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Membrane potential depolarization causes alterations in neuron arrangement and connectivity in cocultures.

Özkucur N, Quinn KP, Pang JC, Du C, Georgakoudi I, Miller E, Levin M, Kaplan DL - Brain Behav (2014)

Bottom Line: The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm.Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures.Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155 ; Biology Department, Tufts University 200 Boston Avenue, Suite 4600, Medford, Massachusetts, 02155.

ABSTRACT

Background: The disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown.

Methods: Neuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins.

Results: Data show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions.

Conclusion: Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

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Related in: MedlinePlus

Ivm causes membrane hyperpolarization in 1 day in vitro cultures of E18 rat primary cortical cells. (A) Cell membrane potential was detected colorimetrically using a specific dye Di-8-ANEPPS. **P ≤ 0.01. N = 60. (B) 1-day cultures of E18 rat primary cortical cells were stained with GFAP.FITC and vimentin. Alexa 568 was used to characterize the cultures (neural progenitor and stem cells). Cell nuclei were visualized using Hoechst. Connectivity is suppressed in immature neurons under hyperpolarizing conditions in 1 day in vitro cultures of E18 rat cortical cells. Aggregates were excluded. (C and D) The total number of somas versus isolated somas (highlighted in yellow squares) was counted to define connectivity in cells before and after ivm exposure. (E) Ratio of total somas to isolated somas was plotted to represent the changes in neural connectivity under normal (control) and hyperpolarizing conditions (ivm). N = 10 microscopic fields from two separate dishes were tracked, and a total of 2500 cells were counted for each control and drug group. (F) Beta-III tubulin labeling showing thin, discontinuous, broken membrane projections (arrows) in ivm-treated cells, whereas control cells have continuous, thick bundles of projections. (G) Synaptophysin labeling showing control cells with large, dense synaptic areas (arrows), whereas lack of those in ivm-treated cells. (H) GLRA labeling showing strong production of the protein along their membrane projections as well as in their somas (arrows), whereas ivm-treated cells have GLRA distribution limited to their somas only. P ≤ 0.05 versus controls at each time points.
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fig05: Ivm causes membrane hyperpolarization in 1 day in vitro cultures of E18 rat primary cortical cells. (A) Cell membrane potential was detected colorimetrically using a specific dye Di-8-ANEPPS. **P ≤ 0.01. N = 60. (B) 1-day cultures of E18 rat primary cortical cells were stained with GFAP.FITC and vimentin. Alexa 568 was used to characterize the cultures (neural progenitor and stem cells). Cell nuclei were visualized using Hoechst. Connectivity is suppressed in immature neurons under hyperpolarizing conditions in 1 day in vitro cultures of E18 rat cortical cells. Aggregates were excluded. (C and D) The total number of somas versus isolated somas (highlighted in yellow squares) was counted to define connectivity in cells before and after ivm exposure. (E) Ratio of total somas to isolated somas was plotted to represent the changes in neural connectivity under normal (control) and hyperpolarizing conditions (ivm). N = 10 microscopic fields from two separate dishes were tracked, and a total of 2500 cells were counted for each control and drug group. (F) Beta-III tubulin labeling showing thin, discontinuous, broken membrane projections (arrows) in ivm-treated cells, whereas control cells have continuous, thick bundles of projections. (G) Synaptophysin labeling showing control cells with large, dense synaptic areas (arrows), whereas lack of those in ivm-treated cells. (H) GLRA labeling showing strong production of the protein along their membrane projections as well as in their somas (arrows), whereas ivm-treated cells have GLRA distribution limited to their somas only. P ≤ 0.05 versus controls at each time points.

Mentions: Neural connectivity was assessed based on the quantification of neurite outgrowth and the establishment of contact between neuron somas. One day cultures of E18 rat primary cortical neurons were assessed under normal physiological (no Ivm)- and depolarizing (after Ivm exposure, 1 μmol/L, 24 h) conditions between 0 and 24 h, with 2 h interval initially. The relative Vmem of cells was measured using di-8-ANEPPS dye (Fig.5A). Immature neurons were characterized using specific markers vimentin and GFAP (Fig.5B). Phase-contrast images were collected of the same cells before- and after Ivm exposure at given time points for quantification. Images taken from cells before and after Ivm exposure were shown here (Fig.5C,D). The ratio of the total number of soma to the number of isolated soma was manually counted by two researchers independently.


Membrane potential depolarization causes alterations in neuron arrangement and connectivity in cocultures.

Özkucur N, Quinn KP, Pang JC, Du C, Georgakoudi I, Miller E, Levin M, Kaplan DL - Brain Behav (2014)

Ivm causes membrane hyperpolarization in 1 day in vitro cultures of E18 rat primary cortical cells. (A) Cell membrane potential was detected colorimetrically using a specific dye Di-8-ANEPPS. **P ≤ 0.01. N = 60. (B) 1-day cultures of E18 rat primary cortical cells were stained with GFAP.FITC and vimentin. Alexa 568 was used to characterize the cultures (neural progenitor and stem cells). Cell nuclei were visualized using Hoechst. Connectivity is suppressed in immature neurons under hyperpolarizing conditions in 1 day in vitro cultures of E18 rat cortical cells. Aggregates were excluded. (C and D) The total number of somas versus isolated somas (highlighted in yellow squares) was counted to define connectivity in cells before and after ivm exposure. (E) Ratio of total somas to isolated somas was plotted to represent the changes in neural connectivity under normal (control) and hyperpolarizing conditions (ivm). N = 10 microscopic fields from two separate dishes were tracked, and a total of 2500 cells were counted for each control and drug group. (F) Beta-III tubulin labeling showing thin, discontinuous, broken membrane projections (arrows) in ivm-treated cells, whereas control cells have continuous, thick bundles of projections. (G) Synaptophysin labeling showing control cells with large, dense synaptic areas (arrows), whereas lack of those in ivm-treated cells. (H) GLRA labeling showing strong production of the protein along their membrane projections as well as in their somas (arrows), whereas ivm-treated cells have GLRA distribution limited to their somas only. P ≤ 0.05 versus controls at each time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321392&req=5

fig05: Ivm causes membrane hyperpolarization in 1 day in vitro cultures of E18 rat primary cortical cells. (A) Cell membrane potential was detected colorimetrically using a specific dye Di-8-ANEPPS. **P ≤ 0.01. N = 60. (B) 1-day cultures of E18 rat primary cortical cells were stained with GFAP.FITC and vimentin. Alexa 568 was used to characterize the cultures (neural progenitor and stem cells). Cell nuclei were visualized using Hoechst. Connectivity is suppressed in immature neurons under hyperpolarizing conditions in 1 day in vitro cultures of E18 rat cortical cells. Aggregates were excluded. (C and D) The total number of somas versus isolated somas (highlighted in yellow squares) was counted to define connectivity in cells before and after ivm exposure. (E) Ratio of total somas to isolated somas was plotted to represent the changes in neural connectivity under normal (control) and hyperpolarizing conditions (ivm). N = 10 microscopic fields from two separate dishes were tracked, and a total of 2500 cells were counted for each control and drug group. (F) Beta-III tubulin labeling showing thin, discontinuous, broken membrane projections (arrows) in ivm-treated cells, whereas control cells have continuous, thick bundles of projections. (G) Synaptophysin labeling showing control cells with large, dense synaptic areas (arrows), whereas lack of those in ivm-treated cells. (H) GLRA labeling showing strong production of the protein along their membrane projections as well as in their somas (arrows), whereas ivm-treated cells have GLRA distribution limited to their somas only. P ≤ 0.05 versus controls at each time points.
Mentions: Neural connectivity was assessed based on the quantification of neurite outgrowth and the establishment of contact between neuron somas. One day cultures of E18 rat primary cortical neurons were assessed under normal physiological (no Ivm)- and depolarizing (after Ivm exposure, 1 μmol/L, 24 h) conditions between 0 and 24 h, with 2 h interval initially. The relative Vmem of cells was measured using di-8-ANEPPS dye (Fig.5A). Immature neurons were characterized using specific markers vimentin and GFAP (Fig.5B). Phase-contrast images were collected of the same cells before- and after Ivm exposure at given time points for quantification. Images taken from cells before and after Ivm exposure were shown here (Fig.5C,D). The ratio of the total number of soma to the number of isolated soma was manually counted by two researchers independently.

Bottom Line: The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm.Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures.Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155 ; Biology Department, Tufts University 200 Boston Avenue, Suite 4600, Medford, Massachusetts, 02155.

ABSTRACT

Background: The disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown.

Methods: Neuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins.

Results: Data show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions.

Conclusion: Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

Show MeSH
Related in: MedlinePlus