Reduced SNAP-25 alters short-term plasticity at developing glutamatergic synapses

Abstract

SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.

Figure 1

Enhanced evoked glutamatergic transmission in het cultures at 14 div. (A–B) Traces of mEPSCs and mIPSCs from wt or het neurons followed by the analysis of frequency and amplitude ((A) frequency, t-test, P=0.399; N=4; amplitude: KS-test not significant; (B) frequency, t-test, P=0.49, N=4; amplitude: KS-test not significant). (C–D) Traces of eEPSCs (C) and eIPSC (D) from wt or het neurons. (E) Analysis of eEPSCs: wt (n=10) versus het (n=10), t-test, P<0.01, N=4; eIPSCs: wt (n=13) versus het (n=8), t-test, P<0.01, N=3). Error bars indicate s.e.m. KS-test, Kolmogorov–Smirnov test; wt, wild type. **P<0.01.

Figure 2

Enhanced glutamatergic transmission in het cultures does not depend on synapse number or RRP size. (A) Traces and quantification of calcium responses on: (a) KCl: wt (n=76) versus het (n=63), t-test, P=0.007; (b) AMPA: wt (n=22) versus het (n=22), t-test, P=0.33; (c) NMDA: wt (n=84) versus het (n=52) t-test, P=0.15; N=3. (B) Staining of neurons for SV2A (red) and β_III tubulin (green) (top panels) and for β_III tubulin (green), vGAT (red) and vGlut (blue). Calibration bar=10 μm. (C) SV2 puncta/20 μm: wt (n=24) versus het (n=26), t-test, P=0.82. (D) Measurement of vGAT/vGlut puncta: wt (n=34) versus het (n=54), t-test, P=0.89. (E) Sucrose-evoked responses from wt and het neurons and charge response: wt (n=12) versus het (n=12), t-test, P=0.55, N=3). (F) Charge transfer in calcimycin experiment: wt (n=13) versus het (n=11) t-test, P=0.06, N=3. Error bars indicate s.e.m. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartic acid; wt, wild type.

Figure 3

Shift from paired-pulse facilitation to paired-pulse depression at 14 DIV hippocampal het synapses. (A–B) Traces of short-term plasticity experiments. (C) PPR at glutamatergic synapses: wt (n=10) versus het (n=10), t-test, P<0.001, N=4; GABAergic synapses: wt (n=13) versus het (n=8), t-test, P<0.001, N=4. (D) PPR plotted against different interpulse intervals. PPR ISI 100: t-test, P=0.048; PPR ISI 150: t-test P=0.619. (E) Merged bright field and fluorescence image of a mixed culture (wt-GFP with het neurons). Calibration bar=10 μm. (F) Short-term plasticity in pairs where either the presynaptic [2] or the postsynaptic [1] neuron was het for SNAP-25. (G) PPR: (pre)-wt-GFP=1.44±0.21, (n=5); (pre)-het=0.85±0.2 (n=5) t-test, P=0.001. (H) Quantitive western blotting for SNAP-25 expression: wt versus het 14 DIV, t-test, P=0.042. (I) eEPSC: wt (n=4) versus het (n=9), t-test, P=0.01, N=3. (J) PPR: glut: wt (n=4) versus het (n=9), t-test, P=0.058; GABA: wt (n=4), het (n=5), t-test, P=0.06, N=2. (K) Calcium responses: wt (n=62) versus het (n=55), t-test, P=0.83, N=3. Error bars indicate s.e.m. DIV, days in vitro; GABA, γ-aminobutyric acid; GFP, green fluorescent protein; NS, not significant; PPR, paired-pulse ratio; SNAP-25, synaptosomal-associated protein of 25 kDa; wt, wild type. *P<0.05; **P<0.01; ***P<0.005.

Figure 4

Paired-pulse depression after acute downregulation of SNAP-25 expression in the presynaptic neuron. (A) SNAP-25 labelling of rat neurons co-transfected with either SNAP-25 siRNA plus GFP or scramble siRNA plus GFP. Large arrows point to neurons transfected with either siRNA (top panels) or scramble (bottom panels) constructs. Small arrows point to non-transfected neurons. Calibration bar=20 μm. (B–C) top: cartoons of recording configurations. (B) (1) stimulation of presynaptic siRNA-treated neuron; (2) stimulation of presynaptic scramble neuron. Bottom: traces of experiments described above; (C) bottom: traces of recordings experiments in scramble siRNA-treated postsynaptic neuron. (D) PPR in scramble siRNA presynaptic (n=6) versus SNAP-25 siRNA presynaptic (n=7), t-test, P=0.018. (E) PPR in SNAP-25 siRNA postsynaptic (n=3) versus scramble siRNA postsynaptic (n=5), t-test, P=0.87, N=4. (F) Examples of 14 DIV rat hippocampal cultures immunostained for SNAP-25 (green) and vGlut1 (blue) on reduction of SNAP-25 expression through stealth oligonucleotides. Calibration bar=10 μm. (G) Western blotting showing varied reduction of SNAP-25 levels following stealth oligonucleotide treatment. (H) Plot of PPR versus residual levels of SNAP-25 in rat hippocampal cultures treated with stealth oligonucleotides. The value of PPR relative to SNAP-25 levels recorded in 21 DIV het neurons is pointed by the arrow. Error bars indicate s.e.m. DIV, days in vitro; GFP, green fluorescent protein; PPR, paired-pulse ratio; siRNA, small interfering RNA; SNAP-25, synaptosomal-associated protein of 25 kDa. *P<0.05.