Neuronal survival and membrane resealing of cultured rat septal neurons after laser-induced neurite transection

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Physiology and Biophysics

First Committee Member

John N. Barrett, Committee Chair


Fetal rat septal neurons were grown in culture pre-treated with lung fibroblast conditioned medium at a final plating density of 1000 to 5000 cells per square centimeter. Selected neurites were transected using a nitrogen laser and 24 hours neuronal survival after neurite transection was measured for neurons that had been in culture for 1 to 30 days before the transection. The percent survival of transected neurons in normal culture medium decreased with increasing in neuronal age before the transection: the average survival was 75% for neurons that had been in culture for less than one week; about 50% survival for neurons that had been in culture for two weeks; the survival decreased to 20% when neurons were one month old in culture.Membrane resealing of transected neurons was assayed using a new dye exclusion method.Local disassembly of microtubules and neurofilaments appears to be critical for resealing.A hypothesized mechanism of membrane resealing is proposed, which states that in normal medium, Ca$\sp{\rm ++}$ enhances resealing by activating calpain-like proteases and calmodulin-dependent mechanisms. Resealing may also be promoted by other Ca-dependent mechanisms, such as direct effects of Ca$\sp{\rm ++}$ on microtubules and neurofilaments. Among these mechanisms, the major event appears to be disassembly of microtubules and neurofilaments. The disassembly of microtubules and neurofilaments could remove the protruding cytoskeleton at the cut end of neurite and remove the cytoskeletal restraint on the movement of membrane components required for membrane resealing. Disassembly of microtubules and neurofilaments may also release F-actin tension which exists in the cortical layer of the plasma membrane, and collapse the cut end of the neurite favoring for resealing.Resealing can also achieved in low Ca$\sp{\rm ++}$ medium provided that disassembly of microtubules and neurofilaments occurs. Therefore, the most essential event appears to be disassembly of cytoskeletal elements. (Abstract shortened with permission of author.)


Biology, Neuroscience

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