Hippocampal Neuron Cultures and Cocultures
Rat hippocampi were isolated from E18-19 rats as described previously (Banker and Cowan, 1977
). Syt-IV knockout mice were provided by H. Herschman (University of California, Los Angeles), and hippocampi were isolated from P1-3 mice of either sex from syt-IV knockout heterozygote matings, where syt-IV knockout cultures could be directly compared to wild-type littermate cultures. Conditional BDNF knockout mice and CamKII-Cre mice (provided by Alexei Morozov, NIH, Bethesda) were crossed to generate homozygous forebrain specific BDNF knockouts, and these mice were crossed to syt-IV knockout heterozygotes for electrophysiology experiments comparing BDNF knockout to BDNF/syt-IV double knockout littermate hippocampal cultures. Hippocampi were treated with trypsin for 20 min at 37° C, triturated to dissociate cells, plated at 25,000-50,000 cells/cm2
on poly-lysine coated coverslips (Carolina Biologicals), and cultured in Neurobasal supplemented with 2% B-27 and 2 mM Glutamax (Gibco/ Invitrogen).
For HEK293 cell/neuron co-cultures, HEK cells were first cultured alone in DMEM with 10% fetal bovine serum and were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, these cells were mechanically dissociated in Neurobasal culture medium by gentle trituration, and added to 12 DIV neuronal cultures at a density of 1000-2000 cells/cm2.
All procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health, as approved by the Animal Care and Use Committee of the University of Wisconsin-Madison.
Antibodies, Reagents, and Mammalian Expression Constructs
Antibodies used were syt-IV, VGAT (Synaptic Systems), VGluT1, GluR2, synapsin, MAP2 (Chemicon), BDNF (Promega, Sigma), GFP, syt-17 (AbCam), synaptophysin (provided by R. Jahn, Göttingen, Germany), PSD-95 (Affinity Bioreagents) and bassoon (Stressgen). Cy2 and Cy3 secondary antibodies were from Jackson Immunologicals, and Alexa 647 secondary antibody was from Molecular Probes. Chemicals used were forskolin, glycine, TTX, APV (Sigma) and MG132 (Calbiochem). Mammalian expression constructs used were pEGFP-N1 (Clontech), mCherry (provided by R. Tsien, University of California, San Diego), and Nlg-GFP (provided by T. Dresbach, University of Heidelberg, Germany). Nrx-GFP was made by inserting PCR amplified GFP into a unique SalI site in VSV-tagged ß̃NRX-1 just after the LNS domain (constructed by H. Lee and provided by E. Isacoff, University of California, Berkeley). Nlg-IRES-GFP was made by inserting Neuroligin-1 (provided by P. Scheiffele, Columbia University, New York) into pIRES2-EGFP (Clontech). BDNF-PAGFP was constructed by replacing the GFP in BDNF-GFP previously described (Dean et al., 2009
) with PAGFP (provided by J. Lippincott-Schwartz, NIH, Bethesda). PAGFP-sytIV was constructed by replacing the GFP in previously described GFP-sytIV (Dean et al., 2009
) with PAGFP. GFP-syt17 was made by replacing the pHluorin in pHluorin-syt-1 (provided by T. Ryan, Weill Medical College of Cornell University, New York, NY) with GFP, and the syt-1 with syt-17 (provided by M. Craxton, MRC Laboratory, Cambridge, UK).
Transfection of Hippocampal Neurons
Neurons on 12 mm coverslips in 24-well plates were transfected with lipofectamine or calcium phosphate. For lipofectamine transfection (at 6-9 DIV), medium was removed, saved, and replaced with 500 μl fresh medium. 1 μl Lipofectamine 2000 (Invitrogen) in 50 μl Neurobasal medium and 0.5 μg DNA in 50 μl Neurobasal medium were incubated separately for 5 min, then mixed and incubated for 30 min at room temp. This mixture was added to the well of neurons, incubated for four hours at 37° C and 5% CO2, and then removed and replaced with half saved and half fresh medium.
Neurons were transfected by calcium phosphate at 3-4 DIV as described previously (Dresbach et al., 2003
). Prior to transfection, medium was removed, saved, and replaced with 500 μl 37°C Optimem (Life Technologies) and incubated for 30-60 min. 105 μl transfection buffer (274 mM NaCl, 10 mM KCl, 1.4 mM Na2
, 15 mM glucose, 42 mM Hepes, pH 7.06) was added dropwise to a solution containing 7 μg of DNA and 250 mM CaCl2
, with gentle vortexing. This mixture was incubated for 20 min at room temp, 30 μl was added per well, and neurons incubated for 60-90 min. This medium was then removed, cells were washed 3× in 37°C Neurobasal medium, and saved medium added back to the transfected cells.
For photoactivation experiments, coverslips of neurons cotransfected with sytIV-PAGFP/mCherry or with BDNF-PAGFP/mCherry were transferred to a live imaging chamber (Warner Instruments) containing 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, 25 mM HEPES, pH 7.4. Images were acquired on a Nikon A1Rsi 32 channel spectral imaging inverted confocal laser microscope equipped with a Perfect Focus System using 40× or 60× objectives and a hybrid galvano/ resonant scanner for simultaneous photoactivation and imaging (at the Nikon Imaging Center at the University of Heidelberg).
DIV 6-8 transfected cells were visualized by mCherry fluorescence, and chosen such that axons and dendrites could be clearly identified based on morphology (and verified by retrospective immunostaining for MAP2 to mark dendrites in all cases where axons and dendrites were difficult to distinguish). For each timelapse experiment, a baseline of GFP and mCherry fluorescence was collected for 10 seconds prior to photoactivation of the region of interest with 405 nm laser light. Regions to photoactivate in axons, dendrites, or cell bodies were chosen to include the largest region possible in the compartment of interest, without including any other regions. shows the specificity of photoactivation within defined regions. Regions were photoactivated continuously with the 405 nm laser at 40% laser power and a pixel dwell time of 20 μsec during simultaneous acquisition of GFP and mCherry fluorescence, such that all vesicles transiting through the region during the timelapse were photoactivated. Laser intensity and pixel dwell time were selected to maximize photoactivation and minimize bleaching. The mCherry channel was imaged simultaneously and served as a control for focal drift.
Spatial accuracy of photoactivation and identification of axons versus dendrites
Following image collection, cell bodies, axons and dendrites were selected as regions in NIS Elements software and fluorescence intensity was plotted versus time. Average fluorescence intensity at the end of the timelapse was calculated in 10 μm long × 3 μm wide regions of processes in axons and dendrites proximal to the cell body. For axon photoactivation experiments, fluorescence intensity was calculated in a region within the photo-activated area, in an area outside and just distal to the photoactivated area, in the cell body, and in proximal dendrites, at t=0 and 10 min. For dendrite photoactivation, fluorescence intensity was calculated in a region within the photoactivated area, in a different dendrite (i.e. the vesicles would have to travel from the photoactivated dendrite, into the cell body, and then into another dendrite), in the cell body, and in the proximal region of the axon, at t=0 and 10 min.
Immunocytochemistry, Image Acquisition and Quantitation
Cells were fixed with 4% paraformaldehyde, permeabilized and blocked in 10% goat serum and 0.1% Triton X-100, and immunostained with primary antibodies at room temperature for 4 h, or overnight at 4°C.
Images of immunostained cells were acquired on an Olympus FV1000 upright confocal microscope with a 60× 1.10 NA water immersion lens. 488 nm, 543 nm, and 635 lasers were used, with Olympus FV1000 automated dichroic/ emission settings for sequential scanning of Cy2, Cy3, and Alexa 647 secondary dyes. For quantitation, images were acquired with identical laser and gain settings and imported into Metamorph (Improvision) for analysis. For % colocalization of syt-IV at synapses (), thresholds were chosen for the VGluT channel (green), VGAT channel (blue), and syt-IV channel (red) such that all recognizable puncta were selected. Since the syt-IV antibody cross-reacts with other proteins in western blots of tissue isolated from brain, we used knockout mice as a control for specificity. Confocal gain in all experiments was set such that no syt-IV signal was visible in syt-IV knockout neurons. The average intensity of syt-IV signal within VGluT or VGAT puncta was determined using Metamorph for all conditions and expressed as % of control. For % colocalization, the fraction of syt-IV positive signal within VGluT or VGAT positive-puncta was calculated using Metamorph. Colocalization between VGluT and VGAT was 5% and a positive control double immunostain using an antibody against the luminal domain of syt I, and another antibody against the cytoplasmic domain of syt I showed 90% overlap. Cell bodies and regions 10 μm proximal to cell bodies were excluded from analysis because the syt-IV signal in the soma often overlapped with inhibitory terminals on cell bodies.
For quantitation of immunofluorescence signals on Nlg-IRES-GFP, Nlg-GFP, Nrx-GFP or GFP expressing HEK293 cells, transfected HEK cells were marked by thresholding the GFP signal. The average intensity of syt-IV signal within this thresholded region was then determined. Statistical significance for all quantitation was determined by a Student's t-test where *=p<0.05, **=p<0.01, and ***=p<0.001.
Protein Purification and Western Blots
12-14 DIV hippocampal neurons treated with 200 μM glycine for 15 minutes or one hour, 50 μM forskolin for four hours, 50 μM MG132 for four hours, 1 μM TTX for 48 hours, or in control conditions growing in 10 cm dishes at a density of 50,000 cells/cm2, were washed twice in PBS, and 1 ml of hypotonic buffer (10 mM Tris-HCl pH 7.4) was added per plate for 5 min. Cells were harvested in 0.5 ml homogenization buffer (320 mM sucrose, 1 mM EDTA, 10 mM Tris-HCl pH 7.4) by scraping and passed through a 27-gauge needle ten times. A final concentration of 150 mM NaCl was added to the lysate. Cell lysates were centrifuged at 4000 rpm for 10 minutes to pellet nuclei and cellular debris. The supernatant was collected and the protein concentration determined by BCA assay. 12 μg of total protein per lane was resolved by SDS-PAGE and analyzed by immunoblotting for syt-IV (which runs as a 46 kDa band).
For imaging of syt-IV/synaptophysin or syt-I/ synaptophysin hippocampal neurons were fixed and immunostained with Atto565 and Dy-485XL secondary antibodies (Dianova, Hamburg, Germany). Pulsed excitation of Atto565 (Atto-Tec, Siegen, Germany) was achieved using a high-repetition rate laser diode source (PicoTA, Picoquant, Berlin, Germany) at a wavelength of λexc=532 nm, synchronized with the STED laser via a photodiode (Alphalas, Göttingen, Germany). Dy-485XL (Dyomics GmbH, Jena, Germany) was excited by the 470 nm line from a pulsed high-repetition rate laser diode source (Picoquant, Berlin, Germany). Fluorescence inhibition was accomplished at λSTED=647 nm using an actively mode locked Ar-Kr laser (Spectra Physics, Irvine, USA). The excitation and modulation beams were combined using acousto-optical tunable filters (AOTF) (Crystal Technology, Inc., Palo Alto, USA) and coupled into a microscope stand (DMI 4000B, Leica Microsystems GmbH, Mannheim, Germany) equipped with a three-axis piezo stage-scanner (PI, Karlsruhe, Germany). The AOTFs enabled blanking of the lasers and allowed the power of each laser beam to be controlled independently. They also provided a means for selecting counter-propagating fluorescence returning from the confocal microscope. The collected fluorescence passed through an additional band-pass filter (580/40 for Atto565 and Dy-485XL, AHF Analysentechnik, Tübingen, Germany) and was detected confocally with a photon counting module (SPCM-AQR-13-FC, PerkinElmer, Waltham, USA). The scanner fly backs were blanked via the line signal from the data acquisition software (Imspector, MPI Göttingen, Germany). A vortex phase plate (RPC Photonics, NY, USA) was used in the STED beam path to generate a donut shaped light distribution in the focal plane.
BDNF and Syt-IV were immunostained in fixed hippocampal neurons using Atto590 and Atto647N secondary antibodies (Dianova, Hamburg, Germany) and imaged with a custom-built two-color STED microscope as previously described (Buckers et al., 2011
). The excitation wavelengths (λexc,Atto590
=570 nm and λexc,Atto647N
=650 nm) and the STED wavelengths (λSTED,Atto590
=(720±10) nm and λSTED,Atto647N
=(755±15) nm) were all provided by a single supercontiuum laser source (SC-450-PP-HE, Fianium Ltd., Southampton, UK). Fluorescence was detected with two separate confocal detection units covering the spectral ranges of λfluo,Atto590
=(620±20) nm and in λflluo,Atto647N
=(670±15) nm. The application of a pulse-interleaved acquisition scheme enabled the simultaneous recording of both imaging channels. Thus, no relative shift of the images, i.e. due to a drift of the sample, is expected. In addition, crosstalk, i.e. leakage of signal into the wrong imaging channel, was eliminated by linear unmixing.
Images were preprocessed using a single-step linear deconvolution (LD), i.e. Wiener filter, which was carried out with a theoretical effective point-spread-function of 40 nm FWHM.
Whole cell patch clamp recordings of mEPSCs were made from 15-21 DIV dissociated hippocampal cultures from syt-IV knockout and wild-type littermates (in control conditions and following treatment with 1 μM TTX, or 200 μM glycine/ 10 μM bicuculline for 48 hours), or BDNF knockout and syt-IV/BDNF double knockout littermate cultures. Neurons were voltage clamped at −70 mV in the presence of 1 μM TTX and currents recorded using an EPC-10/2 amplifier (HEKA Electronics, Germany) and Patchmaster software (HEKA). Cells were continuously perfused with extracellular solution consisting of, in mM: 140 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 1 MgCl2, 10 glucose, 50 μM APV, bubbled with 95% O2 and 5% CO2 (pH 7.3, 300 mOsm). Intracellular solution consisted of, in mM: 130 K-gluconate, 10 EGTA, 10 HEPES, 5 Phosphocreatine, 2 Mg-ATP, 0.3 Na-GTP, (pH 7.3, 300 mOsm). For recording mEPSCs, 0.1 mM picrotoxin and 1 μM TTX were added to the bath solution. For recording mIPSCs, picrotoxin was replaced with 20 μM CNQX. Recordings were filtered at 2.9 kHz and digitized at 5 kHz. Events were detected with MiniAnalysis software (Synaptosoft) using a threshold of five times the RMS noise. Statistical significance was determined by a Student's t-test where * = p< 0.05, ** = p< 0.01, and *** = p< 0.001.
FM Dye Destaining Experiments
For FM dye experiments, wild-type and syt-IV knockout mouse neuron cultures in control conditions, or treated with 1 μM TTX for 48 hours, were loaded with 10 μM FM1-43 in depolarizing buffer (100 mM NaCl, 45 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5 mM glucose, 20 mM HEPES, ph 7.3) for 2 min to maximally load all vesicles. Coverslips were then transferred to a 5 mM KCl solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5 mM glucose, 20 mM Hepes, ph 7.3) for 5 min to remove excess dye. Coverslips were then mounted in live imaging chambers (Warner Instruments) in the 5 mM KCl solution. FM-dye labeled boutons were visualized with a 484 nm excitation/605 nm emission filter and images acquired on a Nikon TE300 inverted microscope with a Roper Scientific Photometric Cascade II EMCCD camera, and a Lambda DG-4 high speed wavelength switcher, interfaced with Metamorph software. Images were collected at 1 sec intervals with 200 msec exposure times. A baseline was collected for 10 images before addition of depolarizing buffer (45 mM KCl) to destain neurons. Following image collection, FM dye-labeled boutons were selected as regions in Metamorph and fluorescence intensity was plotted versus time. Destain traces were normalized by setting maximal load to one and complete destain (disappearance of bouton to background levels) to zero, for comparison of rates. Rate of destain (tau) was determined by a single exponential fit of normalized destain curves.
Detection of epileptiform response by voltage imaging was performed as previously described (Chang et al., 2007
). Five-week old mice were euthanized with CO2
. Brains were removed into ice-cold cutting solution consisting of, in mM: 124 NaCl, 3.2 KCl, 26 NaHCO3
, 1.25 NaH2
PO4, 1 CaCl2
, 6 MgSO4
,10 glucose, and bubbled with 95% O2
. Horizontal 350 μm thick hippocampal slices were cut with a tissue slicer (HR2, Sigmann Elektronik, Germany). Slices were incubated in bubbled artificial cerebrospinal fluid (ACSF), identical to the cutting solution but containing 2.5 mM CaCl2
and 1.3 mM MgSO4
, for 1 h, stained with bubbled ACSF containing 0.02 mg/ml of the voltage-sensitive absorbance dye RH482 (NK3630, Hayashibara Biochemical Laboratories, Okayama, Japan) for 1 hr, and then returned to ACSF. During recording, slices were continuously perfused with bubbled ACSF at 29-31°C. Slices were stimulated with 200 μs current pulses delivered by a stimulus isolator (Model A365, World Precision Instruments, Sarasota, FL). The photodiode array instrumentation and software have been previously described (Chang and Jackson, 2006
). Optical signals (average of four consecutive trials) are presented as the change in transmitted light divided by the resting light intensity (ΔI/I), and are proportional to the membrane potential change within the physiological range of voltages.