Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2

Abstract

A major long-term goal of systems neuroscience is to identify the different roles of neural subtypes in brain circuit function. The ability to causally manipulate selective cell types is critical to meeting this goal. This protocol describes techniques for optically stimulating specific populations of excitatory neurons and inhibitory interneurons in vivo in combination with electrophysiology. Cell type selectivity is obtained using Cre-dependent expression of the light-activated channel Channelrhodopsin-2. We also describe approaches for minimizing optical interference with simultaneous extracellular and intracellular recording. These optogenetic techniques provide a spatially and temporally precise means of studying neural activity in the intact brain and allow a detailed examination of the effect of evoked activity on the surrounding local neural network. Injection of viral vectors requires 30-45 min, and in vivo electrophysiology with optogenetic stimulation requires 1-4 h.

Figure 1

AAV DIO ChR2-mCherry gives Cre-dependent and cell-type-specific expression of light-activated channels in vivo. (a) The adeno-associated viral vector AAV DIO ChR2- mCherry carries an inverted version of ChR2 fused to the fluorescent marker mCherry^,. This strategy prevents ChR2 from being expressed in the absence of Cre. In the presence of Cre, ChR2-mCherry is inverted into the sense direction and expressed from the EF-1α (EEF1A1) promoter. EF-1α, elongation factor 1α promoter; ITR, inverted terminal repeat; pA, poly(A); WPRE, woodchuck hepatitis B virus post-transcriptional element. (b) ChR2-mCherry (red) is expressed specifically in PV⁺ interneurons (green) 6 d after injection of AAV DIO ChR2-mCherry into the barrel cortex of an adult PV-Cre mouse. Confocal Z-stack of two adjacent 3 μm sections. Boxed area is shown in higher magnification and detail in c. (c) PV⁺ cells with ChR2-mCherry expression and typical interneuron morphology. Confocal Z-stack of four adjacent 3 μm sections. Bar = 100 μm (b). All procedures were conducted in accordance with the National Institutes of Health guidelines and with the approval of the Committee on Animal Care at MIT.

Figure 2

Neural activity evoked in vivo by activation of cell-type-specific expression of ChR2. (a) Schematic of the placement of the unjacketed optical fiber and extracellular electrode array within the craniotomy (dashed line). Both the optical fiber and the electrodes were placed several hundred micrometers from the original virus injection site (asterisk). (b) Individual 1-ms pulses of 473 nm light at 46 mW mm⁻² through the optical fiber reliably evoked a single action potential from a fast-spiking, PV⁺ inhibitory interneuron in the primary somatosensory cortex in this multiunit recording. (c) A 100-ms light pulse at the same power level evoked a sustained period of elevated firing (~220 Hz) from a fast-spiking interneuron in this single-unit recording. (d) ChR2 can also be used in conjunction with intracellular recordings in vivo. The traces show the membrane potential of a regular spiking, putative excitatory neuron in a PV-Cre mouse expressing ChR2 in fast-spiking inhibitory interneurons. Local inhibitory interneurons were repeatedly stimulated with 1-ms light pulses while the regular spiking cell was held at varying membrane potentials. The inhibitory post-synaptic potentials (IPSPs) resulting from light-evoked activation of presynaptic inhibitory interneurons reversed around − 70 mV, indicating a fast, GABA_A-mediated chloride conductance.

Figure 3

Elimination of light-induced artifacts in recordings of the local field potential. (a) Direct exposure of metal electrodes to the laser beam causes large electrical artifacts. These examples are taken from recordings in a cortical site not transduced with AAV-DIO-ChR2-mCherry. A glass pipette with a nonreflective coating was placed in the superficial cortex ~350 μm away from a tungsten electrode. The shaft and exposed tip of the tungsten electrode were directly in the cone of blue light, as shown by the schematic to the right. Under these conditions, pulses of laser light (68 mW mm⁻²; blue trace) caused a large, repeated artifact in the LFP recording from the metal electrode (upper black trace). The artifact started at the onset of each light pulse and lasted at least 8 ms after the end of the light pulse (see inset). Simultaneous recordings from a glass pipette whose shaft was in the light cone under the optical fiber did not show any light-induced artifacts (lower black trace). (b) Similar artifacts were observed when the LFP was recorded from a cortical site containing interneurons transduced with AAV DIO ChR2-mCherry (red dots). In this case, the LFP recorded on the metal electrode shows a gamma oscillation induced by stimulating the fast-spiking interneurons at 40 Hz, but the oscillation signal is obscured by the light-induced artifact caused by the shaft of the metal electrode intersecting the laser beam (upper trace). In contrast, the simultaneous recording from the glass electrode shows the gamma oscillation in the absence of the artifact (middle trace), as highlighted by the filtered glass electrode LFP (lower trace). (c) Artifacts in the metal electrode recordings can be eliminated by changing the angle of the electrode so that the shaft does not intersect the laser beam, as shown in the schematic to the right. In this example, the signal on the angled metal electrode (upper traces) agrees well with the signal on the glass electrode (lower traces).