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Trans-Synaptic Eph Receptor-Ephrin Signaling in Hippocampal Mossy Fiber LTP
Anis Contractor, Cheryl Rogers, Cornelia Maron, Mark Henkemeyer, Geoffrey T. Swanson, and Stephen F. Heinemann

Supplementary Material

Materials and Methods

Transverse hippocampal slices (350 Greek Letter Mum) were made from P12-18 mice (strain 129SvEv) as previously described (S1). Slices were transferred to a recording chamber, and whole-cell patch clamp recordings made from visually identified pyramidal cells in the CA3 region of the hippocampus. The composition of the internal solution was: 95 mM CsF, 25 mM CsCl, 10 mM Cs-HEPES, 10 mM Cs-EGTA, 2 mM NaCl, 2 mM Mg-ATP, 10 mM QX-314, 5 mM TEA-Cl, 5 mM 4-AP, pH adjusted to 7.3 with CsOH. Peptides were added directly to the internal solution on the day of the experiment from frozen stocks in protease inhibitors made up in phosphate buffered saline (PBS). To avoid leakage of peptides or antibodies in the extracellular space, pipette tips were filled with normal internal solution and then the pipette was backfilled with peptide/antibody containing internal. The final concentration of peptides was 50Greek Letter MuM in all experiments. The final concentration of protease inhibitors was; bestatin 2 Greek Letter Mug/ml, leupeptin 25 ng/ml, pepstatin 35 ng/ml, aprotinin 100 ng/ml. In control experiment, where peptides were not included, internal solutions were made up in exactly the same way with protease inhibitors in PBS but lacking any added peptide. Antibodies (EphB2 carboxy terminal polyclonal; GluR2/3 carboxy terminal polyclonal) were made up in PBS stock and added directly to the internal solution at a final concentration of 20 Greek Letter Mug/ml. The pH and osmolarity of all internal solutions were checked before use. Series resistances (Rs) were generally < 10MGreek Letter Omega and were continuously monitored throughout the duration of the experiment. Recordings in which Rs significant changed were discarded. Experiments using mutant mice were performed from heterozygous breedings blind to the genotype of the animal. Mossy fiber EPSCs were evoked with a monopolar glass electrode positioned in the stratum lucidum. All recordings were made in the continuous presence of bicuculline (10 Greek Letter MuM), picrotoxin (50 Greek Letter MuM) and D-AP5 (50 Greek Letter MuM). The group II mGluR agonist, L-CCG-1 (10 Greek Letter MuM), which selectively depresses mossy fiber transmission, was applied at the end of each experiment. LTP was induced with three 1s stimulation trains at 100 Hz given every 10s. Data are presented as mean ± SEM. Parameters were compared using the Student's unpaired t-test where not specified, and the K-S test, p < 0.05 was considered significant.
In vitro binding assay
The carboxy terminal region of EphB2 corresponding to mouse EphB2 receptor (aa 930-993) was generated by PCR from mouse brain library and subcloned into pGEX2TK. The GRIP(PDZ 4-6) and (PDZ 6) constructs were obtained by PCR from an adult rat hippocampal library. E coli (strain BL21) were transformed with His-GRIP constructs and GST-EphB2 and protein production induced with IPTG. GST-EphB2 protein was purified on glutathione beads and [35S]EphB2 cleaved from GST with thrombin (1U/Greek Letter Mug). GRIP protein was purified on Ni -NTA agarose, and eluted with imadazole. [35S]EphB2 (50 nM) was incubated with (15 Greek Letter Mug) of either His-GRIP(PDZ4-6) or His-GRIP(PDZ6) protein in PBS buffer with protease inhibitors for 2 hrs at 22 °C in the absence or presence of peptides. [35S]EphB2 binding was determined after 45 min incubation with Ni-NTA and vacuum filtration onto Whatman GFB filter circles (pretreated with 3% BSA). Filters were washed with ice-cold PBS buffer, allowed to equilibrate overnight in Ecolume scintillation cocktail, and specific counts measured in a scintillation counter. Non-specific binding was defined as the residual binding measured in the absence of His-GRIP fusion proteins.
GST pull down assay
GST fusion proteins were incubated with 200 Greek Letter Mug of rat brain membranes at 4 °C for 3 hours. GST beads were washed in (4 X 1 ml) of buffer containing 10 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 1 % Triton. Samples were run on SDS PAGE, transferred to PVDF membrane and blotted with a polyclonal antibody for GluR2/3.

S1. A. Contractor, G. Swanson, S. F. Heinemann, Neuron29, 209-16. (2001).