Title: Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases
Abstract: Article20 December 2011free access Source Data Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases Kai Yang Kai Yang Department of Physiology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Catherine Trepanier Catherine Trepanier Department of Pharmacology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Bikram Sidhu Bikram Sidhu Department of Physiology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Yu-Feng Xie Yu-Feng Xie Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Hongbin Li Hongbin Li Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Gang Lei Gang Lei Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Michael W Salter Michael W Salter Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Beverley A Orser Beverley A Orser Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Takanobu Nakazawa Takanobu Nakazawa Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Michael F Jackson Corresponding Author Michael F Jackson Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author John F MacDonald John F MacDonald Department of Physiology, University of Toronto, Toronto, Ontario, Canada Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Kai Yang Kai Yang Department of Physiology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Catherine Trepanier Catherine Trepanier Department of Pharmacology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Bikram Sidhu Bikram Sidhu Department of Physiology, University of Toronto, Toronto, Ontario, CanadaCo-first authors Search for more papers by this author Yu-Feng Xie Yu-Feng Xie Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Hongbin Li Hongbin Li Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Gang Lei Gang Lei Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Michael W Salter Michael W Salter Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Beverley A Orser Beverley A Orser Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Takanobu Nakazawa Takanobu Nakazawa Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tadashi Yamamoto Tadashi Yamamoto Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Michael F Jackson Corresponding Author Michael F Jackson Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author John F MacDonald John F MacDonald Department of Physiology, University of Toronto, Toronto, Ontario, Canada Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada Search for more papers by this author Author Information Kai Yang1, Catherine Trepanier2, Bikram Sidhu1, Yu-Feng Xie3, Hongbin Li1, Gang Lei3, Michael W Salter4, Beverley A Orser1, Takanobu Nakazawa5, Tadashi Yamamoto5, Michael F Jackson 3 and John F MacDonald1,2,3 1Department of Physiology, University of Toronto, Toronto, Ontario, Canada 2Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada 3Molecular Brain Research Group, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada 4Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada 5Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan *Corresponding author. Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8. Tel.:+519 931 5777/ext. 24230; Fax:+519 931 5721; E-mail: [email protected] The EMBO Journal (2012)31:805-816https://doi.org/10.1038/emboj.2011.453 There is a Have you seen? (February 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Metaplasticity is a higher form of synaptic plasticity that is essential for learning and memory, but its molecular mechanisms remain poorly understood. Here, we report that metaplasticity of transmission at CA1 synapses in the hippocampus is mediated by Src family kinase regulation of NMDA receptors (NMDARs). We found that stimulation of G-protein-coupled receptors (GPCRs) regulated the absolute contribution of GluN2A-versus GluN2B-containing NMDARs in CA1 neurons: pituitary adenylate cyclase activating peptide 1 receptors (PAC1Rs) selectively recruited Src kinase, phosphorylated GluN2ARs, and enhanced their functional contribution; dopamine 1 receptors (D1Rs) selectively stimulated Fyn kinase, phosphorylated GluN2BRs, and enhanced these currents. Surprisingly, PAC1R lowered the threshold for long-term potentiation while long-term depression was enhanced by D1R. We conclude that metaplasticity is gated by the activity of GPCRs, which selectively target subtypes of NMDARs via Src kinases. Introduction Various forms of synaptic plasticity at CA1 synapses are dependent upon activation of postsynaptic NMDA receptors (NMDARs; Collingridge and Bliss, 1995; Abraham, 2008), providing mechanisms underlying important aspects of hippocampal learning and memory (Whitlock et al, 2006; Howland and Wang, 2008). Two major subtypes of NMDARs are located at these synapses; they are heterotetramers composed predominantly of GluN1a in combination with either GluN2A (GluN2ARs) or GluN2B (GluN2BRs) subunits. The requirement for receptor subtypes at these synapses is poorly understood. Each subtype is calcium permeable but differences in their gating kinetics and topographical location in the cell result in distinct temporal and spatial intracellular calcium signals (Kohr, 2006; Berberich et al, 2007). For example, GluN2ARs activate and deactivate more rapidly than do GluN2BRs, allowing for a substantive but transient entry of Ca2+ via GluN2ARs versus a much slower, but in total, much larger total charge transfer for Ca2+ via GluNR2BRs (Vicini et al, 1998). Therefore, each receptor subtype might dramatically differ in its contribution to Ca2+-dependent signalling and synaptic plasticity (Cull-Candy and Leszkiewicz, 2004). The relative contributions of NMDAR subtypes to the direction of synaptic plasticity (potentiation versus depression) have been highly controversial, ranging from distinct roles for GluN2ARs in long-term potentiation (LTP) versus GluN2BRs in long-term depression (LTD; Liu et al, 2004; Fox et al, 2006; Brigman et al, 2010), to the assertion that it is the ratio of GluN2AR/GluN2BR, which determines direction (Cho et al, 2009). Just as significantly, whether signal transduction cascades can alter the relative contribution of each receptor subtype, and thus dynamically alter the direction of synaptic plasticity has not previously been explored. Protein tyrosine phosphorylation provides a powerful means of regulating NMDAR function in the CNS (Salter and Kalia, 2004; Chen and Roche, 2007). The phosphorylation status of NMDAR subunits is set by the concerted and opposing activity of specific kinases and phosphatases, and may be dynamically altered by prior neuronal activity or through the engagement of specific intracellular signalling cascades. In this respect, Src family kinases play an essential role in initiating activity-dependent synaptic plasticity without themselves changing synaptic efficacy (Lu et al, 1998; Ali and Salter, 2001; Huang et al, 2001; Salter and Kalia, 2004; Xu et al, 2008). At CA1 synapses, the activity of the tyrosine kinases Src and Fyn is required for the induction of LTP (Lu et al, 1998; Huang et al, 2001). However, it is unclear if and how Src and Fyn might differentially regulate the function of NMDARs in hippocampal neurons (Salter and Kalia, 2004). We hypothesized that different classes of G-protein-coupled receptors (GPCRs) (Gαq versus Gαs) activate either Src or Fyn kinase; and, that Src and Fyn selectively phosphorylate GluN2ARs or GluN2BRs, respectively. By this means each signalling pathway might selectively alter the function of each subtype of NMDARs, and as consequence, govern the direction of synaptic plasticity. Results GluN2A is required for Src regulation of NMDAR-mediated currents in isolated CA1 neurons We previously showed that low concentrations of pituitary adenylate cyclase activating peptide 38 (PACAP38) potentiated NMDA-induced currents (via the PAC1R) in isolated CA1 pyramidal neurons, and this effect was prevented by co-application of the Src interfering peptide Src(40–58) (Macdonald et al, 2005). This peptide does not inhibit the enzymatic activity of Src, but rather interferes with the binding of Src to the scaffolding protein, NADH dehydrogenase subunit 2, thus preventing Src from orienting to a subcellular compartment where it can phosphorylate NMDARs (Gingrich et al, 2004). For whole-cell recordings from isolated CA1 neurons from rats and mice, NMDA (50 μM NMDA and 500 nM glycine) applications of 3 s duration were repeated at a frequency of 1 per minute using one barrel of a rapid perfusion system while a second barrel was used to apply control bathing solution. Tests on cells from transgenic mice were always matched with appropriate controls from wild-type mice. Recovery from GluN2R desensitization was complete between applications. Recombinant kinases, interfering peptides (e.g., Src(40–58) and PKI5−24) were included in the patch recording pipette. In some experiments, where indicated, we doubled the concentration of NMDA (100 μM) and glycine (1 μM) in order to achieve responses of comparable amplitude to those of controls. Receptor agonists such as PACAP38 and SKF81297 were applied together with NMDA in the first barrel as well as the control barrel, for a period of 5 min as indicated in the figures (shaded regions). In contrast, antagonists were applied continuously in both the control barrel and the NMDA-containing barrel of the perfusion system throughout the entire duration of the recordings. We initially examined the effects of intracellular applications of recombinant Src (30 U/ml, patch electrode) upon peak NMDA current (50 μM NMDA and 500 nM glycine). The Src-induced potentiation was insensitive to the highly selective GluN2BR inhibitor, Ro 25-6981 (500 nM) (Fischer et al, 1997; Malherbe et al, 2003), but was strongly inhibited by the GluN2AR competitive antagonist, NVP-AAM077 (50 nM) (Neyton and Paoletti, 2006; Bartlett et al, 2007; Paoletti and Neyton, 2007; Figure 1A and B). Given the relatively limited selectivity of NVP-AAM077, we further tested Zn2+ at a concentration (300 nM) shown to preferentially block GluN2ARs versus GluN2BRs in the hippocampus (Neyton and Paoletti, 2006; Paoletti and Neyton, 2007; Nozaki et al, 2011). Zinc blocked the potentiation of NMDA-evoked currents by Src (Figure 1B). Furthermore, in CA1 neurons from wild-type mice Src enhanced peak currents but these effects were absent (Figure 1B) in cells from GluN2A−/− knockout mice (Ito et al, 1997). We then used a low concentration of the PAC1R agonist, PACAP38 (1 nM, 5 min, perfusion barrels) (Macdonald et al, 2005) to activate endogenous Src in these neurons (Macdonald et al, 2005). The PAC1Rs effects were not blocked by Ro 25-6981 (500 nM) or ifenprodil (3 μM) but were blocked by Zn2+ (300 nM) and NVP-AAM077 (50 nM). Furthermore, its effects were absent in cells from GluN2A−/− mice (Figure 1C and D). Figure 1.PAC1R-Src activation potentiates GluN2A-containing NMDAR currents in acutely isolated CA1 neurons of the rat but not in cells isolated from GluN2A−/− mice. (A) Src-mediated increase of NMDAR currents (INMDA) in hippocampal neurons (number of cells, N=8) is blocked by NVP-AAM077 (N=7) but not by Ro 25-6981 (N=6). Recombinant Src (30 U/ml) was applied via the patch pipette. NVP-AAM077, Zn2+, and Ro25-6981 were applied to the bath and to the perfusion solutions containing NMDA/Glycine. (B) Src potentiation of INMDA is blocked by NVP-AAM077 (N=7), Zn2+ (N=5), and GluN2A deletion (N=6), but not by Ro25-6981 (N=6). Src enhanced currents in cells from wild-type mice (N=6, 1.7±0.2 times) but not from GluN2A−/− mice, 1.0±0.04. Peak currents were measured immediately following break through and were compared with currents averaged for values between 25 and 30 min. (C) NVP-AAM077 (N=6), but not Ro25-6981 (N=5), inhibits the potentiation of INMDA by PACAP38. The application of PACAP38 is indicated by the shaded region and was 5 min. (D) NVP-AAM077 (N=6) and Zn2+(N=6), but not Ro25-6981 (N=5) or ifenprodil (N=6), prevents the enhancement of INMDA by PACAP38. Furthermore, PACAP38 cannot potentiate INMDA in GluN2A−/− mice (N=5) even though wild-type cells (N=5) demonstrated a degree of potentiation similar to that observed in rat neurons (1.4±0.1 times control). Test reagents (PACAP38, 1 nM; NVP-AAM077, 50 nM; Ro25-6981, 500 nM; ifenprodil 3 μM; Zn2+, 300 nM) were co-applied with NMDA/Glycine solutions using the multi-barreled perfusion system and were included in the bath during the entire recording period (not PACAP38). Peak currents were averaged for 5 min prior to applying PACAP and were compared with currents averaged for values between 20 and 25 min. **Indicates P<0.01, one-way ANOVA (Tukey′s post hoc comparison). Calibration bars: 2 s; (A) Src 150 pA, Ro25-6981 200 pA; (B) PACAP38 200 pA, Ro25-6981 200 pA, NVP-AAM077 300 pA. Download figure Download PowerPoint GluN2B and not GluN2A is required for Fyn regulation of NMDAR-mediated currents in isolated CA1 neurons Fyn kinase has been implicated many times in the regulation of NMDARs (Salter and Kalia, 2004). However, whether such regulation is GluN2 subunit dependent has not been shown. We therefore compared the effects of recombinant Fyn kinase (1 U/ml, patch electrode) on NMDAR-mediated currents with those of recombinant Src. Fyn also enhanced these currents and this effect was prevented by Ro 25-6981 (500 nM) but not by NVP-AAM077 (50 nM) (Figure 2A and B). Using an analogous approach to that used to develop Src(40–58), we synthesized a peptide corresponding to a region of the unique domain of Fyn (amino acids 39–57). We speculated that Fyn must locate to the vicinity of the receptor by binding to an unknown scaffolding protein. Fyn(39–57) (25 ng/ml), but not Src(40–58) (25 ng/ml), attenuated the effect of Fyn (Supplementary Figure S1B). Importantly, we confirmed that co-applications of Src(40–58) (25 ng/ml) prevented the enhancement of NMDA-induced currents by recombinant Src (Supplementary Figure S1A) and, conversely, showed that Fyn(39–57) did not alter the potentiation by Src kinase (Supplementary Figure S1B). These findings illustrate that this peptide can distinguish between the Src- and Fyn-dependent regulation of NMDARs. Again, it is important to note that neither peptide serves as an inhibitor of enzymatic activity (Gingrich et al, 2004). Figure 2.D1R-Fyn activation potentiates GluN2B-containing NMDAR currents in acutely isolated CA1 neurons of the rat and also in cells isolated from GluN2A−/− mice. (A) Fyn-dependent upregulation of INMDA (N=6) was blocked by Ro25-6981 (N=6) but not by NVP-AAM077 (N=6). (B) Quantification of normalized INMDA recorded from hippocampal neurons. Fyn potentiation of INMDA (N=6) is blocked by Ro25-6981 (N=6) but not by NVP-AAM077 (N=6) or Zn2+ (N=7). In cells taken from GluN2A−/− mice (N=5) Fyn enhanced NMDAR peak currents. Recombinant Fyn (1 U/ml) was applied via the patch pipette. NVP-AAM077, Zn2+, and Ro25-6981 were applied to the bath and to the perfusion solutions containing NMDA/Glycine. Peak currents were measured immediately following break through and were compared with currents averaged for values between 25 and 30 min. (C) Upregulation of INMDA by SKF81297 (N=9) was blocked by Ro25-6981 (N=7) but not by NVP-AAM077 (N=5). The application of SKF81297 is indicated by the shaded region and was 5 min. (D) Quantification of normalized INMDA recorded from isolated hippocampal neurons treated with SKF81297. D1R-induced potentiation of INMDA (N=9) is prevented by SCH23390 (N=9), PKI5−24 (N=7), Ro25-6981 (N=7), and Fyn(39–57) (N=5) but not by NVP-AAM077 (N=5), Zn2+ (N=8), or Src(40-58) (N=8). In cells from GluN2A−/− mice, currents were enhanced by applications of SCH23390 (N=6). Test reagents (SKF81297, 10 μM; SCH23390, 10 μM; Ro25-6981 500 nM; Zn2+, 300 nM; NVP-AAM077, 50 nM) were co-applied with NMDA/Glycine solutions using the multi-barreled perfusion system and were included in the bath when appropriate (not SKF81297). Fyn(39–57), 25 ng/ml; Src(40–58), 25 ng/ml and PKI5−24, 300 nM were included in the patch pipettes. Peak currents were averaged for 5 min prior to applying SKF81297 and were compared with currents averaged for values between 20 and 25 min. **Indicates P<0.01, one-way ANOVA. Calibration bars: 2 s; (A) Fyn 150 pA, Ro25-6981 200 pA, NVP-AAM077 150 pA; (B) Control 400 pA, Ro25-6981 400 pA, NVP-AAM077 100 pA. Download figure Download PowerPoint GPCRs coupled to Gαs can also potentiate NMDAR currents in hippocampal neurons (MacDonald et al, 2007). We therefore examined the effects of stimulating the dopamine D1 receptor (D1R; Cepeda and Levine, 2006). In these recordings, we did not alter the concentrations of NMDA and glycine (50 and 0.5 μM, respectively). As a consequence, the proportionate enhancement of the contribution of the spared subtype of receptor is much greater. Application of the D1R agonist, SKF81297 (10 μM, 5 min, perfusion barrels), enhanced NMDAR currents in isolated CA1 neurons (Figure 2C). The effect of SKF81297 was blocked by the D1,5 antagonist, SCH23390 (10 μM). Block of GluN2BRs by Ro 25-6981 prevented this enhancement while block of GluN2ARs with NVP-AAM077, rather than just preventing, actually increased the proportional enhancement by D1R (Figure 2C and D). This enhancement is anticipated because blocking GluNR2ARs will result in currents generated in greater proportion by GluNR2BRs. Similar findings were observed with the GluN2AR antagonist Zn2+. More convincingly, the enhancements were also proportionately larger in CA1 neurons taken from GluN2A knockout mice (Figure 2D). In addition, the potentiation by SKF81297 was prevented by the specific PKA inhibitors PKI5−24 (Figure 2D, applied via the patch pipette) and Rp-cAMPs (patch pipette). Similarly, applications of vasoactive intestinal peptide (VIP, 1 nM), acting through the VPAC1,2R (VIP receptors; Yang et al, 2009), enhanced NMDAR-mediated currents by targeting GluN2BRs (Supplementary Figure S2). Therefore, we determined if D1R and VPACR stimulations required Fyn activation rather than Src. Fyn(39–57) blocked the enhancement of NMDAR currents by each of these GPCRs (Figure 2D; Supplementary Figure S2B). Selective phosphorylation of GluN2A and GluN2B We also examined both the biochemical and electrophysiological actions of these GRPCRs in rat hippocampal slices. Slices were prepared as for electrophysiological recording and were similarly treated with agonists and antagonists. We demonstrated that PACAP38 treatment (1 nM for 10 min) increased Src kinase activity (increased Tyr-416 phosphorylation), but not that of Fyn (Tyr-420 phosphorylation unchanged, Figure 3A), and increased the tyrosine phosphorylation of GluN2A, but not GluN2B subunits (Figure 3B). Importantly, increased GluN2A phosphorylation was prevented by applications of TAT-Src(40–58) (Figure 3B). Conversely, treatment of slices with SKF81297 (10 μM for 10 min) increased Fyn kinase activity, but not that of Src (Figure 3C), and increased tyrosine phosphorylation of GluN2B, but not GluN2A subunits (Figure 3D). The SKF81297-induced increase in GluN2B phosphorylation was prevented by a TAT-Fyn(39–57) (Figure 3D) as well as by a D1R antagonist (SCH23390, 10 μM). Figure 3.PACAP38 and SKF81297 activate Src and Fyn leading to phosphorylation of GluN2A and GluN2B. (A) PACAP38 (1 nM) treatment increases the phosphorylation of Src (pSrcY416) but not Fyn (pFynY420). Below, summary of immunoblot analysis shows the averaged relative density of pSrcY416 or pFynY420 for each condition (n=4). (B) PACAP38 treatment increases the tyrosine phosphorylation of immunoprecipitated GluN2A- but not GluN2B-containing NMDARs. The enhancement by PACAP38 was blocked by co-applications of TAT-Src(40–58). Below, the relative density of pTyr for GluN2A and GluN2B was quantified from immunoblots (n=4) for each of the conditions shown. (C) SKF81297 increases the phosphorylation of Fyn (pFynY420) but not Src (pSrcY416). Below, the averaged relative density of pSrcy416 (n=3) or pFynY420 (n=4) from immunoblots obtained for each condition is shown (below). (D) SKF81297 (10 μM) treatment increases the tyrosine phosphorylation of immunoprecipitated GluN2B- but not GluN2A-containing NMDARs. The enhancement by SKF81297 was blocked by co-applications of TAT-Fyn(39–57). The averaged relative density of pTyr for GluN2A (n=8) and GluN2B (n=3) from immunoblots obtained under each of the conditions is shown. *Indicates P<0.05, t-test, **indicates P<0.01, Student's t-test. Figure source data can be found in Supplementary data. Source Data for Figure 3 [embj2011453-sup-0002.pdf] Download figure Download PowerPoint Selectivity of GPCR activation upon NMDAR-mediated EPSCs in rat hippocampal slices Bath application of PACAP38 (10 nM) or SKF81297 (20 μM) increased the amplitude of NMDAR-mediated EPSCs (NMDAREPSC) recorded from rat hippocampal slices (PACAP38: 1.59±0.15, n=5; SKF81297: 1.78±0.23, n=6) (Figure 4A and B). The effects of PACAP38 on NMDAREPSCs, previously shown to be prevented by Src(40–58) (Macdonald et al, 2005), were not affected by pipette applications of Fyn(39–57) (25 ng/ml, patch pipette) (Figure 4A). Conversely, the effect of SKF81297 was prevented by the Fyn(39–57), but not by the pipette applications of Src(40–58) (Figure 4B and C). We also recorded in the continued presence of bath applied Ro 25-6981 (500 nM), to block GluN2BRs, and demonstrated that NMDAREPSCs were still potentiated when PACAP38 (Figure 4A) was applied. In contrast, the potentiation by SKF81297 (Figure 4B and C) was blocked by Ro 25-6981 (500 nM) and by the D1,5 antagonist, SCH 23390 (10 μM). Therefore, PACAP38 preferentially augments the function of synaptic GluN2ARs, but not GluN2BRs, by enhancing Src activity but not Fyn; and, SKF81297 selectively augments the function of synaptic GluN2BRs, but not GluN2ARs, by enhancing Fyn, but not Src, kinase activity. Figure 4.PACAP38 and SKF81297 increase GluN2AR- and GluN2BR-mediated synaptic currents via Src and Fyn, respectively. (A) The increase of NMDAREPSCs by PACAP38 (5 nM; bath applied) (N=8) is unaffected by Ro25-6981 (500 nM; bath applied) (N=6) or by Fyn(39–57) (25 ng/ml, patch pipette) (N=5). (B) The increase of NMDAREPSCs by SKF81297 (20 μM; bath applied) (N=6) is blocked by Ro25-6981 (500 nM; bath applied) (N=6) and by Fyn(39–57) (25 ng/ml patch pipette) (N=4). (C) The potentiation of NMDAREPSCs by SKF81297 (N=6) could be prevented by SCH23390 (10 μM; bath applied) (N=6), Ro25-6981 (500 nM; bath applied) (N=6), Fyn(39–57) (25 ng/ml patch pipette) (N=6) but not by Src(40–58) (25 ng/ml patch pipette) (N=6). Peak currents were averaged for 5 min prior to applying PACAP and were compared with currents averaged for values between 25 and 30 min. **Indicates P<0.01, Student’s t-test Calibration bars: 100 ms; (A) PACAP38 100 pA, Fyn(39–57) 100 pA, Ro25-6981 100 pA; (B) SKF81297 50 pA, Fyn(39–57) 35 pA, Ro25-6981 25 pA. Download figure Download PowerPoint Selectivity of GPCR activation upon synaptic plasticity in hippocampal slices We then examined the consequences of enhancing each receptor subtype on synaptic plasticity in rat hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum in response to electrical stimulation of Schaffer-collaterals in rat hippocampal slices. In control slices, baseline was monitored for a minimum of 20 min before the induction of synaptic plasticity. In drug treated slices, baseline responses were monitored for 10 min before applying either PACAP38 (1 nM) or SKF81297 (10 μM) to the bath for 10 min (shaded regions in Figure 5) and was continued until just after the induction of synaptic plasticity. fEPSP slopes were unaffected by the bath application of either PACAP38 or SKF81297 (Supplementary Figure S3A). In addition, a separate series of recordings confirmed that neither treatment altered paired-pulse facilitation (Supplementary Figure S3B), a form of short-term plasticity used to assess changes in presynaptic function. Figure 5.In rat hippocampal slices, PACAP38 application lowers the threshold for the induction of LTP whereas SKF81297 enhances LTD at stimulus frequencies near the threshold for the induction of LTP. (A) Control (N=9) and PACAP38-treated (N=11) slices (1 nM; bath applied for time indicated by shaded region) are shown. At time zero, the 10-Hz stimulation was performed. Post induction fEPSP slopes from each treatment group were normalized to baseline responses. (B) Control (N=9) and SKF81297-treated (N=9) slices (10 μM; 10 min bath applied for time indicated by shaded region) are shown. At time zero, the 10-Hz stimulation was performed. Post induction fEPSP slopes were normalized to baseline responses. (C) For a series of recordings from control or PACAP38 (1 nM, 10 min bath applied as indicated above) treated slices, fEPSP slopes were normalized to the mean slope measured during a 20-min baseline recording period. At t=0, repetitive stimulation, consisting of 600 pulses, was delivered at frequencies of 1, 10, 20, 50, or 100 Hz. Controls (N=5–11) and PACAP38 (N=5–9). The relative degree of potentiation or depression at the end of the recordings was plotted versus the stimulation frequency (1–100 Hz) used during the induction of plasticity. (D) As in (C) but for applications of control or SKF81297 (10 μM, 10 min bath applied) treated slices. Controls (N=5–9) and SKF81297 (N=5–9). The relative degree of potentiation or depression at the end of the recordings was plotted versus the stimulation frequency (1–100 Hz) used during the induction of plasticity. The average of slope measurements recorded over the last 10-min period (50–60 min) was employed in each case. *Indicates P<0.001, Student's t-test. Download figure Download PowerPoint Changes in plasticity were characterized by varying the frequency of repetitive stimulation (1–100 Hz) used during the induction phase, thus generating frequency–plasticity relationship for each of our treatment conditions. In this way, we could monitor not only the maximum synaptic gain change but also the frequency at which the direction of plasticity changed from LTD to LTP. In control slices, maximum potentiation and depression were achieved following repetitive stimulation at 50 and 1 Hz, respectively; and the direction of plasticity changed from LTD to LTP at induction frequencies ranging from 10 to 20 Hz (Figure 5). Although LTD was consistently observed in control slices with 5–10 Hz stimulation, LTP was now observed at lower frequencies (Figure 5C; PACAP38 group at 10 and 20 Hz). In contrast, the D1R agonist SKF81297 shifted the modification threshold to the right thereby increasing the threshold for LTP induction. As a