Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Device fabrication and assembly. A) SolidWorks rendering of the device design concept. Red box indicates a zoomed in area for visualization. Each well is divided into two compartments separated by a wall containing microchannels. B) Fabrication process involving two rounds of photolithography. The first round creates 3 μm tall microchannels for axonal access (top). The second round (bottom) defines the microwell reservoirs for cell culture. C) Assembly of device, which for a full plate requires two PDMS slabs bonded to a glass plate. The PDMS slabs are then attached to the bottom of a 96 well plate. D) Photograph of the assembled plate with a two-compartment well shown in the inset.

Overview schematic for induction of human iNs, seeding iNs in 96 compartmental devices, and analysis. Human iPS cells are cultured and differentiated toward an excitatory or inhibitory phenotype from day 0 using subtype-specific lentiviruses. Following the addition of doxycycline to induce gene expression on day 1 and antibiotics for further selection on day 2, lentiviruses specific to the desired functional application are introduced to the culture on day 3 – specifically, GCaMP6f vectors for calcium (Ca2+) imaging analysis, hM3Dq-mCherry for subsequent manipulation of neuronal activity, or neuroligin-3 (NL3) for facilitating synapse formation. After device seeding with iNs, a slow transition from Neurobasal to BrainPhys medium begins, each supplemented with growth factors including BDNF, GDNF, and NT3. After 5–7 weeks, human iNs are analyzed with a high-content imaging system, in this case the GE IN Cell Analyzer 6000. For neuronal morphometric analysis, immunocytochemistry (ICC) with specific antibodies is performed after fixation of human iNs prior to IN Cell analysis and quantification.

Axons project through microchannels. Brightfield image shows device structure. Shadows are produced from holes punched in PDMS. GCaMP6f-positive neurons project axons through the microchannels between the two compartments.

Comparison of synaptic puncta number between different neuroligin-3 conditions. A) Images taken from right compartment of microwell. Wells were fixed and stained for synapsin (red) and MAP2 (blue). B) Puncta and neurite traces obtained by Intellicount synapse quantification software. C) Ratio of synaptic puncta in right-hand side (RHS) compartment to left-hand side (LHS) compartment. A total of seven wells in two different plates was used for each condition (7/2). A one-tailed t-test was used to test for significance between groups (p < 0.04).

Spontaneous circuit activity and disruption by CNQX. A and B) Frames taken from calcium imaging of device well. Excitatory neurons cultured on left side, inhibitory neurons on right side. Frames correspond to specific times indicated in C): Black arrow corresponds to time at frame in A, and black/white arrow corresponds to frame B. C and D) Traces acquired from representative cells in two different wells. Average trace for excitatory neurons shown in black, with gray traces indicating the cells averaged. Inhibitory neurons shown in gray, with a red average trace. CNQX or vehicle added after 222 seconds. Traces are represented as ΔF/F0.

Use of DREADDs to stimulate compartmentalized neural circuits. A) One microwell containing excitatory neurons infected with hM3Dq-mCherry in the left compartment. Co-localization of mCherry and GCaMP6f highlight GCaMP6f-positive neurons containing the hM3Dq-mCherry vector. B) Traces obtained from the well in A). The top black lines represent traces from the excitatory (left side) neurons. The bottom red lines are traces of the inhibitory (right side) neurons. 500 nM CNO was added at frame 900 (99 seconds). Time frame of CNO dosing shown by blue bar. CNO was applied to the entire well. C) A well containing excitatory neurons (left side) and inhibitory neurons (right side) without hM3Dq-mCherry. D) Calcium signals obtained from neurons in well C). The top black lines represent traces from the excitatory (left side) neurons. The bottom red lines are traces of the inhibitory (right side) neurons. All calcium traces are expressed as ΔF/(Fsat – F0) where Fsat is the maximum intensity after KCl addition (data not shown).

Quantification of hM3Dq/CNO dose response within excitatory and inhibitory neurons. A) A custom MATLAB script was used to perform signal scaling and baseline subtraction using a least-squares fit. Original signal displayed in blue, the baseline-corrected signal shown in black. The two baselines are shown in red. Bottom graph shows the quantification of area under the curve (AUC) of pre-drug addition (red) and post-drug addition (blue) of a neuron from an hM3Dq-positive well. B) The same process applied to an inhibitory neuron in an hM3Dq-negative well. C) Area ratio quantification for excitatory neurons (black bars) and inhibitory neurons (red bars). Ten cells were processed from 3 to 4 wells over 2 plates. * indicates p < 0.001 for comparisons between excitatory concentrations, # for p < 0.001 for inhibitory concentrations, and & for p < 0.001 for comparisons between excitatory and inhibitory neurons at the same concentration. D) Dose–response curve generated from area data. Black line corresponds to excitatory neuron area ratios and red line to inhibitory neurons.