One-step optogenetics with multifunctional flexible polymer fibers

Abstract

Optogenetic interrogation of neural pathways relies on delivery of light-sensitive opsins into tissue and subsequent optical illumination and electrical recording from the regions of interest. Despite the recent development of multifunctional neural probes, integration of these modalities in a single biocompatible platform remains a challenge. We developed a device composed of an optical waveguide, six electrodes and two microfluidic channels produced via fiber drawing. Our probes facilitated injections of viral vectors carrying opsin genes while providing collocated neural recording and optical stimulation. The miniature (<200 μm) footprint and modest weight (<0.5 g) of these probes allowed for multiple implantations into the mouse brain, which enabled opto-electrophysiological investigation of projections from the basolateral amygdala to the medial prefrontal cortex and ventral hippocampus during behavioral experiments. Fabricated solely from polymers and polymer composites, these flexible probes minimized tissue response to achieve chronic multimodal interrogation of brain circuits with high fidelity.

Figure 1. Fabrication and characterization of multifunctional fibers

(a) A schematic illustrating fabrication process for graphite-doped conductive polyethylene electrodes (gCPE). (b) Fabrication steps involved in assembly of the preform including a polycarbonate (PC) waveguide core, cyclic olefin copolymer (COC) cladding, gCPE electrodes, hollow channels, and protective sacrificial PC cladding. (c) An illustration of the fiber drawing process. The diameter of the resulting fiber is determined by the ratio of the capstan and feed speeds, and is monitored continuously during the process by a laser sensor. (d) Photograph of a bundle of fiber prior to etching of the sacrificial PC cladding. (e) Cross-sectional photograph of the preform prior to thermal drawing. (f) Cross-sectional microscope image of the multimodal fiber produced by thermal drawing of the preform in (e). (g, h) Impedance spectra of gCPE electrodes within the multimodal fiber. The impedance is reduced by 24 hr soak in phosphate buffered saline (PBS). Bending deformation (90°, 2.5 mm radius of curvature) does not appreciably alter impedance spectra. Number of samples n=6, ***p < 0.001 determined by one-way ANOVA. Flat: p < 0.0001, F_{3, 20} = 27.42, Bent: p < 0.0001, F_{3, 20} = 21.86. All error bars and shaded areas in the figure represent standard deviation. (i) Evaluation of microfluidic channels within multimodal fiber via infusion of a dye (BlueJuice) into a phantom brain (0.6% agarose gel). Images are taken at 0 min, 3 min, and 5 min after initiation of injection at a speed of 100 nl/min. Scale bar = 500 μm. (j) Output speed and return rate measured for microfluidic channel in multifunctional fiber. The microfluidic capability was only slightly reduced during 90° bending deformation. Number of samples n=6. Shaded areas represent standard deviation.

Figure 2. Multifunctional fiber probes enable viral delivery, optical stimulation, and recording with a one-step surgery

(a) A schematic comparing a traditional two-step surgery for optogenetic experiments and a one-step surgery enabled by a multifunctional fiber probe. (b) Picture of a multifunctional fiber probe outfitted with an optical ferrule, electrical connector, and an injection tube. The weight of the device varied between 0.3–0.5 g. Scale bar = 10 mm. (c) A wild-type mouse implanted with a multifunctional probe. (d) An illustration of viral delivery (AAV5-CaMKIIα::ChR2-eYFP), optical stimulation, and electrical recording in medial prefrontal cortex (mPFC) of wild-type mice with a fiber probe. (e) Expression of ChR2-eYFP in the mPFC for a wild-type (WT) mouse 2 weeks after viral transfection. Blue: DAPI, green: eYFP. Scale bar = 1 mm. (f) Electrophysiological recordings during optical stimulation in the mPFC using a fiber probe between 2 days and 21 days following transfection with AAV5-CaMKIIα::ChR2-eYFP. (10 Hz, 8.6 mW/mm², 5 ms pulse width). Optically evoked potentials were observed 11±2 days following implantation and injection surgery (orange box, n=8 mice). (g–j) Velocity recorded for WT mice implanted with fiber probes and injected with AAV5-CaMKIIα::ChR2-eYFP (or control virus AAV5-CaMKIIα::eYFP) in mPFC during open field test (OFT). 9-min experiment consisted of 3-min epochs, OFF/ON/OFF optical stimulation: 5 ms pulse-width, power density 16 mW/mm², and frequency 20 Hz and 130 Hz. (g) 20Hz OFF_pre: p = 0.9247, t = −0.0962, ON: p = 0.0352, t = 2.3309, OFF_post: p = 0.8279, t = −0.2215. (h) 130Hz OFF_pre: p = 0.9819, t = −0.023, ON: p = 0.0417, t = 2.2421, OFF_post: p = 0.8857, t = 0.1464. d.f. = 14 for all. Error bars represent standard deviation (number of animals n=8, *p < 0.05; one-way student’s t-test). (i–j) Representative trajectories for a ChR2 transfected mouse during (i) optical stimulation (5 ms pulse width, 20 Hz) ON and (j) OFF.

Figure 3. Optogenetic projection mapping using multimodal fiber probes

(a) An illustration of the basolateral amygdala (BLA) to mPFC projection. AAV5-CaMKIIα::ChR2-eYFP was delivered to BLA, and the optical stimulation and electrical recording were performed in the mPFC. (b–d) Confocal microscope images of a coronal section containing mPFC 6 weeks after viral transfection in the BLA. (c, d) Higher magnification images of prelymbic (PrL) and intralymbic (IL) areas of mPFC. Scale bars are (b) 500 μm and (c, d) 150 μm, respectively. (e) An illustration of the BLA to ventral hippocampus (vHPC) projection. Here virus was delivered to BLA, and the optical stimulation and electrical recording were performed in the vHPC. (f–h) Confocal microscope images of a coronal section containing vHPC 6 weeks after viral transfection in the BLA. (g, h) Higher magnification images of CA2 and CA3 areas of vHPC. Scale bars are (f) 500 μm and (g, h) 150 μm, respectively. Blue: DAPI, green: eYFP. (i) Electrophysiological recording in the BLA during optical stimulation (10 Hz, 8.6 mW/mm², 5 ms pulse width) using a multifunctional fiber performed between 2 and 21 days following transfection with AAV5-CaMKIIα::ChR2-eYFP. Optically evoked potentials were observed 11±2 days (orange box, n=8 mice) following surgery. (j,k) Electrophysiological recordings during optical stimulation using multifunctional fibers implanted in the mPFC (j) and vHPC (k) between 2 and 21 days after viral transfection using fiber probes implanted in the BLA. Primary low-latency optically evoked potentials were observed following 12±1.4 days (orange box, n=8 mice) for mPFC (j) and following 11±2 days (orange box, n=8 mice) for vHPC (k). (j) For mPFC, secondary long-latency evoked potentials were recorded 15±2 days (green box, n=6 mice) after the surgery. (l–o) OFT experiments performed in mice implanted with multifunctional fiber probes in vHPC and BLA. 9-min OFT consisted of three 3-min epochs, OFF/ON/OFF optical stimulation (5 ms, 16 mW/mm², 20 Hz) in the vHPC. (l, m) Representative heatmap images tracing the position of a mouse transfected with ChR2 during (l) ON and (m) OFF optical stimulation epochs. (n) Time spent in the center of the open field for WT mice transfected in the BLA with ChR2-eYFP or eYFP alone in the absence or presence of optical stimulation in the vHPC. OFF_pre: p = 0.5957, t = 0.543, ON: p = 0.012, t = −2.8833, OFF_post: p = 0.7947, t = −0.2653. d.f. = 14 for all. (o) OFT identical to the one in (l–n) performed following delivery of synaptic blocker CNQX (0.1 mM, 0.5 μl) through a fiber probe implanted in vHPC. OFF_pre: p = 0.328, t = 1.0135, ON: p = 0.2319, t = 1.2497, OFF_post: p = 0.824, t = −0.2266. d.f. = 14 for all. Shaded areas represent standard deviation (Number of animals n=8, *p < 0.05; one way student’s t-test).

Figure 4. Investigation of single-unit potential with recorded signal from multimodal fiber device

(a–d) Tracking of isolated single neuron (unit) action potentials (spikes) recorded with a with multifunctional fiber probe in mPFC over a period of 12 weeks following implantation. (a) Clusters revealed by principle component analysis (PCA) of isolated action potentials. (b) Average spike waveforms recorded between 1 and 12 weeks corresponding to clusters in (a). (c) Interspike interval (ISI) histograms for isolated neurons 1 and 2 from (a,b). Maximum histogram interval = 1000 ms and bin size = 40 ms. (d) Average firing frequencies for neurons 1 and 2 obtained from ISI histograms. Significant difference confirmed by one-way student’s t-test (p < 0.001, t = 18.7798, d.f. = 8). Error bars represent standard deviation (Number of samples n=5). (e–g) Electrophysiological recording of optically evoked potentials in the mPFC of WT mice transfected with AAV5-CaMKIIα::ChR2-eYFP performed (e) 1 month, (f) 2 months, and (g) 3 months after the one-step implantation and transfection surgery. Optical stimulation parameters were fixed at 5 ms pulse width, frequency of 10 Hz and power density 4.3 mW/mm².

Figure 5. Evaluation of fiber probe and steel microwire biocompatibility using immunohistochemistry in coronal slices

(a, b) Representative confocal images of glial scarring and blood brain barrier breach surrounding (a) a 200 μm multifunctional fiber probe and (b) a 125 μm stainless steel microwire 1 month after implantation. Scale bar = 100 μm. (c–f) Average fluorescent intensity quantifying the presence of (c) Iba1, (d) ED1, (e) GFAP, and (f) IgG for fiber probes and microwires 3 days (3D), 2 weeks (2W), 1 month (1M), and 3 months (3M) following implantation. Iba1, 3 days: p < 0.001, t = −6.0805. 2 weeks: p < 0.001, t = −6.1953. 1 month: p = 0.0144, t = −2.9547. 3 months: p = 0.1062, t = −1.7752. ED1, 3 days: p < 0.0001, t = −5.1123. 2 weeks: p = 0.0174, t = −2.8459. 1 month: p = 0.1462, t = −1.5756. 3 months: p = 0.37, t = −1.5756. GFAP, 3 days: p = 0.0079, t = −3.3081. 2 weeks: p = 0.0086, t = −3.2564. 1 month: p < 0.001, t = −5.5066. 3 months: p = 0.3508, t = −0.9787. IgG, 3 days: p = 0.0402, t = −2.3565. 2 weeks: p = −0.6106, t = −0.5256. 1 month: p = 0.4494, t = −0.7872. 3 months: p = 0.7569, t = −0.3182. d.f. = 10 for all. Error bars represent standard deviation (Number of samples n=6 for each device and time point, *p < 0.05, **p < 0.01, ***p < 0.001; one way student’s t-test).