Figure 1. Enhanced Presynaptic Ca²⁺ influx following acute or sustained disruption of postsynaptic glutamate receptor function.

(A) Confocal images of boutons filled with Alexa 568 (left) and Oregon Green 488 BAPTA-1 (OGB-1, right) synapsing onto muscle 6 of a Drosophila NMJ. The red line indicates the location of the line scan.

(B) Example traces of single AP-evoked, spatially-averaged Ca²⁺ transients measured by line scans of a wild-type control (left), and a GluRIIA^SP16 mutant synapse (right) (average of 10 scans each). The decays are fit with an exponential function (red).

(C) Average Ca²⁺ transient peak amplitudes of (ΔF/F, see Experimental Procedures; left), and cumulative frequency plot of ΔF/F peak amplitudes (right) of control (n=26 boutons, gray) and GluRIIA^SP16 (n=28 boutons, red). Note the significant (p<0.001) increase in Ca²⁺ transient amplitude in GluRIIA^SP16 mutants compared to control.

(D) Average baseline fluorescence (‘F_base’) and decay time constant (‘τ’) of the groups introduced in (C).

(E) Average mEPSP amplitude (‘mEPSP’), Ca²⁺-transient peak amplitude of (‘ΔF/F’), and quantal content (EPSP amplitude/mEPSP amplitude; see Experimental Procedures) of GluRIIA^SP16 mutants normalized to w¹¹¹⁸ controls. Electrophysiology data of control (av. mEPSP=0.9±0.08 mV; EPSP=52.3±1.4 mV; n=9) and GluRIIA^SP16 group (av. mEPSP=0.4±0.04 mV; EPSP=51.4±2.2 mV; n=7) is in part based on a separate set of experiments.

(F) Average ΔF/F peak amplitudes of control group (n=28 boutons; gray), and PhTX group (n=36 boutons, red). Note the significant (p=0.023) increase in Ca²⁺ transient amplitude after PhTX application.

(G) Average baseline fluorescence (‘F_base’) and decay time constant (‘τ’) of the data set introduced in (F).

(H) Left: Average mEPSP amplitude, Ca²⁺-transient peak amplitude of (‘ΔF/F’), and quantal content after PhTX treatment normalized to controls. Electrophysiology data of control (av. mEPSP=0.9±0.1 mV; EPSP=48.3±1.7 mV; n=13) and PhTX group (av. mEPSP=0.47±0.03 mV; EPSP=51.2±2.6 mV; n=14) is in part based on a separate set of experiments. Right: Average quantal content (corrected for nonlinear summation; see Experimental Procedures) as a function of presynaptic Ca²⁺ transient peak amplitude (ΔF/F) of the indicated conditions and genotypes. Data are the same as in A – G. Data of experimental groups and control groups were collected side by side.