All-Optical Assay to Study Biological Neural Networks

Abstract

We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally, we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.

Keywords: all-optical assay; calcium imaging; connectivity; lentiviral transduction; neural networks; neurons; optogenetics.

FIGURE 1

OptoCaMP enables simultaneous stimulation and calcium recording in neuronal dissociated cultures. (A) Activation spectrum of CheRiff (blue) and excitation/emission spectra of jRCaMP1b (red). The green and blue arrows, respectively, indicate the continuous green illumination at 550 nm and the blue pulses at 470 nm. (B) Schematic of OptoCaMP, bicistronic lentiviral construct designed for the stoichiometric co-expression of the Genetically Encoded Calcium Indicator (GECI) jRCaMP1b (red box) and the Channelrhodopsin variant CheRiff (blue box) to enable simultaneous stimulation and calcium imaging. (C) Single channels and merged fluorescence images of rat cortical neurons expressing OptoCaMP at 14 days in vitro: CheRiff-eGFP is displayed in green and mRuby based GECI jRCaMP1b in red. Scale bar 25 μm. (D) Quantification of lactose dehydrogenase levels (LDH) in primary cortical neurons 6 days after transduction with the OptoCaMP. The lentivirus delivery did not show any significant difference between the cells non-transduced (cell spontaneous LDH release control) and the cell transduced with OptoCaMP. (w = 3, one-way ANOVA, F(2,6) = 69.49, P < 0.0001; followed by Tukey’s multiple comparisons test: OptoCaMP transduction versus Cell spontaneous LDH release control, P = 0.992; Cell maximum LDH release control versus Cell spontaneous LDH release control, ^∗∗∗P < 0.0001; Cell maximum LDH release control versus OptoCaMP transduced, ^∗∗∗P < 0.0001).

FIGURE 2

Optical recordings of light evoked-calcium event in neuronal dissociated cultures. (A) Schematic drawing of the imaging setup for all-optical stimulus and readout. The setup is based on a Nikon Eclipse Ti epi-fluorescence microscope mounting a B2A filter set (without the emission filter) for the blue LED illumination (470 nm) and a modified Cy3/TRITC longpass filter set (excitation filter 565/24 nm, dichroic mirror 562 nm, longpass emission filter >570 nm) for the green light source. Recordings are performed at 10 Hz using a Andor Zyla 5.5 sCMOS Camera. (B) Neuronal dissociated cultures at 14 days in vitro expressing OptoCaMP via lentiviral transduction. CheRiff-eGFP expression in green (eGFP) and mRuby based GECI jRCaMP1b in red (mRuby). Scale bar 100 μm. (C) Schematic of the temporal optical stimuli protocols which consisted in 10 consecutive pulses of blue light (470 nm) of 10 ms, 100 ms, 250 ms, and 500 ms with increasing steps of 7 mW/cm²± 1 mW/ cm² between each pulse (measured at the sample). (D) Typical trace of the fluorescence change of the GECI jRCaMP1b (red trace) of a single-neuron during the 500 ms pulses protocol of increasing blue stimulation intensities (0 to 67mW/cm²). From this trace we can then extract the peak fluorescence ΔF/F (black arrow) for each stimulus intensity for individual neurons of the field of view simultaneously. (E) Amplitude of the calcium events in response to increasing stimuli intensities and pulse duration of 10, 100, 250, and 500 ms. The peak fluorescence from individual neuron at each stimuli intensity was averaged to obtain mean ± s.e.m. values (n = 3, two-way ANOVA, effect of the stimulus intensity F(9,560) = 41.80, P < 0.0001, effect of the stimulation duration F(3,560) = 4470, P<0.0001 followed by Tukey’s multiple comparisons test showing a plateau (red dashed line) for 500 ms pulses protocol, stimulus above 25 mW/cm²).

FIGURE 3

Design and analysis of the spatio-temporal optical stimuli protocols. (A) Map defining the three zones for the spatio-temporal optical stimuli protocols and analysis. Neurons in the blue zone were stimulated, neurons in zone 1 (green) and 2 (red) were not stimulated. (B–E) Average of the peak fluorescence intensities of single-neurons in the same zone ± s.e.m. against the stimulus intensity for the 10 ms (B), 100 ms (C), 250 ms (D), and 500 ms (E) pulses protocols. According to the map in (A), in blue, average peak fluorescence intensity for the stimulated neurons, in green, non-stimulated neurons in the zone 1 and in red, non-stimulated neurons in the zone 2 (n = 3 independent cell culture experiments, one-way ANOVA, (B) F(2,27) = 0.4533, P = 0.6403; (C) F(2,27) = 298.8, P < 0.0001; (D) F(2,27) = 301.7, P < 0.0001; (E) two-way ANOVA, effect of the stimulus intensity F(9,370) = 39.65, P < 0.0001 and effect of distance to stimuli (zones) F(2,370) = 9316, P < 0.0001; (B–E) followed by Tukey’s multiple comparisons test: ns=non-significant, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗∗P < 0.0001). (F–H) Pooled peak fluorescence intensities of each defined zone separately ± s.e.m. (F) stimulated neurons, (G) non-stimulated neurons in zone 1, and (H) non-stimulated neurons in zone 2 for stimuli intensities from 0 to 67 mW/cm² against the pulse duration. (n=3 independent cell culture experiments, two-way ANOVA, (F) effect of the stimulus intensity F(9,520) = 2810, P < 0.01 and effect of the stimulation duration F(3,520) = 296.1, P < 0.0001; (G) effect of the stimulus intensity F(9,520)=0.7990, non-significant and effect of the stimulus duration F(3,520) = 26.21, P < 0.0001; (H) effect of the stimulus intensity F(9,520) = 0.8480, non-significant and effect of the stimulus duration F(3,520) = 2.994, P < 0.05; (F–H) followed by Tukey’s multiple comparisons test: ns=non-significant, ^∗P<0.05, ^∗∗P < 0.01, ^∗∗∗P<0.001, ^∗∗∗∗P<0.0001). (I–K) Typical traces of the fluorescence change of the GECI jRCaMP1b of a single-neuron during the 100 ms pulses protocol of increasing blue stimulation intensities (0 to 67mW/cm²). (I) neuron stimulated; (J) non-stimulated neuron in zone 1 and (K) non-stimulated neuron in zone 2. From these traces we can then extract the peak fluorescence ΔF/F (black arrow).

FIGURE 4

Qualitative and quantitative analysis of neural network connectivity. (A–C) Average of the amplitude of the calcium events of multiple individual neurons in the same zone ± s.e.m.: stimulated neurons (A), non-stimulated neurons in zone 1 (B), and zone 2 (C) during the 100 ms pulses protocol in four conditions: BrainPhys complete medium (BP), complete Neurobasal medium (NB), synaptic blockers (BP + blockers) and caffeine (BP + caffeine) (n=3 independent cell culture experiments for each condition; one-way ANOVA, (A) F(3,36) = 20.16, P < 0.0001; (B) F(3,36) = 66.31, P < 0.0001 (C) F(3,36) = 59.77, P < 0.0001; (A–C) followed by Tukey’s multiple comparisons test: ns=non-significant, ^∗P<0.05, ^∗∗P<0.01, ^∗∗∗∗P<0.0001). (D) Mean global connectivity in BrainPhys complete medium (BP), complete Neurobasal medium (NB), synaptic blockers (BP + blockers) and caffeine (BP + caffeine) conditions. (n = 3 independent cell culture experiments for each condition; one-way ANOVA, F(3,8) = 854, P<0.0001; followed by Tukey’s multiple comparisons test: ns=non-significant, ^∗∗∗∗P < 0.0001 (E–H) Analysis – Connectivity maps and colormaps. Connectivity of the whole network in complete BrainPhys medium (E), complete Neurobasal medium (F), complete BrainPhys medium with caffeine (G) and complete BrainPhys medium with blockers (H) conditions.