05; Figures 1A and 1B). We also confirmed that HFS-LTD is dependent on group I (Gq-coupled) mGluRs by bath application of the group I mGluR antagonist AIDA (96% ± 12%, p < 0.05 compared to control; Figure 1C). Next, we tested potential signaling pathways downstream of Gq. The canonical target of Gq is PLCβ (Hubbard and Hepler, 2006 and Taylor et al., 1991). Surprisingly, we were unable to block HFS-LTD by including the PLC inhibitor U73122 in the intracellular recording solution (54% ± 5%; Figure 1C). This finding was unexpected because other
groups have demonstrated that eCB-mediated depression in MSNs is PLCβ-dependent (Fino et al., 2010, Hashimotodani et al., 2005, Jung et al., 2005 and Yin and Lovinger, 2006) although not in all cases (Adermark and Lovinger, 2007). Therefore, we decided to examine whether the PLCβ-independence of HFS-LTD was unique to that stimulation protocol. As an alternative to HFS-LTD, we applied a low-frequency Selleck Decitabine stimulation (LFS) induction protocol that is qualitatively similar to that used in previous studies of striatal LFS-LTD (Lerner et al., 2010 and Ronesi and Lovinger, 2005). In brief, we repeatedly paired epochs of 20 Hz stimulation with postsynaptic depolarization over several minutes (see Experimental Procedures for details) to induce LTD (56% ± 10%; Figures 1D and 1E). Similar to HFS-LTD, LFS-LTD was blocked
by AM251 and AIDA (95% ± 6% in AM251; 100% ± 8% in AIDA; both p < 0.05 compared to
control; Figures 1E and 1F), indicating a dependence on CB1 receptors and group I mGluRs, respectively. However, LFS-LTD was DAPT also blocked by intracellular U73122 (102% ± 15%; p < 0.05 compared to control; Figure 1F), indicating a role for PLCβ. Thus, both Org 27569 PLCβ-dependent and -independent forms of eCB-LTD can be elicited at excitatory synapses onto striatal indirect-pathway MSNs simply by using different stimulation frequencies and repetitions. PLCβ is an enzyme that produces the intracellular secondary messenger diacylglycerol (DAG), which can be converted to the eCB 2-arachidonylglycerol (2-AG) by the enzyme DAG lipase (DAGL). The sequential activities of PLCβ and DAGL represent a well-defined pathway for 2-AG production that could mediate LFS-LTD. To test whether DAGL is also required for LFS-LTD, we applied the LFS-LTD induction protocol in the presence of the DAGL inhibitor THL and observed that THL blocked LFS-LTD (92% ± 13%; p < 0.05 compared to control; Figure 2A). In addition to DAG, PLCβ produces another important secondary messenger, IP3, which can activate IP3 receptors located on the endoplasmic reticulum and cause release of calcium from internal stores. To test whether internal calcium stores are involved in LFS-LTD, we added thapsigargin, which depletes these stores, to our intracellular recording solution, but this manipulation did not block LFS-LTD (47% ± 7%; Figure 2B).