR2 (ChR2-SFO) in another. Both of these proteins are rapidly activated by blue light, but the ChR2 expressing neurons stop firing when the light turns off, while ChR2-SFO expressing neurons continue firing after the light is shut off (). Using combinations of spectrally and kinetically distinct optogenetic proteins under control of cell-type specific promoters, it may be possible to identify more than two cell types in the same recording.