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

Abstract

BackgroundThe 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.MethodsNeuron 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.ResultsData 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.ConclusionVmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

Automatic neuron segmentation and neural network analysis method for phase contrast microscopy images

“…17. As already indicated in [19], manual results suggest IVM suppresses the neural growth and connectivity. Here we use repeated measures ANOVA test [46] to accommodate the drug and control group and the five time points.…”

“…8. Illustration of morphological operations [19]. The experiment was approved by Tufts University Institutional Animal Care and Use Committee and complies with the NIH Guide for the Care and Use of Laboratory Animals (IACUC No.B2011 − 45).…”

“…As in [19], we categorize somas into isolated somas and connected somas in order to study the neuronal growth. The isolated somas are defined as the somas which are not connected to other somas by dendrites and are further than 10 pixels from other somas [19]. As indicated in the right column of Fig.…”

“…13, we can see that dendrites are growing and connecting more somas as time passes. In addition, the ratio of total soma number to isolated soma number is used as a metric to evaluate weather IVM suppress the neuronal growth as in [19]. Thus we need to perform evaluation on the segmentation of both isolated somas and all somas.…”

“…The feasibility and advantages of our variational segmentation method are demonstrated by synthetic images in Section 5.1. In addition, the whole pipeline has been validated in real PCM neuron images for the neural development study [19]. The remainder of this paper is organized as follows.…”