The secreted form of recombinant FSTL1 was glycosylated and had a molecular mass of ∼37–45 kDa (Figure S3D). Deglycosylated FSTL1 had a molecular weight of ∼34 kDa (Figure S3D). Recombinant glycosylated FSTL1 was used to examine the effect of

exogenous FSTL1 on synaptic transmission between afferent terminals and local neurons in lamina II (substantia gelatinosa), a translucent band in the superficial dorsal horn. We found that among ∼50% (16/31) of recorded neurons, application of FSTL1 (60 or 300 nM) resulted in a reduction of more than 10% in the mean frequency or mean amplitude Vorinostat mw of spontaneous excitatory postsynaptic currents (sEPSCs) (Figure 3B). Similar application of FSTL1 also reduced the amplitude of C-fiber stimulation evoked EPSCs (eEPSCs), which were present in 50% (12/24) of recorded monosynaptic neurons (Figures 3C and 3D). A similar inhibitory effect of FSTL1 (60 nM) was found in polysynaptic neurons

(Figure S3E). Further studies of miniature EPSCs (mEPSCs) in the presence of tetrodotoxin (0.5 μM) showed that FSTL1 also reduced the frequency, but not the amplitude, of mEPSCs (Figure 3E), suggesting that there is presynaptic suppression of glutamate release by FSTL1. The decay kinetics of eEPSCs were unaffected by FSTL1 (Figure 3D), indicating that FSTL1 had no direct effect on postsynaptic glutamate receptor channel properties. Together, these results suggest that FSTL1 acts presynaptically on neurotransmitter release, rather than postsynaptically on glutamate responses. selleck chemicals llc In addition, for neurons that showed FSTL1-induced eEPSC reduction, the synaptic delay after C-fiber stimulation was reversibly increased (Figure 3C),

oxyclozanide suggesting a presynaptic reduction of excitation-secretion coupling and/or impeded action potential (AP) propagation in the C-fiber. Finally, the specificity of FSTL1 function was indicated by the lack of synaptic suppression effects of follistatin or two mutated forms of FSTL1 that either have a pair of EF-hands deleted (FSTL1ΔEF) or a loss-of-function mutation at the Ca2+-binding site Glu165 (FSTL1E165A), which is conserved across species (Figures 3C and 3E and Figures S3F and S3G). Thus, exogenous FSTL1 suppresses afferent synaptic transmission in the dorsal horn through presynaptic inhibition. How does FSTL1 act on presynaptic nerve terminals? To identify the protein’s membrane target, we used the membrane-impermeant bifunctional reagent bis[sulfosuccinimidyl] suberate (BS3) to chemically crosslink recombinant FSTL1 with myc and His tags to components present on the surface of cultured rat DRG neurons. The cell lysate was analyzed by an immunoblot with myc antibodies.