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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

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.
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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.

Mentions: A raster plot of the electrophysiological recording of a typical spontaneous activity within the culture is shown in Figure 1C. The raster plot shows the spontaneous activity of a culture in a closed compartmented device on DIV 14. The observed typical activity is very similar to that of non-compartmentalized networks and it is indeed characterized by both local random spiking and network bursting behavior. The evolution of network activity over the developmental period from DIV 11 to DIV 35 was recorded at regular intervals (on DIV 11, 14, 17, 21, 24 etc.). An increase in network activity was observed from DIV 14, with a maturation phase in the spontaneous activity during the third week in culture and the activity subsided after DIV 25 resulting in very few spiking neurons by DIV 30, similar to those reported in literature (Chiappalone et al., 2006). Disappearance of network bursts signaling the deterioration of the culture was observed after DIV 30 (Mukai et al., 2003). During third week in culture (∼DIV 21), cortical culture in devices showed an extensive network bursting activity. Pharmacological experiments were performed during the most active phase of the culture starting from DIV 14. To ensure the biological origin of the spiking activity, experiments with TTX were performed as explained earlier. When TTX was added to one compartment (i.e., Compartment B), neuronal activity in that compartment was immediately suppressed without any significant impact on the spiking behavior of neuronal population in the other compartment (Figure 2). Neuronal spiking activities were recovered when TTX in the compartment was washed out with supplement enriched neurobasal medium. Recovery of activity from few electrodes after 1× wash cycle and from majority of the electrodes with 3× wash cycles from a sample experiment is as shown in Figure 2.


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)

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

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

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.
Mentions: A raster plot of the electrophysiological recording of a typical spontaneous activity within the culture is shown in Figure 1C. The raster plot shows the spontaneous activity of a culture in a closed compartmented device on DIV 14. The observed typical activity is very similar to that of non-compartmentalized networks and it is indeed characterized by both local random spiking and network bursting behavior. The evolution of network activity over the developmental period from DIV 11 to DIV 35 was recorded at regular intervals (on DIV 11, 14, 17, 21, 24 etc.). An increase in network activity was observed from DIV 14, with a maturation phase in the spontaneous activity during the third week in culture and the activity subsided after DIV 25 resulting in very few spiking neurons by DIV 30, similar to those reported in literature (Chiappalone et al., 2006). Disappearance of network bursts signaling the deterioration of the culture was observed after DIV 30 (Mukai et al., 2003). During third week in culture (∼DIV 21), cortical culture in devices showed an extensive network bursting activity. Pharmacological experiments were performed during the most active phase of the culture starting from DIV 14. To ensure the biological origin of the spiking activity, experiments with TTX were performed as explained earlier. When TTX was added to one compartment (i.e., Compartment B), neuronal activity in that compartment was immediately suppressed without any significant impact on the spiking behavior of neuronal population in the other compartment (Figure 2). Neuronal spiking activities were recovered when TTX in the compartment was washed out with supplement enriched neurobasal medium. Recovery of activity from few electrodes after 1× wash cycle and from majority of the electrodes with 3× wash cycles from a sample experiment is as shown in Figure 2.

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