Upcoming experiments will define the downstream effector proteins activated by MEK and PI3K in mGluR-dependent LTP, and will also help define the role of Homer1c in cognitive impairment associated with neurological disorders toward finding therapeutic strategies based on the gene replacement or pharmaceutical intervention for memory loss. DETAILED METHODS Animal Subjects and Vector Injections The mice used in these experiments have been described previously BMS-986165 (Yuan et al., 2003). of LTP in acute hippocampal slices from KO+H1c. These data show that Homer1cCmGluR5 interactions are necessary for mGluR-dependent LTP, and that mGluR1/5-dependent LTP entails PI3K and ERK activation. 0.0003). This successful transformation of STP into a prolonged LTP in LM-WT mice recapitulates the findings by the previous groups in SpragueCDawley rats (Bortolotto et al., 1994; Cohen and Abraham, 1996; Cohen et al., 1998; Raymond et al., 2000; Piccinin et al., 2008). In addition to the prolonged LTP induced by this protocol, there was an increase in the magnitude of LTP induction during activation in LM-WT exposed to DHPG versus the LM-WT that received only 0.5 TBS (Fig. 1A, 0.001, 2-min poststimulation). Open in a separate window Physique 1 Homer1c restores mGluR-dependent LTP in KO mice. (A) Priming with DHPG prior to 0.5 TBS results in robust LTP in slices from wild-type mice (= 17 slices (from 9 mice)) relative to nonprimed slices (= 8(3)). (B) H1-KO mice injected with GFP show an failure to transform an STP into LTP via activation BMS-986165 of mGluR1/5 (KO+GFP+DHPG, = 13(6)), KO+GFP (= 9(4)). (C) H1-KO expressing TSPAN7 Homer1c (KO+H1c+DHPG; = 5(3)) show an enhanced ability to maintain a strong mGluR-dependent LTP relative to KO+H1c nonprimed slices (= 4(3)). Black horizontal BMS-986165 line indicates time period of 10 M DHPG application. BMS-986165 Half-train of TBS activation is usually applied at a of time 0 min. Top: representative traces at time of 0.5 TBS and at the end of the recording (120 min; vertical level bars 1 mV, horizontal bars, 1 msec). To test the hypothesis that Homer1c plays a role in mGluR1/5-dependent LTP, we investigated this form of plasticity in H1-KO mice in the absence or presence of Homer1c. We have previously shown strong transgene expression in the dorsal hippocampus using rAAV delivery of Homer1c and green fluorescence protein (GFP) in H1-KO mice (Gerstein et al., 2012). We injected H1-KO mice with either rAAVCGFP (KO+GFP) or rAAVCHomer1c (KO+H1c). GFP injection does not impact synaptic plasticity or behavior in these animals and therefore is a good control for surgery and transgene expression (Gerstein et al., 2012). We found that H1-KO mice show deficits in this form of synaptic plasticity. H1-KO+GFP cannot induce LTP when a 0.5-TBS is preceded by mGluR1/5 activation (Fig. 1B). Expression of Homer1c in H1-KO mice resulted in LTP persistence upon priming with DHPG (Fig. 1C; main effect of treatment 0.0001). This plasticity profile is usually highly comparable to LM-WT (LM-WT+DHPG vs. KO+H1c+DHPG, no main effect of genotype, = 0.6269). The maintenance of LTP seen in the KO+H1c was significantly better than that of the KO+GFP slices (Figs. 1B, C; KO+H1c+DHPG vs. KO+GFP+DHPG, main effect of treatment, 0.0021). There was also an increase in the magnitude of LTP induction during activation in KO+H1c+DHPG versus KO+H1c-DHPG (Fig. 1C, 0.001, 2-min poststimulation). Thus, Homer1c expression in the hippocampus of H1-KO is sufficient for mGluR1/5 activation to convert STP into a prolonged LTP. Next, we set to determine whether mGluR1 or mGluR5 is the specific receptor subtype activating this molecular switch in our animal model. The mGluR5-selective noncompetitive antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and mGluR1-selective competitive antagonist “type”:”entrez-nucleotide”,”attrs”:”text”:”LY367385″,”term_id”:”1257996803″,”term_text”:”LY367385″LY367385 were used to block LTP. Preincubation of wild-type hippocampal slices with the MPEP but not “type”:”entrez-nucleotide”,”attrs”:”text”:”LY367385″,”term_id”:”1257996803″,”term_text”:”LY367385″LY367385 blocked LTP in the presence of DHPG (Fig. 2A; main effect of treatment WT+DHPG vs. WT+DHPG+MPEP; 0.0001; WT+DHPG vs. WT+DHPG+”type”:”entrez-nucleotide”,”attrs”:”text”:”LY367385″,”term_id”:”1257996803″,”term_text”:”LY367385″LY367385 0.4139). LTP from H1-KO slices overexpressing Homer1c was also selectively blocked by MPEP and not “type”:”entrez-nucleotide”,”attrs”:”text”:”LY367385″,”term_id”:”1257996803″,”term_text”:”LY367385″LY367385 (Fig. 2B, KO+H1c+DHPG+MPEP vs. KO+H1c +DHPG, 0.0062; KO+H1c+DHPG vs. KO+H1c+DHPG+”type”:”entrez-nucleotide”,”attrs”:”text”:”LY367385″,”term_id”:”1257996803″,”term_text”:”LY367385″LY367385: 0.1766). Together, these results indicate that this form of.
