Optical stimulation, calcium imaging, and optical recording analysis

As proof-of-concept validation for the optically controlled living electrode approach, we established an “all-optical” paradigm enabling the simultaneous optogenetic control and optical monitoring of aggregate-based μTENNs in vitro. Cortical neuronal aggregates were transduced with either channelrhodopsin-2 (ChR2) for light-based neuronal activation (“input”) or the genetically encoded fluorescent calcium reporter RCaMP1b for optical readout of neuronal activity (“output”) via AAV1 transduction as described above (Penn Vector Core). Five- to 6-mm-long bidirectional μTENNs were then plated with one input/ChR2⁺ aggregate and one output/RCaMP1b⁺ aggregate (n = 5). ChR2 and RCaMP have been investigated and used for all-optical electrophysiology in vitro with minimal spectral overlap, reducing the likelihood of false-positive responses in RCaMP⁺ neurons due to photostimulation of ChR2⁺ neurons (18). At 10 DIV, μTENNs were stimulated via an LED optical fiber positioned approximately 1 to 3 mm above the input aggregate, such that the entire aggregate was illuminated. A Plexon Optogenetic Stimulation System with LED modules for each desired wavelength was used to stimulate the μTENNs (Plexon Inc.). Stimulation consisted of a train of 10 100-ms pulses (1 Hz) at 465 nm, within the excitation spectra of ChR2. Each train was repeated three times for a given LED current amplitude (50, 100, 200, 250, 300 mA); amplitudes corresponded to approximate stimulation intensities of 106, 211, 423, 528, and 634 mW/mm² from the tip of the optical fiber and 4.7, 9.3, 18.7, 23.3, and 28.0 mW/mm² at the aggregate, respectively. As a control, μTENNs were stimulated as above at 620 nm (outside of the excitation spectra of ChR2) at 300 mA/28.0 mW/mm². Recordings of the μTENNs’ output aggregates were acquired at 25 to 30 frames per second on a Nikon Eclipse Ti microscope paired with an ANDOR Neo/Zyla camera and Nikon Elements AR 4.50.00 (Nikon Instruments).

Following optical stimulation and/or recording, calcium imaging acquisitions were manually reviewed against phase images of the same μTENNs to identify regions of interest (ROIs) containing neurons and background ROIs. Neuronal ROIs were identified as areas clearly containing one or more neuronal cell bodies that exhibited repeated synchronous changes in pixel intensity, although because of the dense packing of the aggregate, these ROIs also contained axons. Background ROIs were empty square areas containing no neurons (i.e., only culture media). The mean pixel intensities for each ROI were imported into MATLAB for further analysis via custom scripts (MathWorks Inc.). Within MATLAB, the background ROI intensity for each recording was subtracted from active ROIs. Ten such ROIs were randomly selected and averaged to obtain a representative fluorescence intensity trace across each output aggregate. Subsequently, the percent change in fluorescence intensity over time (∆F/F_o) was calculated for each mean ROI, where ∆F equals (F_T − F_o), F_T is the mean ROI fluorescent intensity at time T, and F_o is the average of the lower half of the preceding intensity values within a predetermined sampling window (19). The peak ∆F/F_o for each train was averaged per μTENN for each of the given stimulation intensities. The average maximum ∆F/F_o was then compared across stimulation intensities with a one-way ANOVA, with post hoc analysis performed where necessary with the Tukey procedure. In addition, the peak ∆F/F_o of the output aggregate under 620-nm stimulation was used as a control (620 nm being outside of ChR2’s activation spectra) and compared to that under 465-nm stimulation at 300 mA/28 mW/mm² using an unpaired t test.