Elucidating the Mechanism of the Spinal Effects by VGF-derived Peptide TLQP-62

Abstract ID 92776 Poster Board 120 Introduction: VGF (non-acronymic) is a neurosecretory protein implicated in neuroplasticity associated with depression, learning, memory, and chronic pain. We previously demonstrated that VGF-derived peptides are involved in the establishment of hypersensitivity in mouse models of chronic pain. We have also shown that the peptide TLQP-62 potentiates calcium responses to glutamate (Glu) in a subset of spinal neurons in naïve male mice. Studies in the hippocampus have suggested that the actions of TLQP-62 are mediated in part by BDNF/TrkB signaling, and in spinal dorsal horn BDNF/TrkB signaling has been shown to participate in neuroplasticity mediated by the NMDA receptor (NMDAR) subunit GluN2B. This study aims to determine the mechanism of the spinal effects of TLQP-62. We hypothesized that the potentiation of spinal Glu responses by TLQP-62 requires GluN2B-NMDAR. Methods: Calcium imaging was conducted in transverse spinal cord slices in wildtype C57BL/6J and GluN2Bfl/fl mice, both male and female. Since GluN2B can be deleted in a Cre-dependent manner in GluN2Bfl/fl mice, we injected GluN2Bfl/fl mice intraspinally at the L3 and L4 spinal levels (300 nL/injection) at 3-4 weeks of age with AAV9-hSyn-GCaMP6s-Cre or control AAV9-hSyn-GCaMP6s-ΔCre, in which Cre-recombinase is inactive and does not induce GluN2B deletion. Calcium imaging was performed 4 weeks after viral injections. Fluo-4 was utilized in spinal slices from C57BL/6J mice to visualize spinal calcium transients. Fluo-4 or GCaMP6s fluorescence intensity was captured as time-lapse images by two-photon microscopy in spinal cord dorsal horn. Slices were sequentially exposed to the following treatments: 1) agonist (10-20 seconds of 100 μM Glu, 100 μM NMDA, or 5 μM AMPA); 2) 50 nM TLQP-62 or aCSF (15 min); 3) same agonist as 1; 4) 1mM Glu (10 s). The fold change in response amplitudes to the two agonist treatments was calculated for each cellular profile. The potentiated response was defined as fold change greater than the mean+2SD of profiles from aCSF-treated slices. Results: In C57BL/6J wildtype mice, a subset of spinal profiles showed potentiated Glu response following TLQP-62 incubation in both male and female mice (n = 3-4/sex, potentiated cells in male and female: 5/30 and 6/15). NMDA responses were also potentiated by TLQP-62 (n = 5-6/sex, 5/36 and 6/36), while AMPA responses did not appear to be affected by the peptide in either sex (n = 3-4/sex, 2/24 and 0/22). In virus-injected GluN2Bfl/fl mice, Cre-dependent deletion of GluN2B was confirmed by loss of ifenprodil inhibition of NMDA response (n = 3/sex, P<0.01, Student's t-test). The fold change in Glu response amplitudes was significantly reduced in TLQP-62-exposed cells from Cre- compared to ΔCre-injected male mice (p < 0.05, Student's t-test). Similarly, the proportion of profiles with potentiated responses was significantly reduced in Cre- compared to ΔCre-injected mice (n = 6-8, 4/49 and 18/40). These Cre-dependent differences were not observed in the cellular profiles of female mice (n = 5-7, 7/38 and 3/43). Conclusions and future directions: These results suggest that NMDARs but not AMPARs are involved in TLQP-62-induced potentiation of Glu responses. The GluN2B subunit mediates potentiation by TLQP-62 in male mice but not in female mice. Ongoing experiments are investigating the contribution of TrkB to TLQP-62's spinal action. This work was supported by DOD Grant W81XWH2010509 to L.V. and the College of Pharmacy of the University of Minnesota.