Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture

Abstract

We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

Keywords: compartmented device; functional connectivity; invitro model; neuron cell culture; pharmacological manipulation.

Figure 1

(A) Schematic layout of the dual-compartment device; (B) Planar MEA substrate with dual-compartment PDMS device; (C) Spontaneous activity of a culture on DIV 14 (the electrode number along the y-axis run from 11 through 88 with the first digit representing column and the second digit its row respectively. Each dot represents an action potential recorded by one of the MEA channels); (D) Typical cortical cell culture in a compartment on DIV 4.

Figure 2

Pharmacological manipulation of spontaneous activity in the dual-compartment device. I: spike rate recorded at individual electrodes in compartment A (bottom – red) and compartment B (Top – blue). II: spike rate recorded with 100 nM TTX in compartment B (suppression of network activity in compartment B); III: spike rate after first wash cycle; IV: spike rates after three wash cycles.

Figure 3

Structural connectivity between the two compartmentalized neuronal sub-populations. (A) Phase contrast image of neurite ladder structure intact after the removal of PDMS structures from the MEA surface; (B) Transfection image of a neurite grown across the microchannels connecting the compartments; (C) Immunofluorescence image of neurite structure following the microchannel placement.

Figure 4

Cross-correlation of spontaneous activity in two compartments. (A) Cross-correlograms between a sample electrode in compartment A (Electrode index # 19) and all electrodes in both the compartments; (B) Cross-correlograms with surrogate peak trains in compartment B (between a sample electrode in compartment A (Electrode index # 19) and all electrodes in both the compartments); (C) Comparison of CC between global mean CC averaged over all 60 electrodes (red), mean CC of a sample electrode (Electrode index # 19) with compartment B (green) and CC of most correlated electrodes in both the compartments (Electrode index # 19in compartment A with a sample electrode [(Electrode index # 43) in compartment B (blue)]. Half-window size = 5 ms and Bin size (temporal resolution) = 0.1 ms; (D) Functional connectivity map showing the strongest 100 connections in the network. Red color arrows (intensity coded) represent the functional connections within a compartment; the blue color arrows (intensity coded) represent the functional connectivity between two compartments (inter-compartment connections). Inter-compartment connections are compared with surrogate spike trains and proven to be genuine (Connectivity map with surrogate spike train data not shown). The bidirectionality in network connectivity can be inferred from the direction of the arrows.

Figure 5

Two-step statistical analysis of an individual device. (A) MEA electrode layout and region separation across both the compartments (red color line between electrode column 4 and 5 represents the microchannel separation); (B) In the first step, “median” of C_peak (t = 1 bin) for all the electrodes under four circumstances (i.e., Intra-compartment, Inter-compartment, Both compartments (Global) and Surrogated spike train in the other compartment) is computed and the electrodes are then segregated into separate regions as described earlier. In the second step, another statistical parameter (“median,” in this case) within each region is computed. Positive error bars are equal to the differences between the 75th percentile and the median, while negative error bars are equal to the differences between the median and the 25th percentile. The two percentiles were computed along with the “median” in the second step of the statistical analysis described in the text. Error bars for Surrogated spike trains in the other compartment are not visible because of their very low amplitude.

Figure 6

Statistical analysis of correlation on multiple devices; box plots of intra and inter-compartmental correlation levels, divided per region, in 17 devices (statistical parameters used in the two-step analysis: “median” for both the steps).