Fig. 7
From: Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways
Alterations in endocytic recycling associated with accumulation of postsynaptic AMPA receptors and defective synaptic plasticity, in syt-17 KOs. a Colocalization of syt17 (Halo fusion construct, magenta), early endosomes (Rab5-GFP, green), and AMPA receptors (anti-GluR, blue) in dendritic spines of a 14 DIV hippocampal neuron. The traces to the right represent normalized fluorescence from the indicated 3 µm linescan. b Left: L-Glu was pressure-applied to apical dendrites. Middle: Representative traces of AMPAR Glu response at −70 and +40 mV in WT, KO, and KO+rescue neurons. Right: I–V plot showing current as a function of holding voltage. The amplitude of postsynaptic responses in syt-17 KO neurons was uniformly increased (except near the reversal potential), and this effect was rescued by expression of exogenous syt-17. c Left: GluR2-pHluorin was exogenously expressed in WT and KO neurons. Right: Spine fluorescence of GluR2-pHluorin was quantified in ACSF of pH 7.4 (extracellular pH) and 5.5 (vesicular pH), and in the presence of NH4Cl to unquench all pHluorin. Traces show intensity at indicated linescans. d Surface expression of GluR2-pHluorin was increased in syt-17 KO (t21 = 2.417, p = 0.02, r2 = 0.218, meanwt = 51.95 ± 3.27% surface fraction, meanko = 64.01 ± 3.81, Nwt = 12 and Nko = 11 neurons). e Rab5-GFP was expressed in WT and KO neurons. f KO neurons had significantly fewer early endosomes per unit dendrite (t35 = 4.529, p < 0.001, r2 = 0.37, meanwt = 5.75 ± 0.43 early endosomes per 10 μm dendrite, meanko = 3.42 ± 0.29, Nwt = 18 and Nko = 19 neurons). Scale bar indicates 10 μm. All experiments were performed on 3–4 independent preparations of animals. g Chemical long-term depression (LTD) along the Shaffer collaterals in hippocampal slices. Right: Representative field excitatory postsynaptic potentials (fEPSPs, 50% of maximal) before (solid lines) and after (dashed lines) five-minute NMDA application. h KO slices fail to exhibit LTD following the induction protocol (p = .012 two-sample t-test post-induction). Experiments were performed on slices from five animals per genotype. All error bars indicate S.E.M.s
