Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

Background Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. Methods We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl- 1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. Results Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 μm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 μm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 μM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 μM, but not 100 μM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 μM 1-EBIO, and partially reduced by 100 μM 1-EBIO. Conclusion The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.