Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca²⁺ concentration

Abstract

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.

A Model for Sealing Plasmalemmal Damage in Neurons and Other Eukaryotic Cells

“…For a given transection distance, cells with longer PT times, up to 10 min PT, did not seal at a significantly ( p Ͼ 0.05, CMH 2 ) different frequency compared to cells with shorter PT times (Table 1). These and all other data in Table 1 confirmed that the frequency and rate of sealing in B104 cells was determined by PC time and not the time of neurite transection, as was previously reported for this and other preparations (Blanchette et al, 1999;Detrait et al, 2000b;Yoo et al, 2004). Hence, unless otherwise stated, all measures of sealing by B104 cells in this article are given in PC times.…”

“…We then added 3 kDa Texas Red dextran at 0 min PC (i.e., after neurite transection) to assess sealing. As reported previously (Yoo et al, 2003(Yoo et al, , 2004Nguyen et al, 2005), cells with transected neurites identified by their relationship to the score mark were always filled with fluorescein dextran (Fig. 1 B, E), whether or not they excluded Texas Red dextran.…”

“…"Sealing frequency" is defined as the percentage of a set of individually-transected and uniquely identified cells that exclude 3 kDa Texas Red dye (sealed) at a given PC time. The CMH 2 test for independence was used to determine whether the differences between the sealing frequency at a given PC time for different experimental treatments was statistically significant ( p Ͻ 0.05), as described previously (Agresti, 1996;Detrait et al, 2000b;Yoo et al, 2003Yoo et al, , 2004.…”

“…Therefore, identifying plasmalemmal sealing pathways in neurons may provide insights into sealing in other cell types. (Bottenstein and Sato, 1979) have often been used as a model system to study neuronal function in vitro (Toda et al, 1999;Tan et al, 2003;Yoo et al, 2003Yoo et al, , 2004Nguyen et al, 2005;Miller et al, 2006). B104 cells extend neurites that have properties typical of nerve axons such as generation of action potentials, smooth (but not rough) endoplasmic reticulum, release of neurotransmitter, and regeneration of severed neurites.…”

“…B104 cells extend neurites that have properties typical of nerve axons such as generation of action potentials, smooth (but not rough) endoplasmic reticulum, release of neurotransmitter, and regeneration of severed neurites. These cells have easily identifiable cell bodies and neurites, allowing precise identification of individually damaged cells at their injury site (Detrait et al, 2000b;Yoo et al, 2003Yoo et al, , 2004. Unlike some other model neuronal cell lines (e.g., PC12 cells), B104 cells do not require growth factor supplements to fetal bovine serum to proliferate (Detrait et al, 2000b;Yoo et al, 2003).…”