Volume 64, Issue 2, Pages 227-239 (October 2009) ELKS2α/CAST Deletion Selectively Increases Neurotransmitter Release at Inhibitory Synapses  Pascal S. Kaeser, Lunbin Deng, Andrés E. Chávez, Xinran Liu, Pablo E. Castillo, Thomas C. Südhof  Neuron  Volume 64, Issue 2, Pages 227-239 (October 2009) DOI: 10.1016/j.neuron.2009.09.019 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Generation of the ELKS2α Mutant Mice (A) Schematic representation of the active zone protein complex containing RIMs and ELKS as central components that connect to synaptic vesicles and to all other active-zone proteins. (B) Targeting strategy for the ELKS2 gene, showing (from top to bottom) the ELKS2 gene, a map of the targeting vector, the mutant allele of the founder line, the flp recombined KI allele, and the cre recombined KO allele. N, neomycin resistance cassette; DT, diphtheria toxin expressing cassette; (∗), tetracysteine tag; E1–4, exons 1–4. Coding exons are colored in blue, and 5′UTR exons are open rectangles. (C) Survival analysis of offspring from heterozygous matings of the KI line (top panel) and the KO line (bottom panel). The gray shaded area represents a Mendelian distribution. (D) Force-plate actometer analysis of male littermate KO and wild-type mice from a continuous single trial recording of 30 min. Data were analyzed in three 10 min frames, and two-way ANOVA and Bonferroni post hoc tests were used for statistical and pairwise analysis (see Table S1 for detailed values), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Protein Composition of Brains of ELKS2α KO and Littermate Wild-Type Control Mice (A) Western blotting using chemiluminescence for detection of ELKS1, ELKS2, and multiple active zone proteins in ELKS2α KO and wild-type littermate brain homogenates. (B) Quantitation of brain proteins of ELKS2α KO and wild-type littermate control mice at P50–P55 using 125I-labeled secondary antibodies (n = 3; also see Figures S6A and S6B). (C) Sample western blot (left) with 125I-labeled secondary antibodies and quantitation (right) of the relative expression levels of ELKS isoforms in 50-day-old ELKS2α KO mice and wild-type littermate controls (n = 3; also see Figure S3D). Protein levels are expressed as the percentage of total ELKS1+ ELKS2α in wild-type mice. All data are shown as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Excitatory Synaptic Transmission at Schaffer Collateral-CA1 Pyramidal Cell Synapses in Wild-Type and ELKS2α KO Mice (A) Sample traces (left) and quantitative analysis (right) of mEPSC activity in wild-type and ELKS2α KO mice. (B) Input/output function of ELKS2α KO and wild-type mice, showing the EPSP slope as a function of stimulus intensity. (C) Paired-pulse responses superimposed after subtraction of the first pulse at 10, 20, 30, 100, and 300 ms interstimulus intervals (ISI). (D) Synaptic responses evoked by a burst of 25 stimuli at 14 Hz. All quantitative analyses are reported as means ± SEM, and no statistically significant difference was observed in (A)–(D). Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Inhibitory Synaptic Transmission at CA1 Pyramidal Cells in Wild-Type and ELKS2α KO Mice (A) Sample traces (left) and summary data (right) of mIPSC activity in wild-type and ELKS2α KO mice. (B) Evoked IPSC amplitudes as a function of stimulus intensity plotted as input/output curves in inhibitory synapses. (∗∗∗), sample values of statistical significance: stimulus intensity 1V, p < 0.002; 20V, p < 0.001; 60V, p < 0.001. (C) Paired-pulse responses superimposed after subtraction of the first pulse at 20, 50, 100, and 300 ms ISIs. Statistical significance: ∗p < 0.05, ∗∗p < 0.01. (D) Synaptic responses and normalized summary data evoked by a burst of 20 stimuli at 10 Hz. (∗∗∗), statistical significance of IPSC train ratio, 20th/1st peak: p < 0.001. (E) Excitatory postsynaptic potential-spike coupling (E-S coupling) in wild-type and ELKS2α KO mice, where the input is the excitatory synaptic response and the output is the population-spike. All graphs show means ± SEMs, ∗∗∗p < 0.001. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Ultrastructural Analysis of Synaptic Morphology in Area CA1 of the Hippocampus in ELKS2α KO and Littermate Control Mice (A) Representative images (top) and quantitative analysis (bottom) of asymmetric, excitatory synapses. (B) Analysis of symmetric, inhibitory synapses. All data are shown as means ± SEMs, ∗∗p < 0.01. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Increased Solubility of RIM1α in ELKS2α KO Brain Homogenates (A) Sample western blots with 125I-labeled secondary antibodies of the crude synaptosomal pellet fraction (P2) and the synaptosomal supernatant (S2). (B) Quantitation of protein levels in S2 of three ELKS2α KO mice and wild-type littermate controls. For a complete analysis see Figure S6C and Table S4. All data are shown as mean ± SEM, ∗p < 0.05. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Inhibitory Synaptic Transmission in Conditional ELKS2α KO Synapses in Mixed Hippocampal Cultures (A) mIPSC recordings in cultured ELKS2α KI neurons after lentiviral cre recombinase infection (conditional KO, ELKS2αf/f:cre) or infection with a inactive control lentivirus (control, ELKS2αf/f:control). (B) Evoked IPSC recordings of conditional KO neurons infected with control lentivirus, cre-recombinase expressing lentivirus, or lentivirus coexpressing both cre-recombinase and full-length ELKS2α rescue protein. One-way ANOVA with Newman-Keuls multiple comparison was used for pairwise comparisons; ∗p < 0.05, ∗∗p < 0.01. (C) Paired-pulse recordings in KO and control neurons at various ISIs. (D) Evoked inhibitory synaptic transmission measured as a function of the extracellular Ca2+ concentration measured at 1, 2, and 5 mM extracellular Ca2+. ∗∗p < 0.01 for genotype variation (two-way ANOVA). (E) Network activity in cultured control and ELKS2α KO neurons. Neurons were categorized in “silent” and “active” groups based on the absence or presence of spontaneous action potentials (APs), respectively. All data are shown as means ± SEMs, ∗∗p < 0.01. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 8 Conditional ELKS2α KO Increases the RRP in Inhibitory Synapses (A) Quantitative analysis of the RRP size in inhibitory synapses of conditional ELKS2α KO neurons infected with either control or cre-recombinase expressing lentivirus. The RRP size was measured by application of three concentrations of hypertonic sucrose; synaptic charge transfer was then quantified during the first 10 s (left) and during the steady-state phase (15–30 s, right). Absolute (top) and normalized (bottom) values are shown. All data are reported as mean ± SEM, ∗p < 0.05. (B) Active zones consist of a dense protein network (represented in blue; see Figure 1A for protein constituents) with depressions that form “priming slots” for synaptic vesicles. Our data advocate a hypothetical model wherein these slots can be closed/gated, and upon slot opening the vesicles become available for priming and fusion. ELKS2α and syntaxin-1 in its open confirmation (Gerber et al., 2008) are negative and positive regulators, respectively, of slot opening, keeping the vesicles from joining the pool of vesicles available to the RRP. The exact molecular mechanism by which ELKS2α decreases inhibitory synaptic transmission remains to be elucidated. Neuron 2009 64, 227-239DOI: (10.1016/j.neuron.2009.09.019) Copyright © 2009 Elsevier Inc. Terms and Conditions