Gap junctions do not underlie changes in whole-cell conductance in anoxic turtle brain

Area of Science:

• Neuroscience
• Cell Biology
• Physiology

Background:

• Acute reduction in cell membrane permeability is a potential strategy to enhance anoxic survival.
• Previous research indicated a decrease in whole-cell neuronal conductance (G(w)) in western painted turtles during anoxia, possibly linked to adenosine A(1) receptors and calcium.
• This reduction in G(w) was hypothesized to result from ion channel closure or gap junction closure.

Purpose of the Study:

• To investigate the role of gap junctions in the anoxic decrease of whole-cell neuronal conductance (G(w)) in the western painted turtle.
• To assess neuronal connectivity changes under various conditions, including anoxia, high calcium, and adenosine perfusion.
• To visualize alterations in gap junction permeability using dye propagation experiments.

Main Methods:

• Antibody staining for connexin 32 and 43 (Cx32 and Cx43) to identify gap junction components in the turtle cortex.
• Measurement of whole-cell capacitance (C(w)) to assess neuronal connectivity during perfusion with normoxic, anoxic, high calcium, and adenosine artificial cerebrospinal fluid (aCSF).
• Lucifer yellow dye loading into turtle neurons to visualize gap junction permeability under various aCSF conditions (normoxic, anoxic, 0 calcium, hypo-osmotic, cold shock, etc.).

Main Results:

• No significant change in whole-cell capacitance (C(w)) was observed during normoxic, anoxic, high calcium, or adenosine perfusion.
• A significant decrease in C(w) was observed during hypo-osmotic aCSF perfusion, reaching a minimum of 50+/-10.4 pF at 30 minutes.
• Lucifer yellow dye propagation, indicating gap junction permeability, was only observed in 3 out of 20 cold shock experiments (4°C).

Conclusions:

• Gap junctions are not involved in the acute reduction of whole-cell neuronal conductance (G(w)) observed during anoxia in the western painted turtle.
• The findings support the hypothesis that ion channel arrest, rather than gap junction closure, is responsible for the observed decrease in G(w) during anoxia.
• Hypo-osmotic stress significantly reduces neuronal connectivity, as indicated by decreased whole-cell capacitance.