Rationale: Whole cell patch recordings of P7-P10 calyxes at 22-24 oC were given a 20 ms depolarization from -80 mV to +10 mV. The initial rate of decay of the capacitance change was significantly slower (~33% of control) in the Actin, Gamma KO suggesting a slower rate of endocytosis following vesicle fusion (Fig 2. A-C). Similar results were observed when the stimulus was changed to a 20 action potential equivalent stimulus (Fig. 2 D-F), performed at 34-37 oC (Fig. 2 G) and in older mice (P13-P14) (Fig. 2 H).
Hippocampal neurons were cultured from the Actg LoxP/LoxP mice transfected with Synaptophysin-pHluorin2X (SypH) with or without a Cre containing plasmid (Fig. 6 A and D). SypH can be used to study vesicle recycling as it is non-fluorescent in relatively low pH environment of vesicles but is fluorescent in the higher pH extracellular environment. Upon stimulation the decay of fluorescence was significantly slower than the control (Fig. 6 E-G). This fluorescence was quenchable by a low pH solution applied externally indicating it was not a deficit in vesicle reacidification, but instead suggesting that the vesicles had not been endocytosed (Fig. 6H). This effect was rescued by transfecting Actg in the KO cultures (Fig 7 A,B). Taken together this data suggests that Actin, Gamma is important for endocytosis.
.. Experimental description: Krox20 Cre/+;Actg LoxP/LoxP mice were created, which knocked out Actin, Gamma in the Calyx of Held. 200 uM parasaggital brain stem slices were cut for capacitance recordings. Actg LoxP/LoxP were used as controls.
Mouse hippocampal cultures were prepared using Actg LoxP/LoxP mice. Mice were transfected with a Cre-mCherry plasmid to create knockout.
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The species of the over-expression construct (Fig.7) is not relevant for species selection since serving as control for specificity of loss of native mouse protein.