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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

Depolarized cell membrane causes more projections to be formed in mature neurons; 3 week cocultures of rat primary cortical neurons and astrocytes were stained with beta tubulin III and Alexa Fluor 488 to visualize the neurons, (A) under control and (B) Ivm-treated (1 μmol/L, 24 h) conditions. (C) Ivm-caused depolarization of the cell membrane detected using ratiometric dye Di-8-ANEPPS. N = 12. (D) The numbers of neural projections of single cells (arrows) were counted. N = 18. (E) Cell viability at 1, 2, 3, and 4 weeks in control (no drug) and Ivm-treated cultures. 10% DMSO was used as negative control. *P ≤ 0.05, **P ≤ 0.01. N = 18.
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fig02: Depolarized cell membrane causes more projections to be formed in mature neurons; 3 week cocultures of rat primary cortical neurons and astrocytes were stained with beta tubulin III and Alexa Fluor 488 to visualize the neurons, (A) under control and (B) Ivm-treated (1 μmol/L, 24 h) conditions. (C) Ivm-caused depolarization of the cell membrane detected using ratiometric dye Di-8-ANEPPS. N = 12. (D) The numbers of neural projections of single cells (arrows) were counted. N = 18. (E) Cell viability at 1, 2, 3, and 4 weeks in control (no drug) and Ivm-treated cultures. 10% DMSO was used as negative control. *P ≤ 0.05, **P ≤ 0.01. N = 18.

Mentions: The total number of projections emerging from single neurons (arrows) was counted manually in the fluorescent (beta III tubulin/AlexaFluor488) images taken from control (Fig.2A) or Ivm-treated 3w cocultures (Fig.2B). Depolarization of neural Vmem was verified by ratiometric measurement of Vmem-specific dye di-8-ANEPPS in control (no Ivm) and in Ivm-treated 3w cocultures. Accuracy of di-8-ANEPPS dye was verified by whole-cell patch clamp measurements as shown in the methods section. When incubated with 1 μmol/L Ivm for 24 h, the di-8-ANEPPS ratio (450 nm/510 nm) signal measured from cocultures was significantly increased (by 29%), indicating a depolarized Vmem (Fig.2C). The Vmem of glial cells in homotypic cultures did not change upon Ivm exposure. Differentiated neurons had a significantly increased (133%, P ≤ 0.01) number of projections when their Vmem was depolarized using 1 μmol/L Ivm for 24 h. Control cells had fewer projections (Fig.2D). Cell toxicity was examined using the WST-1 assay, which measures cell health via readout of mitochondrial activity (Hoper 1997; Ngamwongsatit et al. 2008). We found that 1 μmol/L Ivm was not toxic to E18 cortical cells during 4 weeks in vitro. In contrast, cells treated with the drug exhibited significantly higher mitochondrial activity than cells without the drug (Fig.2E). We conclude that depolarized Vmem causes mature neurons to form more membrane projections.


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)

Depolarized cell membrane causes more projections to be formed in mature neurons; 3 week cocultures of rat primary cortical neurons and astrocytes were stained with beta tubulin III and Alexa Fluor 488 to visualize the neurons, (A) under control and (B) Ivm-treated (1 μmol/L, 24 h) conditions. (C) Ivm-caused depolarization of the cell membrane detected using ratiometric dye Di-8-ANEPPS. N = 12. (D) The numbers of neural projections of single cells (arrows) were counted. N = 18. (E) Cell viability at 1, 2, 3, and 4 weeks in control (no drug) and Ivm-treated cultures. 10% DMSO was used as negative control. *P ≤ 0.05, **P ≤ 0.01. N = 18.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Depolarized cell membrane causes more projections to be formed in mature neurons; 3 week cocultures of rat primary cortical neurons and astrocytes were stained with beta tubulin III and Alexa Fluor 488 to visualize the neurons, (A) under control and (B) Ivm-treated (1 μmol/L, 24 h) conditions. (C) Ivm-caused depolarization of the cell membrane detected using ratiometric dye Di-8-ANEPPS. N = 12. (D) The numbers of neural projections of single cells (arrows) were counted. N = 18. (E) Cell viability at 1, 2, 3, and 4 weeks in control (no drug) and Ivm-treated cultures. 10% DMSO was used as negative control. *P ≤ 0.05, **P ≤ 0.01. N = 18.
Mentions: The total number of projections emerging from single neurons (arrows) was counted manually in the fluorescent (beta III tubulin/AlexaFluor488) images taken from control (Fig.2A) or Ivm-treated 3w cocultures (Fig.2B). Depolarization of neural Vmem was verified by ratiometric measurement of Vmem-specific dye di-8-ANEPPS in control (no Ivm) and in Ivm-treated 3w cocultures. Accuracy of di-8-ANEPPS dye was verified by whole-cell patch clamp measurements as shown in the methods section. When incubated with 1 μmol/L Ivm for 24 h, the di-8-ANEPPS ratio (450 nm/510 nm) signal measured from cocultures was significantly increased (by 29%), indicating a depolarized Vmem (Fig.2C). The Vmem of glial cells in homotypic cultures did not change upon Ivm exposure. Differentiated neurons had a significantly increased (133%, P ≤ 0.01) number of projections when their Vmem was depolarized using 1 μmol/L Ivm for 24 h. Control cells had fewer projections (Fig.2D). Cell toxicity was examined using the WST-1 assay, which measures cell health via readout of mitochondrial activity (Hoper 1997; Ngamwongsatit et al. 2008). We found that 1 μmol/L Ivm was not toxic to E18 cortical cells during 4 weeks in vitro. In contrast, cells treated with the drug exhibited significantly higher mitochondrial activity than cells without the drug (Fig.2E). We conclude that depolarized Vmem causes mature neurons to form more membrane projections.

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