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


CONCLUSIONS
Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.
Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155 ; Biology Department, Tufts University 200 Boston Avenue, Suite 4600, Medford, Massachusetts, 02155.
Nurdan
N
Özkucur
Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155.
Kyle P
KP
Quinn
Department of Electrical and Computer Engineering, Tufts University 161 College Avenue, Medford, Massachusetts, 02155.
Jin C
JC
Pang
Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155 ; Department of Neuroscience, Tufts University 136 Harrison Ave, Boston, Massachusetts.
Chuang
C
Du
Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155.
Irene
I
Georgakoudi
Department of Electrical and Computer Engineering, Tufts University 161 College Avenue, Medford, Massachusetts, 02155.
Eric
E
Miller
Biology Department, Tufts University 200 Boston Avenue, Suite 4600, Medford, Massachusetts, 02155.
Michael
M
Levin
Department of Biomedical Engineering, Tufts University 4 Colby St., Medford, Massachusetts, 02155.
David L
DL
Kaplan
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000502:physiology*; D002540:Cerebral Cortex / Q000166:cytology; D018118:Chloride Channels / Q000502:physiology; D018920:Coculture Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007559:Ivermectin / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D051381:Rats
D018118:Chloride Channels; D007559:Ivermectin
Automated cluster analysis; cell resting membrane potential; connectivity; cortical neurons; neuron assemblies