Fig 2: Vti1a and VAMP7 loss elicits scaling of unitary event amplitudes without changes in excitatory network activity.(a) Representative traces showing background network activity recordings from control and vti1a/VAMP7 DKD neurons cultured from rat hippocampus. (b) No differences were seen in average total charge transfer between control and vti1a/VAMP7 DKD neurons from 3 min of recording (control: n=9 neurons from three independent cultures; vti1a/VAMP7 DKD: n=10 neurons from three independent cultures; P=0.55). (c) No differences were seen in average burst number between control and vti1a/VAMP7 DKD neurons during background network activity analysed in b (P=0.82). (d) Average AMPA-mEPSC amplitudes from vti1a/VAMP7 DKD neurons were significantly increased compared with control neurons from recordings analysed in Fig. 1d (P=0.0005). (e) Cumulative probability histograms of AMPA-mEPSC amplitudes from recordings analysed in Fig. 1d (Kolmogorov–Smirnov test; P=0.0001). (f) Rank-order plot of AMPA-mEPSC amplitudes from recordings analysed in Fig. 1d. The slopes of the linear fits indicate a multiplicative increase (70% scaling) in vti1a/VAMP7 DKD neurons. (g) Representative traces showing evoked asynchronous unitary EPSC recordings in the presence of Sr2+ and absence of Ca2+ in control and vti1a/VAMP7 DKD neurons. The top scale refers to the evoked bulk events and the bottom scale refers to the enlarged insets showing asynchronous unitary EPSCs. (h) Rank-order plot of asynchronous evoked unitary AMPA-EPSC amplitudes from control and vti1a/VAMP7 DKD neurons (control: n=5 recordings from three independent cultures; vti1a/VAMP7 DKD: n=4 recordings from two independent cultures). The slopes of the linear fits of the two curves indicate a multiplicative increase (64% scaling) of unitary event amplitudes in vti1a/VAMP7 DKD neurons compared with control neurons.

Fig 3: Postsynaptic loss of vti1a and VAMP7 is insufficient to elicit synaptic scaling of excitatory unitary event amplitudes.(a) Representative image of a GFP-positive neuron expressing shRNA constructs against vti1a and VAMP7 overlaid by a brightfield image showing many neighbouring GFP-negative (non-transfected control) neurons. Scale bar, 30 µm. (b) Diagram of experimental design to test the potential postsynaptic effects of vti1a/VAMP7 DKD in rat hippocampal cultures. AMPA-mEPSC recordings were made from both GFP-negative, control neurons and GFP-positive, vti1a/VAMP7 DKD-transfected neurons that receive predominantly control presynaptic inputs due to very low transfection efficiency. (c) Representative AMPA-mEPSC recordings from non-transfected (control) and vti1a/VAMP7 DKD-transfected neurons. (d) AMPA-mEPSC amplitudes did not differ between non-transfected and vti1a/VAMP7 DKD-transfected postsynaptic neurons (non-transfected: n=10 recordings from three independent cultures; vti1a/VAMP7 DKD-transfected: n=11 recordings from three independent cultures; P=0.56).

Fig 4: Presynaptic loss of vti1a and VAMP7 impairs NMDA receptor block-induced synaptic plasticity in Schaffer collateral synapses.(a) Diagram of the experimental design to test the effects of vti1a/VAMP7 DKD in axons of mouse hippocampus. In vivo viral delivery to area CA3 of shRNA constructs against vti1a and VAMP7 with a GFP marker was followed by acute slice preparation and field potential electrophysiology of the Schaffer collateral synaptic pathway in CA1. Dotted line represents removal of area CA3 before recording. DG, dentate gyrus. (b) Composite image at 10 × magnification of GFP expression in a hippocampal slice made 3 weeks after injection of vti1a/VAMP7 DKD virus into area CA3. Scale bar, 500 µm. Top inset: 63 × magnification of the boxed region in the left image. GFP-positive Schaffer collateral axons can be observed. Bottom inset: 4 × optical zoom of the boxed region in the top inset. (c) Input–output responses were not altered by presynaptic vti1a/VAMP7 DKD (control: n=25 slices from 17 mice; vti1a/VAMP7 DKD: n=27 slices from 18 mice; control slope versus vti1a/VAMP7 DKD slope, P=0.76). Inset: representative trace showing that the initial response slope is measured against the amplitude of the presynaptic fibre volley. Scale is 0.4 mV by 2 ms. (d) Paired-pulse facilitation was not significantly different at 20, 30, 50, 100, 200, 400 or 500 ms inter-stimulus intervals after vti1a/VAMP7 DKD (control: n=37 slices from 21 mice; vti1a/VAMP7 DKD: n=36 slices from 20 mice; corrected P>0.05 for all inter-stimulus intervals). Inset: representative traces showing responses at the various inter-stimulus intervals. Scale is 0.5 mV by 100 ms. (e) Ketamine-induced synaptic potentiation was reduced by presynaptic vti1a/VAMP7 DKD (control: n=25 slices from 17 mice; vti1a/VAMP7 DKD: n=21 slices from 14 mice; P=0.019). Solid horizontal lines indicate application of 20 µM ketamine and then ACSF wash. Inset: representative traces from before (solid line) and after (dashed line) ketamine treatment. Scales are 0.3 mV (control) and 0.2 mV (vti1a/VAMP7 DKD) by 2 ms. (f) Long-term potentiation (LTP) was not altered by presynaptic vti1a/VAMP7 DKD (control: n=11 slices from seven mice; vti1a/VAMP7 DKD: n=12 slices from eight mice; P=0.69). Arrow indicates when theta burst stimulation (TBS) was applied to Schaffer collateral axons. Inset: representative traces showing increased response (dashed line) after LTP induction. Scales are 0.3 mV (control) and 0.2 mV (vti1a/VAMP7 DKD) by 4 ms.

Fig 5: Vti1a and VAMP7 loss in hippocampal area CA1 is insufficient to alter neurotransmission in Schaffer collateral synapses.(a) Diagram of the experimental design to test the effects of vti1a/VAMP7 DKD in area CA1 of mouse hippocampus. In vivo viral delivery to area CA1 of shRNA constructs against vti1a and VAMP7 with a GFP marker was followed by acute slice preparation and field potential electrophysiology of the Schaffer collateral synaptic pathway. Dotted line represents removal of area CA3 before recording. DG, dentate gyrus. (b) Left: composite image at 10 × magnification of GFP expression in a hippocampal slice made 3 weeks after injection of vti1a/VAMP7 DKD virus into area CA1. Scale bar, 500 µm. Right: 40 × magnification of areas indicated by the white boxes. Scale bar, 100 µm. (c) Input–output responses were not altered by vti1a/VAMP7 DKD (control: n=10 slices from 7 mice; vti1a/VAMP7 DKD: n=12 slices from 10 mice; control slope versus vti1a/VAMP7 DKD slope, P=0.99). Inset: representative trace showing that the initial slope of the excitatory postsynaptic potential (EPSP) is measured against the amplitude of the presynaptic fibre volley. Scale is 0.3 mV by 2 ms. (d) Paired-pulse facilitation was not significantly different at 20, 30, 50, 100, 200, 400 or 500 ms inter-stimulus intervals after vti1a/VAMP7 DKD (control: n=14 slices from 7 mice; vti1a/VAMP7 DKD: n=18 slices from 11 mice; corrected P>0.05 for all inter-stimulus intervals). Inset: representative traces showing responses at the various inter-stimulus intervals. Scale is 0.5 mV by 100 ms. (e) Ketamine-induced synaptic potentiation was not altered by vti1a/VAMP7 DKD (control: n=10 slices from 7 mice; vti1a/VAMP7 DKD: n=6 slices from 6 mice; P=0.77). Field potential responses are normalized to the average baseline slope. Solid horizontal lines indicate application of 20 µM ketamine and then ACSF wash. Inset: representative traces from before (solid line) and after (dashed line) ketamine treatment. Scales are 0.2 mV (control) and 0.3 mV (vti1a/VAMP7 DKD) by 2 ms.