Animal care and procedures were approved by the University of Iowa Animal Care and Use Committee, and performed in accordance with the standards set by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996. On postnatal Days 0–1, pups of either sex of the knock-in model of DYT1 dystonia (Goodchild et al.,2005) were genotyped according to the fast-genotyping procedure (within ∼5 h, EZ Fast Tissue/Tail PCR Genotyping Kit, EZ BioResearch LLC, St, Louis, MO), using a published protocol (Goodchild et al.,2005).
Individual newborn pups were genotyped and cultured separately. Primary hippocampal neurons were cultured by a method described in our previous work (Harata et al.,2006), with some modification. Briefly, the CA3-CA1 regions of the hippocampus were dissected on postnatal Days 0–1, trypsinized and dissociated. The cells were plated on 12-mm coverslips pre-seeded with a rat glial feeder layer (Garcia-Junco-Clemente et al.,2010), in 24-well dishes and at a density of 12,000 cells per well. The feeder layer had been seeded in plating medium with the following composition: MEM (Invitrogen, Carlsbad, CA) plus 5 g/l glucose, 0.2 g/l NaHCO3, 100 mg/l bovine transferrin (EMD Chemicals, Gibbstown, NJ), 2 mM GlutaMAX (Invitrogen), 25 mg/l insulin, and 10% fetal bovine serum (FBS, Invitrogen). Feeder layers were maintained in a 1:1 mixture of plating medium and growth medium. The latter had the following composition: MEM plus 4 μM cytosine β-D-arabinofuranoside, 0.5 mM GlutaMAX, NS21 (Chen et al.,2008), and 5% FBS. The hippocampal neurons were used on Days 11–14 of culture. The experimental data were obtained from 4 or 5 different culture batches (animals) for each genotype (wild-type, heterozygous and homozygous littermates).
Patch-clamp recording of miniature postsynaptic currents
Electrical recordings were carried out on an inverted microscope (Eclipse-TiE, Nikon, Melville, NY). Membrane currents were monitored by the voltage-clamp mode of conventional whole-cell patch-clamp recording. Patch-pipettes were fabricated from borosilicate glass tubes (1.5-mm outer diameter, PG52151-4, World Precision Instruments, Sarasota, FL) using a pipette puller (P-97 Flaming/Brown Micropipette Puller, Sutter Instrument, Novato, CA), and fire polished (Micro Forge MF-830, Narishige International USA, East Meadow, NY). The resistance of the recording electrode was 2–3 MΩ. The patch-pipettes were positioned with high precision and minimal drifting using a manipulator (PatchStar Micromanipulator, Scientifica, East Sussex, UK). The current and voltage were measured with a patch-clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA), which was controlled by the pCLAMP software (Molecular Devices). The membrane currents were acquired at 10 kHz, and filtered at 5 kHz with a built-in 4-pole Bessel filter.
Miniature postsynaptic currents were recorded in the continued presence of 0.5 μM TTX (Tocris Bioscience, Ellisville, MO) in the external solution. Recording of miniature events was started at least 3–4 min after establishing the whole-cell recording, to allow enough time for the internal pipette solution to equilibrate with the intracellular condition. Miniature events were recorded for 2–4 min. In one series of experiments (Supporting Information Fig. S1), glutamate-mediated mEPSCs and GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) were also isolated in the presence of GABAA receptor antagonist (20 μM (-)-bicuculline methochloride, Tocris Bioscience, Ellisville, MO) and AMPA receptor antagonist (10 μM, 6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, Tocris Bioscience), respectively. In a separate experiment, we confirmed the effectiveness of TTX treatment in our assay, demonstrating that it completely eliminated the inward Na+ currents induced by a 2-ms step depolarization to 0 mV from a holding potential (VH) of −70 mV. Series resistance was measured based on the response to a 10 mV depolarizing voltage step from a holding potential of –70 mV for 100 ms, and was compensated at ∼70%. The recordings were discarded if the series resistance before compensation exceeded 20 MΩ or if the membrane resistance was below 100 MΩ. All experiments were performed at room temperature (23–25°C).
The Tyrode (external) solution had the following composition (in mM): 125 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 30 glucose, 25 HEPES, 310 mOsm, pH 7.4. Two internal solutions were used. One, with an intra-pipette Cl− concentration ([Cl−]pipette) of 75 mM, had the following composition (in mM): 60 K-gluconate, 70 KCl, 5 NaCl, 1 EGTA, 4 MgATP, 0.3 GTPNa2, 10 HEPES, 10 phosphocreatine, 5 unit/mg creatine phosphokinase, pH 7.2, 305 mOsm. The second internal solution had the [Cl−]pipette of 9 mM and the following composition (in mM): 130 K-gluconate, 8 NaCl, 1 EGTA, 4 MgATP, 0.3 GTPNa2, 10 HEPES, 10 phosphocreatine, 5 unit/mg creatine phosphokinase, pH 7.2, 305 mOsm. The calculated Cl− equilibrium potentials (ECl) were −15.0 and −69.1 mV for [Cl−]pipette of 75 and 9 mM, respectively. ECl was calculated based on the Nernst equation at 23°C: ECl = −58.8*log(135/[Cl−]pipette) (Kakazu et al.,1999).
It is possible to record mIPSC as an outward current by having ECl more hyperpolarized than the holding potential (e.g. Li et al.,2011), but we did not use this method because the overriding, outward mIPSC would have complicated the analysis of inward mEPSCs.
Analysis of mEPSCs
Miniature events were analyzed using the Mini Analysis Program (version 6.0.7, Synaptosoft, Fort Lee, NJ). Events were selected for analyses using criteria based on threshold amplitude (5x IRMS) and area under the curve (1.5x amplitude). In addition, all the traces were visually examined to protect against software errors. The average baseline noise (IRMS) was 2.94 ± 0.34 pA for wild-type neurons (n = 16), and 3.90 ± 0.49 pA for heterozygous neurons (n = 14) over 51.2 ms, with no statistically significant difference (P = 0.11, mean ± SEM, t-test).
Interevent interval was defined as the time elapsing between measured peaks for two consecutive events. The amplitude of a mEPSC event was calculated by subtracting the pre-event baseline current (averaged over 1 ms) from the peak amplitude. If the rise of an event occurred during the falling phase of a previous event, as might occur during a burst, the baseline was estimated by extrapolating the decay of the first peak using a single exponential function. The amplitude of the event was then calculated by subtracting the extrapolated baseline from the peak amplitude. Rise time was defined as the time for the signal to increase from the onset (0.5% of the peak amplitude) to the peak (100%). Decay time was defined as the time for the signal to decrease from the peak (100%) to 40%. This parameter is susceptible to changes in noise level, especially near the end of the decay period, when the signal amplitude approaches that of the noise. Also multiple overriding events were not excluded in measuring this parameter. Thus the decay phase was also analyzed using another parameter, which is less susceptible to changes in noise level. The decay time constant was measured by fitting a single exponential curve to the 80–20% decay phase of an isolated event. Curve fitting was applied to the first 100 events, in each recorded neuron, that started from a stable baseline and decayed back to baseline (i.e., no other events overrode the decay phase). An area of an event (unitary mEPSC charge) was defined as the area delimited by the pre-event baseline level, onset time (0.5% of peak amplitude) and the time at which the signal decreased to 40% of peak amplitude.
Live-cell and fixed-cell imaging of FM dye
For live-cell imaging, the neurons were subjected to staining by spontaneous activity (basal activity) (Kakazu et al., 2012). They were incubated in 2.5 μM FM4-64 (in MEM, Invitrogen) for 10 min, at 37°C, in the absence of neurotransmitter receptor antagonists and TTX. After staining, neurons were transferred to an imaging chamber (RC-21BRFS, Warner Instruments, Hamden, CT) and washed with dye-free Tyrode solution for 6–7 min, at room temperature. Spontaneous loss of FM dye due to action potentials was suppressed by the application of TTX, CNQX, and the NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (50 μM, Tocris Bioscience).
After washing, the nerve terminals were imaged for 120 s for the miniature FM release, in the continued presence of TTX, CNQX, and D,L-AP5. They were destained afterwards, by applying the Ca2+ ionophore, ionomycin (5 μM) for 120 s in the continued presence of TTX, CNQX and D,L-AP5 at room temperature. For each coverslip, only a single imaging field was analyzed. This eliminates the possibility that any of the imaged fields was subject to ionomycin-induced FM destaining during an earlier experiment.
For fixed-cell imaging of FM dye, the live neurons were stained with a modified Tyrode solution containing 45 mM KCl and 2.5 μM aldehyde-fixable FM4-64 (FM4-64FX, Invitrogen) for 1 min. Thereafter the neurons were washed for 1 min and chemically fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and 4% sucrose in Tyrode solution for 30 min at 4°C, washed for 10 min in Tyrode solution and imaged. The fluorescence intensity of fixable FM4-64 was retained after paraformaldehyde fixation.
All experiments with FM dyes were carried out using extracellular solutions lacking GABAA receptor antagonists.
Immunocytochemistry was used to detect the glutamatergic presynaptic marker, vesicular glutamate transporter 1 (VGLUT1), and the dendritic marker, microtubule-associated protein 2 (MAP2). The cultured neurons were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and 4% sucrose in Tyrode solution for 30 min at 4°C. After being rinsed with Tyrode solution twice for 5 min each at 4°C, the cells were blocked and permeabilized with 2% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) and 0.4% saponin in phosphate-buffered saline (PBS, pH 7.4, Invitrogen) (blocking solution), for 60 min at room temperature. Thereafter they were treated with polyclonal, guinea-pig anti-VGLUT1 antibody (AB5905, Chemicon-Millipore, Billerica, MA) (1000x dilution in blocking solution), and polyclonal, rabbit anti-MAP2 antibody (AB5622, Chemicon-Millipore) (400x dilution) overnight (15-21 hours) at 4°C. Following rinsing with PBS, 3 times for 7 min each, the neurons were incubated with goat anti-guinea-pig IgG antibody conjugated with Alexa Fluor 594 (Invitrogen) (1000x dilution in blocking solution), and goat anti-rabbit IgG antibody conjugated with Alexa Fluor 405 (Invitrogen) (1000x dilution) for 60 min at room temperature. They were rinsed with PBS at least five times for 20 min each, and observed directly in PBS.
Fluorescence imaging system
Cells were imaged using an inverted microscope (Eclipse-TiE, Nikon). For imaging FM dyes, we used an EMCCD camera (DU-860, Andor Technology, Belfast, UK). The camera was continuously perfused with chilled water (Oasis 160 liquid recirculating chiller, Solid State Cooling Systems, Wappingers Falls, NY) to maintain a temperature of −80°C, and to thereby reduce noise.
In live neurons, FM dye was excited using a 490-nm light-emitting diode (LED, CoolLED-Custom Interconnect, Hampshire, UK) with 10% intensity, and imaged with an objective lens (Plan Fluor, 40x, NA1.30, Nikon), a filter cube (490/20-nm ex, 510-nm dclp, 650-nm-LP em) and 0.7x coupler. 16-bit images were acquired at 1 frame/s, 20-ms exposure time of camera, EM gain of 50, 10 MHz pixel readout rate, without binning, using the Solis software (Andor). Images were stored in a SIF format (Andor). Neuronal exposure time to excitation light was minimized to 20 ms, by turning the LED on only during image capture (triggered by “Shutter” output of the camera).
In fixed neurons, fixable FM dye was visualized using largely the same set of imaging parameters used for FM4-64 in live neurons, including the 20-ms exposure time of camera. The exceptions were the use of either (1) 10% LED intensity and a 20-ms exposure to LED light (the same as in live-neuron imaging), (2) 100% LED intensity and a 20-ms exposure to LED, or (3) 100% LED intensity and continuous exposure to LED (still with the same 20-ms exposure time of camera).
For immunocytochemical observation, we used an interline CCD camera (Clara, Andor Technology). The camera was cooled at -45°C by an internal fan. Alexa Fluor 594 was excited using a 595-nm LED (CoolLED-Custom Interconnect) with 100% intensity, and imaged with a filter cube (590/55-nm ex, 625-nm dclp, 665/65-nm em), and 2-s exposure. Alexa Fluor 405 was excited using a 400-nm LED (CoolLED-Custom Interconnect) at 100% intensity, and imaged with a filter cube (405/40-nm ex, 440-nm dclp, 470/40-nm em), and 2-s exposure. 16-bit images were acquired with an objective lens (Plan Fluor, 40x), without a coupler (i.e., 1x) and without binning, in a single-image capturing mode of the Solis software (Andor). Images were stored in a SIF format (Andor). For immunocytochemical imaging, differential interference contrast (DIC) optics was first applied to identify neurons of normal morphology; fluorescence images were acquired only afterward, and thus neurons were selected for analysis without regard to fluorescence information.
Image analysis for FM experiments
FM signal was quantified using Image J (v1.43m, W. S. Rasband, NIH) and the associated plug-ins. Acquired images in a time series (“Stack”) were aligned, using the ImageJ-Image Stabilizer plug-in (Kang Li) to correct for small movements in FM signals, and saved in a TIFF format.
Functional nerve terminals (boutons) in live neurons were identified based on differences in fluorescence intensity between the 5-frame averages taken immediately before and after ionomycin-induced destaining. Regions-of-interest (ROIs, 3x3 pixels, 2.4x2.4 μm) were assigned on isolated, fluorescent puncta in the difference image, if five or more of the 9 pixels showed an intensity above 30 arbitrary units (a.u.), which was approximately the standard deviation of the background intensity obtained from the bare coverslip area. Changes in ROI intensity were measured using ImageJ-Time Series Analyzer V2.0 (Balaji Jayaprakash), and were exported to Microsoft Excel. ROIs were excluded if they exhibited any of the following changes in intensity: an increase during recording, a sudden decrease before application of ionomycin or a long latency after application of ionomycin.
Miniature FM release was quantified as the cumulative change in the absolute intensity of FM4-64 between times 0 and 120 s of imaging (ΔFMMinis). It should be noted that individual release events cannot be visualized under the conditions used here, since the signal intensity from a single vesicle is low compared to the high noise level created by other stained vesicles in the same terminal (Aravanis et al.,2003).
The FM dye photobleaching was assessed in the fixed neurons stained with fixable FM4-64. ROIs of the same size were chosen from isolated fluorescent puncta that corresponded to nerve terminals. Changes in FM intensity in the ROIs during imaging were normalized by setting the absolute intensity at the start of imaging experiments as “1” and setting the absolute intensity when the camera shutter was closed as “0.” The normalized curves were fit with single exponential functions: FM(t) = exp(-t/τPB), where FM(t) is the relative intensity after time t of imaging; t is time in seconds after start of imaging; τPB is a time constant of photobleaching. Our measurement shows that our imaging system caused only minimal photobleaching (Supporting Information Fig. S5), and thus we did not need to correct for this.
For figure production, the contrast and brightness of the FM images were changed linearly using ImageJ, such that the parameters remained the same for all images in a single figure.
Image analysis for counting the numbers of somata and nerve terminals
Images were analyzed using Image J. Neuronal density was measured by counting the number of somata of live neurons, in a field imaged with 4x phase-contrast objective lens. Each field encompassed 3.74 mm2.
The number of glutamatergic nerve terminals in our preparation was counted, based on positive immunocytochemical staining for VGLUT1. Positive signals were identified after the intensity of image background was subtracted, and the pixels were selected for the intensity above a threshold. A cluster of contiguous positive pixels was counted as a potential glutamatergic terminal. To focus on the population of VGLUT1 in nerve terminals that make synapses on postsynaptic dendrites, we limited our analysis to the isolated VGLUT1 signals making contact with the MAP2-positive region.
For figure production, the images of VGLUT1 and MAP2 were binarized, and overlaid on gray-scale DIC images using ImageJ.
The distributions of the parameters of miniature events were compared for statistical significance using the nonparametric Kolmogorov–Smirnov test (Figs. 1, 2, and 4) (Sulzer and Pothos,2000; Van der Kloot,1996) because they were not Gaussian (normal) (Supporting Information Fig. S1, panel E). Specifically, a cumulative histogram was created by (Sulzer and Pothos,2000; Van der Kloot,1996): sorting the data from a single experiment in ascending order; assigning each data point a rank number and a fractional value based on that rank (i.e., fractional value = rank number/total number of events); and plotting the fractional values of all events cumulatively, with the y-axis indicating the fractional value for each data point and the x-axis indicating the specific value of the parameter for the data point (e.g., mEPSC amplitude). This normalizes the distributions between y = 0 and y = 1. Each curve of the final cumulative histogram was then obtained by averaging the cumulative histograms obtained from individual neurons of the same genotype, in order to eliminate giving different weights to neurons based on the occurrence of different numbers of events.
Figure 1. The interevent intervals of miniature excitatory postsynaptic currents (mEPSCs) were shortened in ΔE-torsinA neurons. A: Phase-contrast image of a patch-clamped hippocampal neuron in primary culture, obtained from a wild-type mouse. Patch pipette is visible in the lower-right quadrant. B: Representative traces from mEPSC voltage-clamp recordings in neurons of different genotypes. Arrows indicate interevent intervals in the wild-type trace. The miniature release events were recorded in the presence of the voltage-dependent Na+-channel blocker tetrodotoxin (TTX). The holding potential (VH) was −70 mV, and the Cl− concentration in the internal, pipette solution ([Cl−]pipette) was 9 mM. These recording conditions allow for the detection of mEPSCs without interference from miniature inhibitory postsynaptic currents (mIPSCs), even in the absence of a GABAA receptor antagonist (see Supporting Information Figs. S2 and S3). C: Cumulative histograms of the interevent intervals of mEPSCs. The interevent intervals were significantly shorter in heterozygous (red) and homozygous neurons (green) than in their wild-type counterparts (black). In a black-and-white printing, the distribution for heterozygous neurons is in darker gray, and that for homozygous neurons is in lighter gray. The numbers of analyzed mEPSC events (n) were 10,440, 17,463, and 13,049, obtained from (N) 16 wild-type, 14 heterozygous, and 12 homozygous neurons, respectively. Asterisks indicate statistically significant shifts to shorter intervals, as assessed by Kolmogorov-Smirnov test, based on “n”. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 2. Miniature release of FM dyes from nerve terminals was enhanced in ΔE-torsinA neurons. A: Live-cell imaging of miniature FM release. Live neurons were loaded with FM4-64 through spontaneous activity, by incubating them in the dye-containing solution for 10 min at 37°C (in the absence of TTX). The neurons were visualized using differential interference contrast optics (DIC) before fluorescence imaging (a), and using fluorescence optics at: the start of imaging (b), 120 s after the start of imaging (c), and 120 s after fluorescence loss (destaining) induced by application of the Ca2+ ionophore ionomycin (d). Nerve terminals were identified based on positive responses to ionomycin, and signals from only those nerve terminals were analyzed. TTX was present throughout the imaging period. The intensity of FM4-64 fluorescence in nerve terminals is plotted for the whole experiment (e), and in an expanded form for the period of miniature FM release (f). The total amount of FM lost in the presence of TTX (ΔFMMinis) was determined by plotting the data using the end point of the 120-s observation as a baseline (dotted lines in e and f). Thus the signals above the dotted horizontal lines represent the miniature releases. The signals under the dotted horizontal lines represent the evoked intensity changes that we had dealt with in our previous publication (Kakazu et al.,2012). The curves represent averages ± sem of data from the wild-type (black), heterozygous (red), and homozygous neurons (green). The numbers of analyzed nerve terminals (n) were 328, 387, and 380, obtained from (N) 3 wild-type, 4 heterozygous and 5 homozygous neurons, respectively. The arrow shows ΔFMMinis with averaged wild-type signals as an example. Thin continuous curve represents the estimated amount and time course of photobleaching, based on the data obtained in Supporting Information Figure S5. The photobleaching was small and therefore was not used for correcting the positive signals. B: Cumulative histograms of ΔFMMinis obtained from the data in panel A. The raw data (a) show that the absolute values of ΔFMMinis in heterozygous (red) and homozygous (green) neurons are larger than those in wild-type neurons (black). The asterisks indicate statistical significance (wild-type vs. heterozygotes, P = 9.4 x 10−6; wild-type vs. homozygotes, 5.3 x 10−9, Kolmogorov-Smirnov test based on “n”). In the bottom panels, good fits to the heterozygous (b) and homozygous distributions (c) were achieved by scaling the wild-type distribution (broken curve) in the x-direction. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Means were compared using the unpaired Student's t test (Fig. 3). All significance values provided are two-tailed P values.
Figure 3. Effects of the ΔE-torsinA mutation on the number of glutamatergic nerve terminals. A–C: Immuno-fluorescence images of the glutamatergic marker, vesicular glutamate transporter 1 (VGLUT1, red), and of a dendritic marker, microtubule-associated protein 2 (MAP2, green). They were overlaid on a DIC image of the same field. The images were obtained from representative wild-type (A), heterozygous (B), and homozygous cultures (C). For the purposes of illustration, only VGLUT1 signals in close association with MAP2 signals have been selected. In addition, the images of VGLUT1 and MAP2 are shown on a binary scale, whereas the DIC image is shown in gray scale. D: Numbers of VGLUT1 puncta normalized to averaged values of wild-type neurons in the same batch of cultures. The numbers were 1.00 ± 0.09 in wild-type neurons, 0.88 ± 0.07 in heterozygous neurons, and 1.34 ± 0.10 in homozygous neurons (mean ± sem, obtained from N = 37, 28, and 38 images, respectively). The value was higher in homozygous than in wild-type neurons (P < 0.02, t-test), but there was no difference between heterozygous and wild-type neurons (P > 0.05, t-test). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 4. Effects of the ΔE-torsinA mutation on individual mEPSC events. A: Representative traces of mEPSCs obtained from wild-type (a, black), heterozygous (b, red) and homozygous neurons (c, green). They were recorded in the presence of TTX and absence of GABAA receptor antagonists, at VH = −70 mV and [Cl−]pipette = 9 mM, as in Figure 1. The scale bars in panel c apply to panels a–c. Overlay of the traces is shown without (d) and with normalization of the peak amplitudes (e), illustrating that mEPSCs of different genotypes differ to some extent. B: Cumulative histograms of amplitude. C: Cumulative histograms of decay time. D: Cumulative histograms of decay time constants. E: Cumulative histograms of rise time. F: Cumulative histograms of synaptic strength (unitary mEPSC charge or an area under an event). The methods for measuring these parameters are indicated schematically in figure insets, and also described in the Materials and Methods section. The numbers of analyzed events (n) were 10,457, 17,479, 13,071 in all panels, except for panel D where the numbers were 1600, 1400, 1200 for wild-type, heterozygous and homozygous neurons, respectively. They were obtained from (N) 16 wild-type, 14 heterozygous and 12 homozygous neurons. The asterisks and the number signs indicate statistical significance at P = 10−30 and 10−10, respectively, as assessed using the Kolmogorov-Smirnov test (based on “n”). N.S. represents a non-significant comparison. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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