Presynaptic Deletion of GIT Proteins Results in Increased Synaptic Strength at a Mammalian Central Synapse

Abstract

A cytomatrix of proteins at the presynaptic active zone (CAZ) controls the strength and speed of neurotransmitter release at synapses in response to action potentials. However, the functional role of many CAZ proteins and their respective isoforms remains unresolved. Here, we demonstrate that presynaptic deletion of the two G protein-coupled receptor kinase-interacting proteins (GITs), GIT1 and GIT2, at the mouse calyx of Held leads to a large increase in AP-evoked release with no change in the readily releasable pool size. Selective presynaptic GIT1 ablation identified a GIT1-specific role in regulating release probability that was largely responsible for increased synaptic strength. Increased synaptic strength was not due to changes in voltage-gated calcium channel currents or activation kinetics. Quantitative electron microscopy revealed unaltered ultrastructural parameters. Thus, our data uncover distinct roles for GIT1 and GIT2 in regulating neurotransmitter release strength, with GIT1 as a specific regulator of presynaptic release probability.

Figure 1. GIT proteins regulate AP-evoked release and P_r

A) Representative traces of single AP-evoked EPSCS from WT, Git2^−/−, Git1^−/−, or Git1^−/−/Git2^−/− calyces. B) Summary data showing average EPSC amplitudes. C) Cumulative frequency histogram of EPSC amplitudes. D) Average normalized EPSC amplitudes. E) Example traces in response to a 100Hz stimulus train from WT, Git2^−/−, Git1^−/−, or Git1^−/−/Git2^−/− calyces. F) Cumulative plots of EPSC amplitudes with back-extrapolated linear fits. G) Average values for the calculated RRP size. H) Cumulative frequency histogram of RRP size. I) P_r obtained by dividing the amplitude of the first EPSC from the 100Hz train by the calculated RRP size. J) Cumulative frequency histogram of P_r. *p<0.05, **p<0.01, ***p<0.001 One-way ANOVA with a post hoc Dunnett’s test.

Figure 2. Synaptic plasticity is altered by the loss of GIT expression

A) Example traces of the EPSC obtained by fiber stimulation after applying two consecutive pulses at the frequencies indicated (a1, 10Hz; a2, 50Hz; a3, 100Hz). Black and white arrow heads indicate the 1^st and 2^nd EPSC, respectively. B) PPR was calculated by dividing the amplitude of the second EPSC by the amplitude of the first EPSC at 10Hz (b1), 50Hz (b2) and 100Hz (b3). C) Summary plot of normalized EPSC amplitude to the first EPSC during train stimuli using 10Hz (c1), 50Hz (c2) and 100Hz (c3) against the stimulus number. D) Steady state depression level measured and plotted as a function of the stimulation frequency (d1, 10Hz; d2, 50Hz; d3, 100Hz). **p<0.01, ***p<0.001 One-way ANOVA with a post hoc Dunnett’s test.

Figure 3. Loss of GIT proteins results in no changes of Ca²⁺ current and activation kinetics

A) Stimulus protocol used for the experiment from B to F. B) Representative I_Ca traces for I/V curve analysis from −80mV to +60mV from WT, Git2^−/−, Git1^−/− and Git1^−/−/Git2^−/− calyces. Insets show in detail the tail currents. C) I/V plot representing the average steady state I_Ca amplitudes plotted against voltage. D) Normalized average raw I_Ca amplitudes to Imax. E) Analysis of voltage dependent activation of VGCCs. F) Normalized tail currents to Imax. G) Stimulus protocol used for the experiment from H to L. H) Representative I_Ca obtained from WT, Git2^−/−, Git1^−/− and Git1^−/−/Git2^−/− calyces. I) Average values of I_Ca charge. J) Cumulative frequency of I_Ca charge. K) Average values of capacitance (C_slow). L) Cumlative frequency of C_slow.

Figure 4. Ablation of GIT expression does not affect the number of docked SV or SV distribution

A) EM sample images taken from WT, Git2^−/−, Git1^−/− and Git1^−/−/Git2^−/− calyces. Top panels: calyx of Held is demarcated in yellow, and the nanogold GFP labeling in green (scale bar=500 nm). Bottom panels: detail of the AZ analyzed. SVs closest to the AZ are shown in red shade and AZ length is been denoted by a red line (scale bar=200 nm). B) Summary of normalized distribution of SV distance from AZs. C) Average number of SVs localized within 5 nm relative to the AZs. D) Cumulative frequency histogram of the AZ length. E) Example of 3D reconstructions from WT, Git2^−/−, Git1^−/− and Git1^−/−/Git2^−/− calyces. F) Average values of surface area and the corresponding cumulative frequency distribution in G). H) Summary data showing average values of volume and the corresponding cumulative frequency distribution in I).