This system allowed us to monitor synaptic transmission in the same neuron before and after selective Thr-induced cleavage of NLG1. Recordings from neurons expressing GFP-NLG1 lacking a Thr cleavage site were used to control for possible off-target effects of Thr activity. We first examined the effect of NLG1 cleavage on miniature excitatory postsynaptic currents (mEPSCs; Figure 6A).

Application of Thr for 30 min reduced mEPSC frequency (44% ± 10% of baseline; Figures 6B and S6A) with no significant effect on amplitude (91% ± 8% of baseline; Figures 6C and S6A), indicating that NLG1 ectodomain cleavage does not change postsynaptic AMPA receptor number or function, but rather reduces neurotransmitter release or decreases the number of functional excitatory synapses. By contrast, Thr had no effect on mEPSCs in neurons expressing GFP-NLG1 (mEPSC frequency, 100% ± GDC-0068 chemical structure 3% of baseline; amplitude, 102% ± 4% of baseline; Figures 6A–6C and S6B). Importantly, there was no difference in baseline mEPSCs between neurons expressing GFP-NLG1 (frequency: 1.74 ± 0.44 s−1; amplitude: 11.66 ± 1.70 pA) and GFP-Thr-NLG1 (frequency: 2.00 ± 0.36 s−1; amplitude: 10.74 ± 0.61 pA). Consistent

with the predominant localization of NLG1 to glutamatergic synapses (Chubykin et al., 2007; Graf et al., 2004), NLG1 cleavage failed to alter miniature inhibitory postsynaptic current (mIPSC) frequency MycoClean Mycoplasma Removal Kit or amplitude (mIPSC frequency: this website 103 ± 3%; mIPSC amplitude: 98% ± 8% of baseline; Figures S6C–S6F), indicating that NLG1 cleavage does not acutely alter GABAergic transmission. The reduction in mEPSC frequency (Figures 6A, 6B, and S6A) together with the destabilization of NRX1β (Figures 5C–5G) suggested a possible alteration in presynaptic function. To test this further, we examined the effects of Thr-induced NLG1 cleavage on evoked excitatory synaptic currents (eEPSCs) and their paired-pulse responses.

Evoked responses were elicited by stimulating nearby cells with an interstimulus interval of 100 ms. Neurons expressing GFP-NLG1 or GFP-Thr-NLG1 exhibited similar baseline paired-pulse ratio (PPR) responses (GFP-NLG1: 1.42 ± 0.12; GFP-Thr-NLG1: 1.59 ± 0.46). Recordings were then made on the same cell before and 30 min after thrombin application. Thrombin treatment of neurons expressing GFP-Thr-NLG1 reduced the amplitude of the first response and increased PPR (eEPSC amplitude: 50% ± 18% of baseline; PPR: 139% ± 8% of baseline; Figures 6D–6F), strongly suggesting that NLG1 cleavage decreases the probability of neurotransmitter release. This effect was not attributable to Thr treatment per se, as there was no change in eEPSC amplitude or PPR in neurons expressing GFP-NLG1 lacking a Thr cleavage sequence (eEPSC amplitude: 95% ± 8% of baseline; PPR: 106% ± 6% of baseline; Figures 6D–6F).