The amplitude (Figures 5A and 5B) and frequency (Figures 5C and 5D) of mEPSCs were markedly reduced by TSPAN7 knockdown, both with siRNA14 and siRNA47. These effects were reversed by expressing siRNA14 together with

rescue WT (rescue, Figures 5A–5D). Next, we tested whether TSPAN7 knockdown affected AMPAR subunit composition by using philanthotoxin-433 (PhTx), a specific blocker of AMPARs lacking GluA2. PhTx had no effect on mEPSC amplitude in control neurons (Figure S5A), as reported previously (Thiagarajan et al., 2005). Likewise, PhTx did not reduce mEPSC amplitude in neurons transfected with siRNA14 or siRNA47 (Figure S5B), suggesting that TSPAN7 knockdown does not preferentially Ivacaftor in vitro deplete synapses of AMPARs containing GluA2, consistent with our finding that both GluA1 and GluA2/3

staining is reduced (Figure 4). These findings therefore show that TSPAN7 knockdown results in markedly impaired postsynaptic excitatory transmission. Because reduced mEPSC frequency in TSPAN7 knockdown pyramidal cells (Figures 5C and 5D) could be due to either Selleckchem I BET151 reduced release probability or reduced number of functional synapses, we discriminated between these possibilities by studying evoked synaptic AMPAR and NMDAR currents between pairs of primary hippocampal pyramidal neurons (Figure S6). As in the mEPSC recordings, only the postsynaptic neuron was transfected with siRNA14, whereas the presynaptic cell expressed normal levels of TSPAN7. Under these conditions, evoked AMPAR currents were strongly reduced (Figure S6A, bottom), consistent with the effects on mEPSCs (Figure 5). By contrast, evoked NMDAR currents were not significantly affected by TSPAN7 knockdown (Figure S6A, top), consistent with our findings on GluN1 immunolocalization ADAMTS5 (Figure 4). As a consequence, the NMDA/AMPA ratio was

markedly and significantly increased in TSPAN7 knockdown neurons (Figure S6B, right). By contrast, the paired-pulse ratio—a measure related to presynaptic release probability—was not significantly affected (Figures S6A and S6B, left). These findings therefore suggest that postsynaptic loss of TSPAN7 compromises excitatory synaptic transmission by selectively impairing AMPAR over NMDAR currents, with negligible effects on presynaptic release probability. As a consequence, TSPAN7-deprived neurons have more silent synapses. To gain insights into how TSPAN7 influences postsynaptic organization and synaptic transmission, we next identified proteins that bind TSPAN7. We used a yeast two-hybrid system, with the cytoplasmic C terminus of TSPAN7 (aa 234–249; Figure 6A) as bait, to screen a human fetal brain cDNA library. Four prey cDNA clones were isolated (clones 40, 48, 15, and 28, Figure 6A), all of which encoded PICK1.