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

Kanagasabapathi TT, Ciliberti D, Martinoia S, Wadman WJ, Decré MM - Front Neuroeng (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Minimally Invasive Healthcare Department, Philips Research Laboratories Eindhoven, Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus

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 Cpeak (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.
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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 Cpeak (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.

Mentions: The electrode columns were segregated into four regions as in Figure 5A. The 16 values obtained through the two-step statistical analysis previously described can be plotted as in Figure 5B. It may be noted that: (i) the intra-compartmental connectivity is higher than inter-compartmental connectivity (ii) intra compartment and inter-compartment connectivity are higher than the non-genuine (namely due to randomness) connectivity contribution obtained with surrogate spike trains and (iii) no clear differences can be observed among the different spatial regions (R1–R4). To assess the robustness of our observations, we analyzed 17 devices that contained cortical cells in both compartment plated with similar cell densities but from different DIV (from DIV 17 to DIV 32). Each device had at least 52 active electrodes (an electrode was considered to be active if the average spiking frequency was at least 0.2 Hz; Shahaf and Marom, 2001).


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

Kanagasabapathi TT, Ciliberti D, Martinoia S, Wadman WJ, Decré MM - Front Neuroeng (2011)

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 Cpeak (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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198030&req=5

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 Cpeak (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.
Mentions: The electrode columns were segregated into four regions as in Figure 5A. The 16 values obtained through the two-step statistical analysis previously described can be plotted as in Figure 5B. It may be noted that: (i) the intra-compartmental connectivity is higher than inter-compartmental connectivity (ii) intra compartment and inter-compartment connectivity are higher than the non-genuine (namely due to randomness) connectivity contribution obtained with surrogate spike trains and (iii) no clear differences can be observed among the different spatial regions (R1–R4). To assess the robustness of our observations, we analyzed 17 devices that contained cortical cells in both compartment plated with similar cell densities but from different DIV (from DIV 17 to DIV 32). Each device had at least 52 active electrodes (an electrode was considered to be active if the average spiking frequency was at least 0.2 Hz; Shahaf and Marom, 2001).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Minimally Invasive Healthcare Department, Philips Research Laboratories Eindhoven, Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus