Combining Microfluidics, Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

International audienceIn this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations

Compatibility of ChR2 and Calcium Orange.

<p>A- Quantification of background stimulation in ChR2(ET/TC) transduced cultures during calcium imaging with Calcium Orange at DIV12. The abscissa indicates the intensity of excitation light (549 nm) used during imaging relative to the recording conditions used in the rest of the experiments (1x); 2x is twice that level and 4x is 4 times that level. The different bursting rates at 2x and 4x were normalized by the bursting rates recorded at 1x for each culture so as to remove inter-culture variability for initial bursting rates. For information purpose, average absolute bursting rate at 1x was 5.9 ± 2.2 bursts per minute (n = 9 cultures). B- Absorption spectra of two ChR2 variants, including the ChR2(ET/TC) used in this study, as well as those of the commonly used Fluo-4 and its red-shifted counterpart Calcium Orange indicator. The figures of merit of each combination are indicated in % of maximal absorption.</p

Optogenetic induction of bursting events.

<p>A- Typical responses of a transduced culture (DIV 13) to different stimulation durations (1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 ms). Vertical blue bars represent stimulation times and durations. No fluorescence data was recorded during the stimulations as the photodiode, recording directly the stimulated spot, was saturated. B- Same experiment as in A after addition of 10 μM CNQX (AMPA receptor antagonist).</p

Design of a simple neuronal device.

<p>A- Scheme showing the neuronal device featuring two micro-wells seeded with hippocampal neurons (DIV 15) and connected by an array of axon diodes. The neurons colored in green correspond to neurons that are transduced by ChR2. Neurons colored in purple are not transduced. B- Image of a neuronal device obtained by confocal fluorescence microscopy showing neurons expressing ChR2-YFP (false color).</p

Local stimulation and burst propagation (DIV15).

<p>A- Picture of a symmetrical two-compartments device with only one side transduced (left). Green and red channels are for ChR2-YFP and Calcium Orange indicator, respectively. This dual marking reveals that 70% of cells are expressing ChR2 inside the transduced compartment. B and C- Averaged response after stimulation of the transduced (B) and non-transduced (C) compartments. The solid colour lines correspond to the peristimulus fluorescence signal averaged over 30 stimulations (0.1 Hz in alternation) while the filled surfaces indicate associated standard deviations. Vertical dashed lines indicate stimulations. Diagrams are presented for clarity, with matching line and population colors. Green neurons are transduced neurons.</p

Burst transmission in asymmetrical networks (DIV15).

<p>A—Explanatory diagrams for graphs presented on the left and right column respectively, with the same color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120680#pone.0120680.g004" target="_blank">Fig 4</a>. B—Typical example of a device showing unidirectional transmission. C—Results from a device where bursts also propagate in the reverse direction, albeit with a greater delay. Addition of 5 μM CNQX to the latter device yields the typical unidirectional transmission (D). Delays obtained by normalized cross correlation.</p