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. 2011 Dec 4;15(1):163-70.
doi: 10.1038/nn.2992.

Optetrode: A Multichannel Readout for Optogenetic Control in Freely Moving Mice

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Free PMC article

Optetrode: A Multichannel Readout for Optogenetic Control in Freely Moving Mice

Polina Anikeeva et al. Nat Neurosci. .
Free PMC article

Abstract

Recent advances in optogenetics have improved the precision with which defined circuit elements can be controlled optically in freely moving mammals; in particular, recombinase-dependent opsin viruses, used with a growing pool of transgenic mice expressing recombinases, allow manipulation of specific cell types. However, although optogenetic control has allowed neural circuits to be manipulated in increasingly powerful ways, combining optogenetic stimulation with simultaneous multichannel electrophysiological readout of isolated units in freely moving mice remains a challenge. We designed and validated the optetrode, a device that allows for colocalized multi-tetrode electrophysiological recording and optical stimulation in freely moving mice. Optetrode manufacture employs a unique optical fiber-centric coaxial design approach that yields a lightweight (2 g), compact and robust device that is suitable for behaving mice. This low-cost device is easy to construct (2.5 h to build without specialized equipment). We found that the drive design produced stable high-quality recordings and continued to do so for at least 6 weeks following implantation. We validated the optetrode by quantifying, for the first time, the response of cells in the medial prefrontal cortex to local optical excitation and inhibition, probing multiple different genetically defined classes of cells in the mouse during open field exploration.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Optetrode design. (a) Vertical cross section of the optetrode. The body of the device consists of a plastic housing and a thumbscrew, held tightly in place with two friction-fit plastic pins, mechanically driving a vented screw into which a protective tube containing the four tetrodes and the multimode fiber is glued. The head of the screw is epoxied to both the metal ferrule optical connector end of the fiber and an electronic interface board, which connects the tetrode microwires to an 18-pin electrical connector. Inset, horizontal cross section of the optical fiber with four tetrode bundles affixed. (b) Vertical cross section of the mechanical drive perpendicular to the cross section in a. The bottom half of the vented screw is thinned such that it tightly fits the tunnel of the plastic housing in only one possible orientation and thus does not rotate during vertical motion. (c) Three-dimensional view of the mechanical drive used in the optetrode. As the interference pins prevent the vertical translation of the thumbscrew, turning in the counterclockwise direction results in a downward motion of the vented screw (red arrows). (d) Shown is a 10-week-old wild-type male mouse 2 weeks after the optetrode implantation and virus injection (mouse was 8 weeks old at the time of surgery). (e) Action potential amplitudes on two channels of a tetrode bundle demonstrate five amplitude clusters (color coded). Average action potential waveforms from the maximum amplitude channel are shown next to each cluster. Unclustered spikes are not shown.
Figure 2
Figure 2
Optetrode-facilitated electrophysiology during broad optogenetic stimulation during the OFT. (ae) Wild-type (WT) mice were transduced with AAV5-hSyn::eNpHR3.0-EYFP. Average MUA was binned at a rate of 1 Hz (a). Shaded area represents s.e.m. (n = 30 tetrode recording sites, 14 OFTs). The L-ratio (left) and isolation distance (right) were plotted for the clusters without and during light stimulation (n = 23 clusters, b). Raster plots (top) and corresponding normalized firing rate profiles (bottom) for are shown for a neuron robustly inhibited by green light (c), a neuron initially inhibited by green light, but for which activity recovered over the duration of the stimulation epoch (d), and a neuron excited by green light (e). (fk) Wild-type mice were transduced with AAV5-hSyn::ChR2-EYFP. Average MUA was binned at 1 Hz or 10 Hz (n = 40 tetrode recording sites, 10 OFTs; f, g). Shaded area represents s.e.m. The L-ratio (top) and isolation distance (bottom) were plotted for the clusters without and during light stimulation (n = 21 clusters, h). Examples of a neuron that maintained high cluster quality and coherence with light pulses during 5-Hz (top) and 20-Hz (bottom) stimulation are shown (i). Raster plots (top) showing the spike times of the example neuron relative to the onset of each light pulse during 5-Hz and 20-Hz stimulation are shown in j and k, as well as the corresponding pulse-triggered average firing rates (bottom). Only clusters classified as being well-isolated without light stimulation (see Online Methods) were plotted in be and hk. Horizontal dashed lines represent the cut-off for well-isolated clusters.
Figure 3
Figure 3
Optetrode-facilitated electrophysiology during cell type–specific optogenetic stimulation in the context of the OFT. (af) Wild-type mice were transduced with AAV5-CaMKIIα::ChR2-EYFP. Average MUA was binned at a rate of 1 Hz (a) and 10 Hz (b). Shaded areas represent s.e.m. (n = 84 tetrode recording sites, 33 OFTs). The L-ratio (top) and isolation distance (bottom) were plotted for clusters without and during light stimulation (n = 14 clusters, c). An example neuron is shown that maintained high cluster quality and coherence with light pulses during 5-Hz (left) and 20-Hz (right) optical stimulation (d). Raster plots of spike times (left) and pulse-triggered average firing rates (right) are shown for an example neuron relative to the onset of each light pulse during 5-Hz (e) and 20-Hz (f) stimulation. (gk) PV::Cre transgenic mice were transduced with AAV5–DIO-EF1α::ChR2-EYFP. Average MUA was binned at 1 Hz (g). Shaded areas represent s.e.m. (n = 45 tetrode recording sites, 29 OFTs). The L-ratio (left) and isolation distance (right) were plotted without and during light stimulation (n = 50 clusters, h). An example neuron is shown that maintained high cluster quality and decreased firing rate in response to 5-Hz (left) and 20-Hz (right) stimulation of parvalbumin-expressing cells (i). Raster plots of spike times (left) and pulse-triggered average firing rates (right) are shown for the example neuron relative to the onset of each light pulse during 5-Hz (j) and 20-Hz (k) stimulation. Only clusters classified as well-isolated without light stimulation (see Online Methods) were plotted in cf and hk. Horizontal dashed lines represent cut-off for well-isolated clusters.
Figure 4
Figure 4
Behavioral and neural activity effects of optogenetic stimulation during the OFT. Mice were subjected to a 22-min OFT and were exposed to 30-s light stimulation epochs every 2 min. We used 5 Hz, 20 Hz, 130 Hz with 5-ms pulse width and 130 Hz with 2-ms pulse width for stimulation parameters. Each stimulation type was used exactly twice and the order was randomized. (a) Velocity of CaMKIIα::ChR2-EYFP (n = 84 tetrode recording sites across 33 OFTs in 2 mice) and CaMKIIα::EYFP mice (n = 119 tetrode recording sites across 43 OFTs in 3 mice) in mPFC during and after each stimulation epoch. *P < 0.05, ***P < 0.001. (b) Example OFT traces showing the path of the mice during the 30 s immediately before stimulation (top) and then during 30 s of 20-Hz stimulation (bottom). (c, d) Data are presented as in a for normalized average multi-unit firing rate (c) and the percent time spent in the center of the open field (d). All error bars indicate s.e.m.
Figure 5
Figure 5
Optetrode projection targeting: driving axonal inputs from BLA into mPFC. (a) Schematic showing BLA-to-mPFC projection targeting. (b) Spontaneous activity of a PrL neuron is shown over a 1-s time window. (c) Average action potential shape is shown for the recording in b. Shaded area indicates the s.e.m. from the mean. (d) Autocorrelation analysis of the firing rate of the unit in b revealed no clear periodicity of the firing pattern on the scale of ≤1 s. a.u., arbitrary units. (e) Light stimulation of the BLA-to-mPFC projection evoked action potentials of the unit in b and also a complex multi-unit response (data not shown). (f) Average action potential shape is shown for the recording in e. (g) Autocorrelation analysis revealed that the unit fired with periodicity of 10 Hz coherent with applied optical stimulation during freely moving open field exploration. Laser light was employed at λ = 473 nm, 5-ms pulse width, 10-Hz rate and power density of 80 mW mm−2 at the tip of the implanted fiber (6 mW mm−2 at 0.5 mm and 1 mW mm−2 at 1 mm below the fiber, at the tetrode tips).

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