Hippocampal cultures and viral infection
Hippocampal primary cultures were prepared from embryonic day 18 Sprague-Dawley rats as per Osten et al., 1998 with the exception of coverslip coating. Here, hippocampi were triturated 15 min after trypsinization and plated on coverslips coated with poly D-lysine (50 μg/ml, Sigma) and laminin (5 μg/ml, Invitrogen). Dissociated neurons were cultured in neurobasal medium supplemented with B27 (Invitrogen). To eliminate proliferative glia cells, 5 μM cytosine arabinoside (AraC, Sigma), a specific inhibitor of DNA synthesis during meiosis and mitosis, was included after 8 DIV. Primary neurons were infected with a normalized infectious titer of modified Sindbis virus resulting in 10–20% infection of neurons for imaging and electrophysiological experiments one day before use and ~30% for biotinylation. Attenuated Sindbis viruses expressing EGFP-tagged Kv4.2 and EGFP alone were produced using the SINrep(nsP2S726
) viral vector and DH-BB(tRNA/TE12) helper plasmid as described (Kim et al., 2004
). The improved red fluorescent protein (tdTomato, provided by Dr. Tsien, (Shaner et al., 2004
)), a tandem dimer variant of DsRed, was transfected by Nucleofection (Amaxa Biosystems) in accordance with the manufacturer’s protocol. Other constructs used in present experiments were described previously (Kim et al., 2005
). Hippocampal organotypic slice cultures (400 μm thick) were prepared from postnatal day 7–8 Sprague-Dawley rats after (Shi et al., 1999
). Hippocampal CA1 neurons were infected on 4 DIV by microinjection (Nanoliter 2000, World Precision Instruments). All animal procedures were conducted with accordance of the National Institutes of Health Guide for the Care and Use of Laboratory Animals under a protocol approved by the National Institutes of Child Health and Human Development’s Animal Care and Use Committee.
Biotinylation assay and immunoblots
Hippocampal neurons (18–24 DIV) or acute slices (postnatal day 14) were incubated with freshly made sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin, 1 mg/ml, Pierce) in ice-cold PBS for 30 min at 4°C, followed by 10 min incubation in ice-cold glycine (100 mM) at 4°C, and lysed with lysis buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.05% SDS and a protease inhibitor mixture tablet (Roche). The lysate was incubated at 4°C for 20 min and spun at 13,000 rpm for 10 min. Biotin-labeled surface proteins were precipitated with 20–30 μl of Immobilized Streptavidin beaded agarose (Pierce) at 4°C overnight. The beads were washed five times with lysis buffer and proteins were eluted in 2x SDS loading buffer. Immunoblotting was performed as described (Kim et al., 2005
). Antibodies: anti-GFP (Molecular Probes, 1:5,000), anti-GluR1-C (Chemicon, 1:200), anti-Kv4.2 (K57/1, NeuroMab, 1:1,000), and anti-rab4 (BD Transduction Laboratories, 1:1,000).
Hippocampal neurons (18–24 DIV) were stimulated for imaging and biotinylation assays in culture media with either KCl (50 mM, Sigma), Glutamate (50 μM, Sigma), NMDA (20 μM, Tocris) or (S)-AMPA (100 μM, Tocris) at 37°C for 15 min. Due to activity-dependent filamentous structural change of F-actin in these experiments (Halpain et al., 1998
; Hering and Sheng, 2003
), a more mild stimulation was achieved by reducing the concentration of stimulants (25 mM KCl, 50 μM AMPA). All blockers were applied 15 min prior to stimulation. The blockers used in the experiments were 100 μM D,L-APV, 50 μM CNQX, 1 μM TTX, 20 nM Tetanus toxin (TeTN, Sigma) and 10 μM BAPTA-AM (Molecular Probes). Myristolated DYN peptide (myrs-QVPSRPNRAP) and scrambled DYN (myrs-QPPASNPRVR) were synthesized and purified by Sigma Genosys. Peptides (50 μM) were applied 10 min prior to AMPA stimulation. For the chemical LTP induction, 200 μM glycine was bath-applied in the Mg-free ACSF containing (in mM): 125 NaCl, 25 NaHCO3
, 2.5 KCl, 1.25 NaH2
, 2 CaCl2
, 11 D-glucose, 0.0005 TTX, 0.001 strychnine, and 0.02 bicuculine (pH 7.2) at 37°C for 3-5 min. Neurons were then incubated in the same ACSF without glycine at 37°C for 20 min. Treated neurons were always compared to simultaneously prepared sister culture controls.
Treated neurons were immediately fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS containing 0.12 M sucrose for 8 min on ice and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Actin was labeled with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (0.5 μM, Sigma) in PBS containing 1% BSA for 5 min.
Neurons were fixed/permeabilized as described above for GluR1 and synaptophysin staining or fixed with –20°C methanol for NMDAR1. After preblocking with PBS containing 5% NGS, 0.05% Triton X-100, and 450 mM NaCl for 1 h at 4°C, neurons were incubated with antibodies in the blocking solution overnight at 4°C and followed by incubation with Alexa 546-conjugated secondary antibodies (Molecular Probes) for 2 h at RT. To label surface AMPARs, non-permeabilized neurons were incubated with anti-GluR1-N in PBS containing 1% BSA and 4% NGS at 4°C overnight. Antibodies: Alexa 488-labeled anti-GFP (Molecular Probes, 1:10,000), anti-GluR1-N (Calbiochem, 1:20), anti-GluR1-C (Chemicon, 1:200), anti-NMDAR1 (Chemicon, 1:100), and anti-Synaptophysin (Sigma, 1:200).
Image acquisition and analysis
Fixed cell images were acquired with Leica TCS RS confocal microscope. The same instrument parameter settings were kept for each experiment. Every experiment was repeated a minimum of five times. The two fluorophores were excited with different wavelengths, 488 and 543 nm, and were separately imaged using dual sequential scanning to avoid overlapped emission from one to the other. Acquired images were analyzed using MetaMorph v6.3 (Universal Imaging Corporation) and ImageJ v1.36 (http://rsb.info.nih.gov/ij/
) under the same analytic parameter settings for each channel. To determine the number of Kv4.2g-positive spines or co-labeled clusters, marker-labeled spines or puncta (clusters) were randomly selected on 6–27 neurons (260–1400 spines) using a 1.7 mm diameter circle region of interest (ROI). For dendritic shaft analysis, the ROI was moved to the shaft directly below the spine. Fluorescent signals of Kv4.2g within the ROI were then measured. Pixels above the threshold intensity were counted and logged into Excel (Microsoft). To compare the co-localization of Kv4.2g and GluR1, fluorescent intensity was line-plotted by manually drawing a line either inside or outside of dendrites (range 0–255, arbitrary units).
Live imaging was performed at the NICHD Microscopy & Imaging Core using a Zeiss LSM 510 Inverted Meta with 100 × Zeiss alpha plan-neofluar oil objective (http://mic.nichd.nih.gov/index.htm
). Hippocampal primary neurons transfected with the tdTomato were plated on 25 mm coverslips and then infected with modified Sindbis virus expressing Kv4.2g on 18–24 DIV. Coverslips containing Kv4.2g and tdTomato co-expressed neurons were placed in an Attoflour cell chamber (Molecular Probes) and perfused with Mg-free ACSF containing (in mM): 125 NaCl, 25 NaHCO3
, 2.5 KCl, 1.25 NaH2
, 2 CaCl2
, 11 D-glucose (pH 7.2). The neurons, objective, and stage were heated in a custom-built incubation chamber supplemented with 5% CO2
. Time-lapse images were captured every 1 min for 15–20 min at 37 °C using Zeiss LSM Image Browser software v3.2. The instrument parameter settings were optimized in unstimulated Kv4.2g expressing neurons to avoid photobleaching and image saturation. Each image was a maximal projection of 5–7 z-stacks obtained at 0.5 μm depth intervals. Projection images generated for each time points were analyzed using MetaMorph, ImageJ, and Zeiss software. Spines were identified by tdTomato signal and randomly selected using ROIs. All spines identified in the first time point were followed for all the sequent time images, and thus selection was blind regarding the consequence. Fluorescent changes (ΔF) in Kv4.2g intensity were calculated after normalizing by the tdTomato intensity value in each time point using the initial green fluorescence (F0
) prior to stimulation as a baseline. Hence, fractional fluorescent change in each time trace was represented by Δ; F/F0
=(normalized F–normalized F0
. The data were averaged from 75–195 spines of 4–9 neurons from at least 6 independent experiments. Averaged values were presented as means±standard error of the mean (S.E.M.).
Thick-walled, filamented patch electrodes had tip resistances of 3–6 MΩ. Series resistance varied between 8–30 ΩM and recordings where series resistance varied by more than 10% were rejected. No electronic compensation for series resistance was employed. All electrophysiological data were recorded using an Axopatch200B amplifier (Molecular Devices Corp.). Recordings were filtered at 2 kHz for mEPSCs and at 5 kHz for K+ currents and evoked EPSCs. Command pulse generation, data acquisition and analysis were performed using IGOR Pro (Wavemetrics). MiniAnalysis (Synaptosoft), SPSS (SPSS Inc.) and Excel (Microsoft) software were used for further data and statistical analysis. One-way ANOVA, Wilcoxon Signed Rank, and Student’s t tests were used to examine statistical significance, set to p<0.05.
K+ current recordings
Transient K+ currents were measured in young hippocampal primary neurons (7–8 DIV). These smaller neurons, possessing fewer dendritic processes, were used to limit space-clamp and access resistance errors associated with whole-cell recordings of large (nA) currents. Electrodes were filled with a solution containing (mM): 140 KCl, 2 MgCl2, 10 HEPES, 5 EGTA, 5 ATP, 1 CaCl2, 10 D-glucose (pH 7.3 with KOH). 1 μM TTX was added to the external solution to block voltage-gated Na+ currents except during AMPA stimulation. After recording K+ currents, AMPA (50μM, 5 min) or glycine (200μM, 3 min) containing external solution was applied and currents were recorded every 5 min for 30 min. Transient and sustained K+ currents were digitally separated using a prepulse protocol after the subtraction of leak currents. Peak currents were measured at +120 mV after a 400 ms prepulse to either −120 mV or +30 mV. Non-myristolated DYN peptide (QVPSRPNRAP, 100 μg/ml) or scrambled DYN (QPPASNPRVR, 100 μg/ml) was co-applied in internal solution, 20–25 min prior to AMPA stimulation. During and after AMPA stimulation, resting membrane potential was monitored. Cells depolarizing beyond −30 mV during AMPA stimulation were not analyzed. Glycine application depolarized cells up to −45 mV. Cells that did not recover to −60 mV after 5 min of AMPA or glycine washout were also not analyzed.
Miniature excitatory postsynaptic current recordings
For recording mEPSCs, primary dissociated culture neurons of 14–21 DIV were used. The patch electrode solution contained either the same internal solution listed above for K+
current recordings (mEPSC amplitude ratio experiments, in some cases without MgCl2
) or, for chemLTP experiments (mM): 100 Cs-gluconate, 5 MgCl2
, 0.6 EGTA, 8 NaCl, 40 HEPES, 2 NaATP, 0.3 TrisGTP (pH 7.2 with KOH). TTX (0.5–1 μM), strychnine (1μM) and bicuculine (20μM) were included in the external solution during all mEPSC recordings. After whole-cell formation a 5–10 min recovery period elapsed before data collection. Ten seconds of spontaneous activity were recorded every 30 sec for each holding potential. mEPSC data were then averaged over a 5 min period (total sampling duration of 100 sec/5 min period) for up to 60 min. Only 5 min periods exhibiting more than 10 events and average decay times of less than 10 ms were analyzed. Neurons not recovering to within 10 mV of their resting membrane potential within 20 min after AMPA stimulation (25 μM, 2–3 min) were not analyzed. For amplitude and ratio experiments 2–3 episodes (10–15 min of data) were averaged before and after each condition. For chemLTP analysis, the largest 20% of mEPSC amplitudes for each 5 min period were averaged to reduce the impact of non-specific frequency changes (Stell and Mody, 2002
Hippocampal organotypic slice culture recordings
Hippocampal organotypic slice cultures were transferred to a submerged recording chamber with a continuous flow of ACSF containing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 25 Glucose, 2 CaCl2, 1 MgCl2, 0.005 bicuculine (pH 7.4) and bubbled with 5% CO2/95% O2. In all experiments, 5 μM 2-chloroadenosine was included in the external solution to block recurrent synaptic connections. The patch electrodes were filled with (in mM): 20 KCl, 125 Kglu, 10 HEPES, 4 NaCl, 0.5 EGTA, 4 APT, 0.3 TrisGTP, 10 Phosphocreatin (pH 7.2). To record EPSCs at several holding potentials, whole-cell CA1 pyramidal neuron recordings were made in voltage-clamp mode at 31–32 °C. The pathway between CA1 and CA3 was cut before each experiment. EPSCs were elicited by a test pulse (0.2 ms duration at 0.1 Hz, 30–600 μA amplitude), alternatively at −60 or −80 mV through a glass bipolar electrode (10 μm tip) located at the Schaffer collateral pathway. For LTP induction, 2 Hz stimulation was paired with depolarization to 0 mV for 1 min. In some experiments a second stimulating electrode was used to record EPSCs in a control (unpaired) pathway. Pathway independence was measured using a cross paired-pulse facilitation protocol. LTP-induced changes of EPSC amplitude were monitored for up to 1 hour (but at least 40 min).