We found that FSTL1 mainly appeared in small vesicle fractions that contained synaptoporin, a member of the synaptophysin superfamily and an integral membrane component of synaptic vesicles in afferent terminals (Sun et al., 2006), but not in LDCV fractions labeled by CGRP (Figure 2C). Thus, FSTL1 is localized to small translucent vesicles. The identity of FSTL1 vesicles was further analyzed in the afferent axons of dorsal roots. Double

immunostaining showed that ∼53% of FSTL1 vesicles (n = 2050 in 212 axon profiles) contained vesicular glutamate transporter 2 (VGluT2) in the axons of rats (Figure 2D) and mice (Figure S3A). Also, ∼39% of the vesicles contained vesicle-associated membrane protein 2 (VAMP2) (n = 378 in 35 axon profiles) Cell Cycle inhibitor (Figure 2D). However, only a small number of FSTL1 vesicles contained synaptoporin (∼12%, n = 2624 in 138 axon profiles) or synapsin (∼14%, n = 777 in 64 axon profiles) (Figure 2D). Thus, newly synthesized FSTL1 is mainly transported via VGluT2- and VAMP2-containing vesicles, while only a small amount of FSTL1 is transported via the vesicles carrying synaptoporin and synapsin

(Hannah et al., 1999 and Santos VX770 et al., 2009). We directly examined the potential secretion of FSTL1 in DRG neurons cultured from young rats. Immunoblot analysis showed that the level of FSTL1 in the culture medium was increased in the absence of the stimulus (Figure 2E), indicating spontaneous secretion of FSTL1. Furthermore, FSTL1 secretion was elevated by K+-induced membrane Amisulpride depolarization (55 mM KCl, 1 hr), but only in the presence of extracellular Ca2+ ([Ca2+]o) (Figures 2F and 2G). The FSTL1 level was increased by the TRPV1 channel activator, capsaicin, and that response was abolished by the TRPV1 channel blocker, capsaizepine (Figure 2F). Additionally, high-K+ stimulation for 15 min enhanced FSTL1 secretion from the spinal cord slices of rats (Figure 2H) and mice

(Figure S3B) as well as synaptosomes prepared from the spinal dorsal horn (Figure 2I). As expected, the secretion of CGRP, SP, and glutamate was also increased in the same preparation (Figures 2H and 2I). The stimulus-evoked secretion of FSTL1 from synaptosomes occurred only in the presence of [Ca2+]o (Figure 2I). In contrast, the spontaneous secretion of tenascin-C, an extracellular matrix glycoprotein (Joester and Faissner, 2001), was unaffected by the K+ stimulation (Figure 2I and Figure S3C). Together, these results suggest that FSTL1 is stored in small translucent vesicles and can be secreted either spontaneously or by depolarization in a manner similar to neurotransmitters. To determine whether secreted FSTL1 plays a physiological role in the spinal cord, we performed whole-cell recording of lamina II neurons to monitor afferent synaptic transmission in dorsal root-attached spinal cord slices from rats (Nakatsuka et al., 2000).