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Control of Excitatory Synaptic Transmission by C-terminal Src Kinase

2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês

10.1074/jbc.m800917200

ISSN

1083-351X

Autores

Jindong Xu, Manjula Weerapura, Mohammad Kutub Ali, Michael Jackson, Hongbin Li, Gang Lei, Sheng Xue, Chun L. Kwan, Morris F. Manolson, Kai Yang, John F. MacDonald, Xian‐Min Yu,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca2+, followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca2+ into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus. The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca2+, followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca2+ into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus. Pyramidal neurons of the CA1 hippocampus receive excitatory synaptic input from the axons of CA3 pyramidal neurons, which form the Schaffer Collateral pathway. The induction of synaptic plasticity at these CA3-CA1 synapses requires stimulation of N-methyl-d-aspartate receptors (NMDARs) 4The abbreviations used are: NMDAR, N-methyl-d-aspartate receptor; PTPα, protein-tyrosine phosphatase α; LTP, long-term potentiation; LTD, long-term depression; AMPAR, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential; GST, glutathione S-transferase; Ω, ohm; SH domain, Src homology domain; HEK, human embryonic kidney; Csk, C-terminal Src kinase; CT, C-terminal. located at CA1 pyramidal cell dendritic spine synapses. It is widely accepted that spine depolarization relieves the Mg2+ block of NMDARs, causing a concomitant increase in the influx of Ca2+. Both longterm potentiation (LTP) and long-term depression (LTD) are triggered by the subsequent accumulation of intracellular Ca2+ in the spines. For example, induction of LTP increases Ca2+ up to 10μm, whereas LTD is associated with lower but more sustained increases (1Sobczyk A. Scheuss V. Svoboda K. J. Neurosci. 2005; 25: 6037-6046Crossref PubMed Scopus (216) Google Scholar). Appreciable NMDAR-dependent Ca2+ influx occurs at spines even at resting membrane potential due to the relative incompleteness of the Mg2+ block (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar). Therefore an NMDAR-dependent influx of Ca2+ occurs even at low, baseline frequencies of stimulation that induce neither LTP nor LTD. This raises a fundamental question: why does this baseline influx of Ca2+ fail to induce synaptic plasticity? Part of the answer to this question may lie in the observations that the induction of LTP at these synapses also requires activation of the Src family tyrosine kinases, Fyn and Src (3Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar). Src family kinases control the induction of LTP by potentiating the NMDAR-dependent influx of Ca2+ (3Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar) and activation of Src is "necessary and sufficient" to induce LTP at CA3-CA1 neurons (4Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Crossref PubMed Scopus (277) Google Scholar). Src facilitates NMDAR function likely by increasing tyrosine phosphorylation of the NR2A subunit, whereas Fyn does so by phosphorylating the NR2B subunit (3Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar). The precise mechanism whereby Src and/or Fyn are activated during the induction of LTP is poorly understood. The influx of Ca2+ likely enhances the tyrosine kinase Pyk2, which associates with and activates Src (3Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar, 5Seabold G.K. Burette A. Lim I.A. Weinberg R.J. Hell J.W. J. Biol. Chem. 2003; 278: 15040-15048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Therefore, activation of NMDARs and an influx of Ca2+ are coupled to stimulation of Src, which is in turn responsible for increasing both NMDAR activity and the subsequent influx of Ca2+. During high frequency stimulation, relief of the Mg2+ block of NMDARs is insufficient in itself for induction of long-term potentiation at CA3-CA1 synapses. This early Src-dependent positive feedback amplifies the NMDAR-dependent Ca2+ signal (6Dineley K.T. Weeber E.J. Atkins C. Adams J.P. Anderson A.E. Sweatt J.D. J. Neurochem. 2001; 77: 961-971Crossref PubMed Scopus (42) Google Scholar), allowing the activation of the appropriate downstream pathways (e.g. calcium/calmodulin-dependent kinase II) responsible for enhancing the surface expression (7Groc L. Choquet D. Cell Tissue Res. 2006; 326: 423-438Crossref PubMed Scopus (137) Google Scholar) and/or function of AMPARs (8Soderling T.R. Derkach V.A. Trends Neurosci. 2000; 23: 75-80Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Surprisingly, during baseline stimulation intracellular applications of selective inhibitors of Pyk2 or Src are without effect on the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) and thus fail to modify AMPAR-mediated EPSCs. However, they are highly effective in preventing the induction of LTP (4Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Crossref PubMed Scopus (277) Google Scholar, 9Huang Y.Q. Lu W.Y. Ali D.W. Pelkey K.A. Pitcher G.M. Lu Y.M. Aoto H. Roder J.C. Sasaki T. Salter M.W. MacDonald J.F. Neuron. 2001; 29: 485-496Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). These results are not consistent with a NMDAR-mediated Ca2+ entry into postsynaptic spines during the baseline stimulation (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar). Indeed, such elemental Ca2+ signals might be expected to be amplified by Src-mediated facilitation of NMDARs (positive feedback), which would subsequently and progressively facilitate AMPAR-mediated EPSPs. Intuitively, such a scheme would be undesirable for the maintenance of a stable neuronal network. A regulatory mechanism may therefore exist that holds Src in balance while leaving NMDARs unaffected until they are required for the induction of synaptic plasticity (e.g. LTP). Csk family kinases (Csk, COOH-terminal Src kinase and Chk, Csk-homologous kinase) are cytosolic kinases that inhibit Src family kinases and as such are possible candidates for such a regulatory mechanism. Reports showing the down-regulation of Csk in the adult brain and a parallel up-regulation of Chk suggest that Chk would be the most likely candidate (10Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar, 11Kuo S.S. Moran P. Gripp J. Armanini M. Phillips H.S. Goddard A. Caras I.W. J. Neurosci. Res. 1994; 38: 705-715Crossref PubMed Scopus (38) Google Scholar). However, we present evidence that Csk is present at CA3-CA1 synapses and that it interacts directly with the NMDAR complex to regulate the ability of Src to enhance NMDARs. Csk prevents an inappropriate activation of Src by the NMDAR-dependent influx of Ca2+ that occurs during baseline stimulation frequencies and, in turn, inhibits the induction of LTP. Ours is the first demonstration that Csk plays a critical role in regulating the balance between baseline synaptic excitatory transmission and plasticity in the adult central nervous system. All animal experiments were conducted following the guidelines of the Canadian Council on Animal Care and the NIH, and were approved by the Animal Care Committee of the University of Toronto and the Animal Care and Use Committee of Florida State University. Acutely Isolated CA1 Pyramidal Neurons and Electrophysiological Recordings—Methods for acute neuron isolation from young adult rats have been described previously (12Lu W.Y. Xiong Z.G. Lei S. Orser B.A. Dudek E. Browning M.D. MacDonald J.F. Nat. Neurosci. 1999; 2: 331-338Crossref PubMed Scopus (351) Google Scholar). In brief, the hippocampi of postnatal (14-22 days) Wistar rats were dissected out and sliced into ∼1-mm thick slices in ice-cold oxygenated extracellular solution and incubated for 45 min followed by digestion with 2-3 mg/ml crude papain extract for 30 min at room temperature. The slices were then washed and incubated at room temperature for an additional 1-2 h prior to use (see supplementary data). Acutely isolated CA1 pyramidal neurons present both advantages and disadvantages as a preparation for examining the function of NMDAR-mediated currents. The isolation procedure shears off much of the dendritic tree leaving only a few major primary and secondary dendritic branches. Applications of NMDA during whole cell recordings will therefore induce currents mediated by the activation of channels largely found within the extrasynaptic compartment. Nevertheless, some synaptic NMDARs are likely stimulated as many synapses are located on the large dendritic processes that come along with the cell soma. The predominant subtype of NMDAR in the extrasynaptic compartment of CA1 pyramidal neurons is NR1/NR2B. However, it is estimated that ∼25% of the receptor compliment in this compartment is composed of NR1/NR2A receptors. This contrasts with the ∼75% compliment of this receptor subtype at the synapse (13Groc L. Heine M. Cousins S.L. Stephenson F.A. Lounis B. Cognet L. Choquet D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18769-18774Crossref PubMed Scopus (277) Google Scholar). Furthermore, the peak open probability of NR1/2A receptors is about 2 to 5 times greater than for NR1/NRB receptors (14Chen N. Luo T. Wellington C. Metzler M. McCutcheon K. Hayden M.R. Raymond L.A. J. Neurochem. 1999; 72: 1890-1898Crossref PubMed Scopus (170) Google Scholar). It is therefore anticipated that populations of both NR1/NR2A and NR1/NR2B receptors will contribute substantially to the peak whole cell currents recorded from isolated CA1 pyramidal neurons. Of course, we cannot rule out the possible presence of triheteromeric receptors. Whole cell-clamped pyramidal neurons are lifted into the stream of a rapid perfusion system (see below). One of the significant advantages to this preparation is the ability to rapidly apply agonists and antagonists to the entirety of each cell and to attain near equilibrium conditions that are similar to those used to study recombinant NMDAR currents expressed in HEK293 cells (15Priestley T. Laughton P. Myers J. Le Bourdellés B. Kerby J. Whiting P.J. Mol. Pharmacol. 1995; 48: 841-848PubMed Google Scholar, 16Chen N. Luo T. Raymond L.A. J. Neurosci. 1999; 19: 6844-6854Crossref PubMed Google Scholar). It is far more difficult, if not impossible, to achieve such conditions with cultured hippocampal neurons or with CA1 pyramidal neurons in situ in the slice preparation. For whole cell recordings, acutely isolated neurons were bathed with the extracellular solution containing (mm): NaCl (140), KCl (5.4), 1.3 CaCl2 (1.3), HEPES (25Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1029) Google Scholar), glucose (33Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (364) Google Scholar), tetrodotoxin (0.001), and glycine (0.003), pH 7.4, osmolarity 310-320 mOsm. Recording pipettes were filled with intracellular solution containing (mm): CsCl or CsF (140), HEPES (10Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar), MgCl2 (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (1Sobczyk A. Scheuss V. Svoboda K. J. Neurosci. 2005; 25: 6037-6046Crossref PubMed Scopus (216) Google Scholar), and K2ATP (4Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Crossref PubMed Scopus (277) Google Scholar), pH 7.2, and osmolarity 290-300 mOsm. DC resistances of recording electrodes were 4-6 MΩ. Input resistance was monitored by applying a voltage step of -10 mV and any recordings with more than a 20% change during the recording were rejected. Only recordings with a pipette-membrane seal >2GΩ were included. For evoking NMDAR-mediated whole cell responses, the standard and NMDAR agonist-containing extracellular solutions were rapidly applied through double-barrel glass to neurons using the SF-77B fast-step perfusion system (Warner Instruments). Whole cell NMDAR-mediated currents were recorded using an Axopatch 1D or Axopatch 200B amplifier (Molecular Devices) under voltage-clamp and a holding potential of -60 mV except where otherwise indicated. For recordings from acutely isolated CA1 pyramidal neurons NMDA was applied at 50 μm in the continuing presence of 0.5 μm glycine. On-line data acquisition and off-line analysis were performed using pClamp8 software (Molecular Devices). Hippocampal Slice Preparation and Electrophysiological Recording—Techniques used for the hippocampal slice preparation and electrophysiological recording have been previously described (9Huang Y.Q. Lu W.Y. Ali D.W. Pelkey K.A. Pitcher G.M. Lu Y.M. Aoto H. Roder J.C. Sasaki T. Salter M.W. MacDonald J.F. Neuron. 2001; 29: 485-496Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17Lei G. Xue S. Chery N. Liu Q. Xu J. Kwan C.L. Fu Y.P. Lu Y.M. Liu M. Harder K.W. Yu X.M. EMBO J. 2002; 21: 2977-2989Crossref PubMed Scopus (68) Google Scholar). In brief, hippocampal slices (400 μm) were prepared from 3-4-week-old Wistar rats. Slices were recovered for 1 h in oxygenated artificial cerebral spinal fluid containing (mm): NaCl (124), KCl (3Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar), CaCl2 (2.6), MgCl2 (1.3), NaHCO3 (26Neet K. Hunter T. Mol. Cell. Biol. 1995; 15: 4908-4920Crossref PubMed Scopus (70) Google Scholar), NaH2PO4 (1.25), and glucose (10Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar), osmolarity 310-320 mOsm, pH 7.4. Recording electrodes were filled with an intracellular solution containing (mm): potassium gluconate (135.5), KCl (17.5), HEPES (10Kuo S.S. Armanini M.P. Phillips H.S. Caras I.W. Eur. J. Neurosci. 1997; 9: 2383-2393Crossref PubMed Scopus (15) Google Scholar), EGTA (0.2), MgATP (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar), GTP (0.3), and QX 314 (5Seabold G.K. Burette A. Lim I.A. Weinberg R.J. Hell J.W. J. Biol. Chem. 2003; 278: 15040-15048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) with osmolarity 290-300 mOsm, pH 7.2. DC resistances were 3-5 mΩ. Series resistance (10-30 megohms), monitored throughout the recording period, was estimated from the series resistance compensation of currents generated in response to 5-mV voltage steps. Cells were rejected if the resistance values changed by more than 20%. During recording, slices were continuously perfused at 3-5 ml/min with oxygenated and heated (30-32 °C) artificial cerebral spinal fluid. Synaptic responses were evoked with electrical stimulation at the border of the stratum radiatum-stratum lacunosum region. Stimuli were delivered at 0.05 or 0.1 Hz with the intensity adjusted to 25-30% of the maximal synaptic responses. Whole cell EPSCs or EPSPs were recorded from the CA1 cell body layer using Multiclamp 700A amplifier and Clampex 9 software (Molecular Devices). During whole cell recordings, field EPSPs (fEPSPs) were simultaneously recorded with electrodes filled with artificial cerebral spinal fluid and placed within the stratum radiatum. Whole cell EPSC recordings were conducted at -90 mV. After recording baseline synaptic activity for 10-30 min, tetanic stimulations (two 100 Hz stimulus trains; 500 ms with 10-s intertrain interval) were applied. The peak amplitudes of EPSCs and the slopes between 10 and 70% of the peak responses of EPSPs and fEPSPs were measured. Pharmacologically isolated NMDAR-mediated EPSPs were also recorded in some experiments with cells held at -60 mV in the presence of CNQX (10 μm) to block AMPARs and bicuculline to block GABAAR responses. Biochemical Experiments-Subcellular Fractionation and PSD Isolation—Subcellular fractionation of adult male rat (40 to 60 days) forebrains were performed as previously described (18Kalia L.V. Salter M.W. Neuropharmacology. 2003; 45: 720-728Crossref PubMed Scopus (89) Google Scholar, 19Dunah A.W. Standaert D.G. J. Neurosci. 2001; 21: 5546-5558Crossref PubMed Google Scholar, supplemental data). The approaches used for PSD isolation are described elsewhere (20Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (606) Google Scholar, 21Petralia R.S. Sans N. Wang Y.X. Wenthold R.J. Mol. Cell. Neurosci. 2005; 29: 436-452Crossref PubMed Scopus (188) Google Scholar, 22Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). Briefly, the synaptosome fraction was isolated by discontinuous sucrose gradient centrifugation and solubilized in 0.5% Triton X-100, then centrifuged to obtain the PSD-I pellet. This pellet was re-suspended, solubilized in 0.5% Triton X-100, and centrifuged to obtain the PSD-II pellet. Extracts from individual fractions were finally solubilized in 1% SDS and centrifuged at 15,000 × g for 5 min for Western blot analysis. Immunoprecipitation and Western Blot Analysis—Experiments were performed as described previously (9Huang Y.Q. Lu W.Y. Ali D.W. Pelkey K.A. Pitcher G.M. Lu Y.M. Aoto H. Roder J.C. Sasaki T. Salter M.W. MacDonald J.F. Neuron. 2001; 29: 485-496Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17Lei G. Xue S. Chery N. Liu Q. Xu J. Kwan C.L. Fu Y.P. Lu Y.M. Liu M. Harder K.W. Yu X.M. EMBO J. 2002; 21: 2977-2989Crossref PubMed Scopus (68) Google Scholar). In brief, proteins from the P2 (or P1) fraction of adult rat forebrain were dissolved in a lysis buffer containing (mm): Tris-HCl (50, pH 9.0), NaCl (150), 0.5% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, EDTA (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar), and sodium orthovanadate (1Sobczyk A. Scheuss V. Svoboda K. J. Neurosci. 2005; 25: 6037-6046Crossref PubMed Scopus (216) Google Scholar). For attached cell homogenates, cells were mechanically dissociated and cell pellets were re-suspended in HNTG buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, and 1 mm sodium orthovanadate). Solubilized proteins (600-800 μg) were incubated overnight at 4 °C with antibodies, as indicated. The immune complexes were collected with protein G-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C. The blotting analysis was performed by repeated stripping and successive probing with antibodies as indicated. Antibodies used in these experiments include anti-NR1 (Pharmingen), NR2A C-terminal (Upstate), NR2A N-terminal (Santa Cruz), protein-tyrosine phosphatase α (PTPα) (17Lei G. Xue S. Chery N. Liu Q. Xu J. Kwan C.L. Fu Y.P. Lu Y.M. Liu M. Harder K.W. Yu X.M. EMBO J. 2002; 21: 2977-2989Crossref PubMed Scopus (68) Google Scholar), Src (Oncogene), Src-Tyr(P)-416 (Cell Signaling), Src-Tyr(P)-527 (Cell Signaling), Chk (Santa Cruz), phosphotyrosine (Upstate), kv3.1b (BD Bioscience), and GluR2/3 (Upstate). Monoclonal ("anti-Csk," mouse, BD Biosciences) and polyclonal (rabbit, Santa Cruz) antibodies against Csk were used to avoid signal overlapping of Csk with IgG heavy chain in Western blotting. To avoid confusion, only the monoclonal Csk antibody raised against the SH2 and SH3 domains of Csk was labeled as anti-Csk in this paper. Non-selective mouse or rabbit IgG was utilized for immunoprecipitation as negative control. cDNA Transfection—HEK293 or COS7 cells were transfected using the calcium phosphate method (Invitrogen) as per the manufacturer's instruction with expression vectors (pcDNA3 or pRcCMV) containing cDNAs encoding NR1-1a, full-length or C-terminal truncated NR2A, v-Src (a gift of Dr. T. Pawson, University of Toronto), Csk (a gift of Dr. K. Harder, Royal Melbourne Hospital), wild-type or mutant neuronal Src (n-Src, Y535F or K303R/Y535F, provided by Dr. S. Hanks, Vanderbilt University), and Src-(40-58) (a gift from Dr. M. W. Salter, Hospital For Sick Children, Toronto). To avoid confusion, the n-Src mutants of chicken c-Src used in our experiments are referred to as Y527F and K295R/Y527F. Transfected cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum for 24 h before lysis. GST Pull-down Assay—pGEX2th-Csk-ST plasmid was kindly provided by Dr. G. Sun at the University of Rhode Island. Csk S109C mutation was made using QuikChange XL Site-directed Mutagenesis Kit (Stratagene) as described by the manufacturer. Point mutation was confirmed by DNA sequencing. The GST and GST fusion proteins conjugated with Csk or Csk S109C were produced in Escherichia coli (BL-21 strain, Invitrogen) with isopropyl β-d-thiogalactopyranoside (0.1 mm) induction. The bead-bound GST fusion proteins or GST alone (15-20 μg) were each incubated with solubilized membrane proteins (600-800 μg) extracted from the adult rat brain tissue or 5 μl of [35S]methionine-labeled NR1 C-terminal (CT) peptide and NR2A CT peptide in binding buffer (2% bovine serum albumin in phosphate-buffered saline) overnight at 4 °C. Bound proteins were separated by electrophoresis and detected by Western blotting or autoradiography. [35S]Methionine-labeled NR1 CT peptide and NR2A CT peptide were synthesized in the TnT T7 Quick Coupled Transcription/Translation System (Promega) following the protocol recommended by the manufacturer. For dephosphorylation treatment, 5 μl of [35S]methionine-labeled NR2A CT peptide was incubated with 100 units of λ-protein phosphatase (New England Biolabs) in the reaction buffer prior to the pull-down assays (see supplemental data). Due to the lack of highly selective inhibitors of Csk kinases we employed a Csk antibody (anti-Csk; mouse monoclonal antibody, 2.5 μg/ml, BD Biosciences) that prevents the ability of Csk to interact with NR2 subunits (see Fig. 7B) thereby inhibiting the ability of Csk to regulate the phosphorylation of NMDARs by Src. Such an approach is analogous to the use of anti-Src to selectively inhibit Src kinase activity in CA1 neurons (4Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Crossref PubMed Scopus (277) Google Scholar). Intracellular applications of anti-Csk caused a time-dependent increase in the magnitude of the EPSP slope to 189 ± 11% of that recorded immediately after breakthrough (Fig. 1, A and B). The slope of the CA3-CA1 EPSP largely reflects the underlying AMPAR conductance when extracellular Mg2+ is present (e.g. 1.3 mm). The enhancement of EPSPs induced by the anti-Csk antibody application subsequently occluded the induction of LTP (Fig. 1B). Furthermore, the potentiation of EPSPs by anti-Csk was blocked by pre-treating slices with the NMDAR antagonist AP5 (50 μm) or with the Src family kinase inhibitor PP2 (10 μm, 1 h) (Fig. 1A), indicating that endogenous Csk regulates synaptic plasticity through a Src family kinase and NMDAR-dependent mechanism.FIGURE 1Csk limits Src-dependent LTP at CA1 synapses. A, the slopes of EPSPs were recorded from neurons during intracellular applications of anti-Csk (2.5 μg/ml, closed squares, n = 6) or during applications of the antibody in slices pre-treated with NMDAR antagonist AP5 (50 μm, open squares, n = 7) or SFK inhibitor PP2 (10 μm, open circles, n = 5). Slopes of EPSPs were normalized with respect to the values recorded at breakthrough. Anti-Csk substantially enhanced EPSP slopes during baseline stimulation (p < 0.01, Mann Whitney test). B, in two groups of cells anti-Csk (2.5 μg/ml, closed squares, n = 6) or non-selective mouse IgG (mIgG, 2.5 μg/ml, open squares, n = 6) was applied and subsequently LTP was induced. C, summary data (mean ± S.E.) showing the peak amplitudes of EPSCs (normalized with the mean values of EPSC peak amplitudes during the first 2 min: It/Im) recorded from neurons during intracellular application of Csk (0.1 μg/ml, closed squares, n = 6) or no Csk (open squares, n = 9) and the attenuation of LTP (p < 0.01, Mann-Whitney test). Insets show examples of EPSCs recorded before (1Sobczyk A. Scheuss V. Svoboda K. J. Neurosci. 2005; 25: 6037-6046Crossref PubMed Scopus (216) Google Scholar) and after (2Bloodgood B.L. Sabatini B.L. Curr. Opin. Neurobiol. 2007; 17: 345-351Crossref PubMed Scopus (119) Google Scholar) the tetanus from neurons as indicated. D, slopes of fEPSPs normalized to the mean values of fEPSP slopes during the first 2 min. The fEPSPs were recorded simultaneously with the EPSC recordings shown in C with application of Csk (closed squares, n = 6) or no Csk (open squares, n = 9). Insets show examples of fEPSPs recorded before (1) and after (2 and 3) the tetanus. Large open arrows in C and D indicated tetanic stimulations. Time zero corresponds to the first evoked synaptic response (EPSP or EPSC as the case may be) recorded within 2 min after breakthrough, this brief delay being necessary to stabilize the recording.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also examined the effects of recombinant Csk supplied through our patch pipette on EPSCs recorded in the whole cell voltage-clamp configuration. Intracellular applications of Csk had no time-dependent effect on the amplitude of baseline EPSCs (Fig. 1C), suggesting that endogenous Csk is sufficient to limit Src activity and prevent plasticity of baseline EPSCs. In contrast, recombinant Csk substantially attenuated the induction of LTP. In control recordings lacking Csk, the amplitude of EPSCs were significantly enhanced to 285 ± 32% (mean ± S.E., n = 9) after tetanic stimulation (Fig. 1C). However, application of recombinant Csk (0.1 μg/ml) into neurons through the recording pipettes substantially attenuated LTP of EPSCs (Fig. 1C). Recordings of fEPSPs performed simultaneously with whole cell recordings of EPSCs were used to confirm that the magnitude of LTP did not differ in slices from each treatment group (Fig. 1D). We next considered if intracellular applications of Csk or its inhibitor (anti-Csk) would alter pharmacologically isolated NMDAR EPSCs. Csk caused no change in the amplitude of these NMDAR-mediated synaptic currents (Fig. 2A). In contrast, intracellular applications of anti-Csk caused a substantial and time-dependent increase in the amplitude of these currents that was not mimicked by a non-selective IgG (Fig. 2B). This anti-Csk induced enhancement was prevented by including the selective Src kinase inhibitory peptide (Src-(40-58)) in the patch pipette together with the antibody (Fig. 2B). These results imply that endogenous Csk kinases in CA1 neurons constitutively inhibit the Src-dependent potentiation of NMDAR EPSCs. Furthermore, this inhibition is likely complete, as supplementing the neurons with exogenous Csk kinase were without effect on these baseline responses. To investigate the inhibitory effect of endogenous Csk on excitatory synaptic transmission, we first determined the subcellular distribution of Csk family kinases in the brain. Sequential fractionation of forebrain homogenates was performed from adult rats (40-60-days old). The NR1 subunit, PSD-95 (9Huang Y.Q. Lu W.Y. Ali D.W. Pelkey K.A. Pitcher G.M. Lu Y.M. Aoto H. Roder J.C. Sasaki T. Salter M.W. MacDonald J.F. Neuron. 2001; 29: 485-496Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 18Kalia L.V. Salter M.W. Neuropharmacology. 2003; 45: 720-

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