Animals were kept and used in accordance with the European Council Directive of 24 November 1986 (86/609/EEC) and the Hungarian Animal Act, 1998 and associated local guidelines. All efforts were made to reduce animal suffering and the number of animals used.
Buffers contained in mM ACSF: 129 NaCl, 3 KCl, 1.6 CaCl2, 1.8 MgSO4, 1.25 NaH2PO4, 21 NaHCO3, 10 glucose (pH 7.4); nominally Mg2+-free ACSF was prepared as control ACSF with no added Mg2+ (based on the Mg2+ contamination of the Ca2+ salts, we estimated the Mg2+ concentration of this buffer to be approximately 1 μM).
Transverse, 300 μm thick hippocampal-entorhinal slices from 10- to 18-day-old Wistar rats (Toxicoop, Budapest, Hungary) were prepared in modified ACSF (75 mM sucrose, 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2
, 7 mM MgSO4
, 0.5 mM CaCl2
, 25 mM NaHCO3
, 25 mM glucose, continuously bubbled with 95% O2
+ 5% CO2
gas mixture) at 4°C, as described before [80
]. In most of the experiments (158 out of 163), animals between P11-15 were used, while in measurements of recurrent seizure like activity (SLE) the age range was further reduced to P11-13 since they are more susceptible to developing seizures [35
]. Slices were incubated in an interface-type chamber that was continuously circulated with ACSF for one hour at 37°C (followed by incubation at room temperature) before performing the experiments. In lowered [Mg2+
] experiments slices were incubated in ACSF containing approximately 100 μM. In experiments where the diamine oxidase inhibitor, aminoguanidine, was applied, aminoguanidine was added to the ACSF before incubating the slices in the interface chamber in order to effectively block the putrescine-GABA synthetic route.
In vitro electrophysiology
Electrophysiological recordings were performed either at room temperature or at 31°C. Signals were recorded with Multiclamp700A amplifiers (Axon Instruments, Foster City, CA, USA), low-pass filtered at 2 kHz and digitized at 10 kHz (Digidata1320A, Axon Instruments). For single cell recording CA1 pyramidal cells were identified visually. Pipettes (5 to 9 MΩ) were filled with a solution containing (in mM) 130 CsMeSO3, 10 NaCl, 0.05 CaCl2, 2 ATP (magnesium salt), 1 EGTA and 10 HEPES (pH set to 7.3 with 1N CsOH). To suppress escape action currents 5 mM QX 314 (Tocris, Bristol, UK) was added. Cells were voltage-clamped at 0 mV (corrected for a calculated junction potential of +15 mV) to record GABAergic (outward) currents. Input resistance was 171 ± 65 MΩ. If signs of seal deterioration or cell closure occurred (> 20% change in the access resistance) the recordings were discarded. Synaptic recordings were made for 10 to 25 minutes in control conditions following 10 to 20 minutes of 100 μMSNAP-5114 application and 10 to 30 minutes washout.
In experiments where local Glu release at CA1 pyramidal cells was evoked, 100 μs, 500 μA stimuli were applied to the Schaffer collaterals by a bipolar Tungsten electrode at 15 s intervals. Experiments were discarded if stimulation of Schaffer collaterals did not evoke Gluergic current in the CA1 pyramidal cell voltage clamped at -45 mV. Sweeps were recorded for 9 s following the stimulus and the ranges between 1.5 to 8 s were used to analyze spontaneous IPSCs and to determine the baseline in order to exclude evoked responses.
Synaptic currents were identified as GABAergic inhibitory postsynaptic currents and baseline currents were validated as a measure of tonic GABAergic currents by adding the GABAA antagonist picrotoxin (100 μM) at the end of 43 of 154 experiments (Figure ). Picrotoxin sensitive tonic current was found to be 53.1 ± 10.7 pA, 57.1 ± 8.4 pA and 63.4 ± 10.7 pA at [Mg2+] = 1, 10 and 30 μM, respectively.
Figure 9 Validation of phasic and tonic currents recorded from CA1 pyramidal neurons at 0 mV holding potential as GABAergic currents. (A) Voltage-clamp recording showing the elimination of both GABAergic IPSCs and GABAergic tonic current in the presence of 100 (more ...)
Holding currents were determined according to Glykys et al.
]. All-point histograms were plotted for each sweep (in episodic recording mode when the Schaffer collaterals were stimulated) or for each 20 s period of experimental traces (in gap-free recording mode). A Gaussian was fitted to the unskewed part of the histogram and the position of the center of the fitted Gaussian was used as the holding current. Values during SLEs were not included in data evaluation. Experiments were discarded if the holding current continuously shifted to either a negative or positive direction during both SNAP-5114 or Glu application and washout, except when the shift was clearly linear, in which case the holding current values were detrended. Spontaneous IPSCs were analyzed by the MiniAnalysis software (Synaptosoft, Decatur, GA, USA) using 10 pA as amplitude threshold. sIPSCs with event frequency values greater than 300 Hz were excluded from histogram plots to avoid duplicate sIPSC detection.
Epileptiform activity was induced in 400 μm thick hippocampal slices by switching the perfusing solution to ACSF with no added Mg2+ ions and [K+] raised to 5 mM. Field potential recordings were performed with glass microelectrodes (3 to 5 MΩ) inserted into the CA3 stratum pyramidale. Slices were discarded if SLE did not appear in 20 minutes starting from the exposure to low-[Mg2+]/elevated-[K+] ACSF. Being not fully developed, the first SLEs were always discarded from data evaluation. Recordings were analyzed after high-pass filtering at 1 Hz. Tonic-to-clonic transitions were identified by the first reappearance of secondary discharges. SLE intensity was calculated as the standard deviation (SD) of the field potential trace containing the whole SLE, normalized to the SD of the preceding period.
In vivo electrophysiology
Rats (N = 6) weighing 250 to 350 g were used for the in vivo
experiments. All procedures followed NIH Guidelines for the Use of Laboratory Animals. In each animal, pressure injections of saline (0.9% w/v) + DMSO (1%), t-PDC (1 mM, in saline containing 1% DMSO) and t-PDC (1 mM) + SNAP-5114 (1 mM) (in saline containing 1% DMSO) were made into the CA1 area of the hippocampus, in approximate stereotactic coordinates AP 3.0, ML 2.0 and DV 3.0 mm [82
]. CA1 was also identified by the electrophysiological recordings, guided by the appearance of the large amplitude gamma oscillations (30 to 80 Hz) in local field potential (LFP) recordings. At the end of the experiments the animals were sacrificed, the brains were removed and histology confirmed the localization of the electrodes in Nissl stained sections [83
Anaesthesia was induced by the intramuscular injection of a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and maintained by repeated (approximately every 30 minutes) intramuscular administration of the same substance. Body temperature was kept at 37°C with a heating pad. The head was held by a rat adaptor affixed to a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Midline incision was made on the scalp exposing the skull, followed by retraction of the skin and craniotomy to expose a part of the left hemisphere. The dura was left intact and room temperature saline solution was used to prevent desiccation.
A 23 channel laminar multi-electrode equipped with two inner cannulae (40 μm diameter glass capillaries) was used to record field potentials and to deliver the testing solutions. The injector electrode was lowered through the intact dura to target the CA1 region using a microdrive. Interelectrode spacing was 150 μm, electrode site diameter was 25 μm, shaft diameter was 350 μm, the drug delivery site was located between contacts 5 and 6 from the top (corresponding to current source density channel 4 and 5 due to the fact that during current source density computation the first and last channel is lost). The cannulae were attached to two calibrated micrometer driven 5 μl Hamilton syringes (Hamilton Company, Reno, NV, USA) via a 250 μm inner diameter Tygon tube (Saint-Gobain, Akron, OH, USA). Separate cannulae were carefully forward- and back-filled with the testing solutions to avoid air bubbles in the tubes.
LFP (0.03 Hz to 5,000 Hz) was recorded from each of the contacts, sampled at 20 kHz/channel rate with 16 bit precision (LabView, National Instruments, Austin, TX, USA) and stored on a hard drive for off-line analysis. Current source density (CSD) analysis identifies synaptic/transmembrane generators of LFP, using high-resolution maps of simultaneously recorded field potentials obtained across a laminated neural structure. Inhomogeneous conductivity was not taken into account, second spatial derivative was calculated by the nearest neighbour method, and high spatial frequency noise was reduced by Hamming-window smoothing [84
]. Artefact free single sweep CSD epochs (256 ms long) were averaged (N = 1,000) in the frequency domain using FFT to obtain the power spectrum for all of the conditions. The CSD power spectra in the str. radiatum of the CA1 before and after the testing solution pressure injection (500 nl) were compared using t-test, significance level was set to P
= 0.005 (t = 2.57).
Double immunolabeling of GAT-3 and EAAT2
In the first step, an affinity-purified polyclonal antiserum (rabbit anti-GAT-3, cat# AB1574, Chemicon, Temecula, CA, USA) was applied to label GAT-3. The epitope for the anti-GAT-3 antiserum was the C-terminus of rat GAT-3 (aa 607 to 627) coupled to keyhole limpet hemocyanin. No cross-reactivity to the C-termini of other transmitter transporters was detected for the anti-GAT-3 antiserum (see the manufacturer's technical data sheet). Subsequently, sections were immunolabeled for EAAT2 using a monoclonal mouse anti-EAAT2 antiserum (mouse anti-EAAT2, cat# ab77039, Abcam, Cambridge, UK).
The immunostaining was performed by using Alexa594 labeled secondary antibody (cat# A21207, Life Technologies, Grand Island, NY, USA) for GAT-3 followed by FITC-tyramide fluorescent amplification immunocytochemistry for EAAT-2. Briefly, free-floating brain sections were pretreated in phosphate buffer (pH = 7.4; PB) containing 0.5% Triton X-100 and 3% bovine serum albumin for one hour. Then, they were incubated with primary antibody against GAT-3 (1:50) in PB containing 0.5% Triton X-100, 3% bovine serum albumin, and 0.1% sodium azide for 48 hours at room temperature. Sections were then incubated in Alexa 594 donkey anti-rabbit secondary antibody (1:400) for two hours. After washing, the sections were incubated overnight in the anti-EAAT-2 antibody followed by incubation in biotin-conjugated donkey anti-mouse secondary antibody at 1:1,000 (Jackson ImmunoResearch, West Grove, PA, USA) for two hours, followed by incubation in avidinbiotin-horseradish peroxidase complex (ABC) at 1:500 (Vectastain ABC Elite kit, Vector, Burlingame, CA, USA) for two hours. Then, sections were treated with fluorescein isothiocyanate (FITC)-tyramide (1:8,000) and 0.003% H2O2 in Tris-HCl buffer (0.05 M, pH 8.2) for eight minutes, washed, mounted on positively charged slides (Superfrost Plus, Fisher Scientific, Fair Lawn, NJ, USA), and cover-slipped in antifade medium (Prolong Antifade Kit, Life Technologies, Grand Island, NY, USA).
Sections were examined by using an Olympus BX60 light microscope (Olympus Corporation, Tokyo, Japan) also equipped with fluorescent epi-illumination. Images were captured at 2,048 × 2,048 pixel resolution with a SPOT Xplorer digital CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) using 4 to 40x objectives. Confocal images were acquired at 1,024 × 1,024 pixel resolution with a Nikon Eclipse E800 confocal microscope (Nikon Corporation, Tokyo, Japan) equipped with a BioRad Radiance 2100 Laser Scanning System (Bio-Rad Laboratories, Hercules, CA, USA) using 20 to 60x objectives at optical thicknesses of 1 to 5 μm. Contrast and sharpness of the images were adjusted by using the levels and sharpness commands in Adobe Photoshop CS 8.0 (Adobe Systems, San Jose, CA, USA). Full resolution was maintained until the photomicrographs were cropped and assembled for printing, at which point images were adjusted to a resolution of 300 dpi.
[Na+] monitoring in astrocytes
We used 250 μm hippocampal slices loaded with sodium-binding benzofuranisophthalate (35 μM, Molecular Probes) in the presence of 0.07% Pluronic-127 (Molecular Probes) in ACSF for one hour at 37°C. Pluronic-127 was dissolved in DMSO. Astrocytes were marked [40
] by applying 1 μM sulforhodamin 101 (SR101) for 10 minutes at 37°C. To validate the morphology of SR101-labelled cells, slices were imaged with an Olympus FV300 laser scanning confocal microscope system. Excitation was performed at 543 nm, emitted light was filtered with a 560 to 600 nm bandpass filter. Images were obtained by summation of optical sections taken in the Z-axis, using ImageJ (NIH) software.
Conventional, wide-field fluorescence imaging was performed using a digital imaging system (Olympus BX-FLA) attached to an upright microscope (Olympus BX50WI, 40x water immersion objective) and a CCD camera as sensor (Princeton Micromax, Princeton Instruments, Trenton, NJ, USA). Fluorescence excitation wavelengths were selected by using a high-speed wavelength switcher (Sutter Lambda DG-4, Sutter Instrument, Novato, CA, USA). Image acquisition at 0.1 Hz and time series were computer-controlled using the software Metafluor.
For wide-field imaging with SBFI-AM at 37°C, fluorescence signals from astrocytes previously identified by sulforhodamine 101 were collected at 525 nm (45 nm bandwidth) after alternate excitation at 340 nm and at 380 nm, and the fluorescence ratio (F340
) was calculated. At the end of some experiments an in situ
calibration was performed [86
] by permeabilizing cells for Na+
using gramicidin (6 μg/ml, Sigma, Sigma-Aldrich, St. Louis, MO, USA) and monensin (10 μM, Sigma) with simultaneous inhibition of the Na+
-ATPase with ouabain (1 mM, Tocris) in a buffer containing 1.23 mM KH2
, 1.8 mM MgSO4
, 1.6 mM CaCl2
, 21 mM KHCO3
and 10 mM glucose. Slices were then sequentially perfused with solutions containing 0, 5, 10, 20 and 50 mM Na+
, keeping (NaCl + choline-chloride) concentration at 150 mM. A five-point calibration curve was computed for each selected cell in the field of view and used to convert fluorescence ratio values into Na+
concentrations. Baseline [Na+
] was 4.3 ± 0.05 mM in average of 29 cells, Kd
for SBFI was found to be 7.52 ± 0.82 mM.
After pre-incubation for three to five hours in an interface-type incubation chamber, seven 300 μm hippocampal slices were placed on the bottom of a well in a 24-well plate. Following one-hour incubation in 300 μl of either normal ACSF or [Mg2+
] = 1 μM ACSF, the bath solution was removed and used as a measure of the extracellular environment. The slices were transferred to a micro tube. The remaining small amount of buffer was removed from the tube and the slices were weighted to obtain the wet tissue weight. In order to circumvent the high background in GABA concentration measurements due to the extreme level of GABA in neurotransmitter vesicles, the slices were then subject to a mild digestion procedure by freezing and thawing three times for 10 minutes per cycle. This protocol is supposed to selectively extract the cytosolic compartment leaving the neurotransmitter vesicles intact. Using [3
H] GABA to selectively label the cytosolic or vesicular pools [87
], we verified that this procedure selectively extracts the cytosolic compartment. After the freezing-thawing cycles, the fragmented membrane homogenate was centrifuged at 61,000 g for 20 minutes and the supernatant was used as a measure of the cytosolic environment. The GABA concentration in these samples was found to be approximately 1 mM, further confirming its cytosolic origin.
The samples were separated prior to mass spectrometric analysis by a Perkin Elmer Series 200 Micro HPLC system (Norwalk, CT, USA) consisting of a binary pump, an autosampler and a column oven compartment. The modified method of Eckstein [88
] was used where the mobile phase A was 1% formic acid and 0.5% heptafluoro-butiric acid (HFBA) in water and mobile phase B was 1% formic acid and 0.5% HFBA in acetonitrile. The column used was a Phenomenex Synergy Hydro-RP 80A (Torrance, CA, USA) (150 × 3 mm, 4 μm). Oven temperature was 45°C. The initial mobile phase composition was 100% A for 3.5 minutes then a linear gradient was applied for 5.5 minutes to 90% B. This was maintained for 1.0 minute and a quick linear gradient back to 100% A for 0.5 minute was followed by a 3.5 minutes equilibrium period. The overall run time was 14.0 minutes. The flow rate of the mobile phase was 500 μl/minute.
For quantitative analysis of compounds of interest an AB Sciex 3200 Qtrap tandem mass spectrometer (AB Sciex, Foster City, CA, USA) was used. The instrument was run in positive electrospray multiple reaction monitoring (MRM) mode. The source conditions were: curtain gas 20 l/minute, GS1 and GS2 50 and 40 l/minute, respectively, temperature of the drying gas 500°C, spray voltage 5000 V and the declustering potential was 20 V. The MRM transitions and collision energies were (Q1/Q3, CE) GABA 104/87, 20 ornithine 133/70, 20 and putrescine 89/72, 25. The dwell time was 100 ms for all transitions. Due to the different expected concentrations of the target compounds in cytosolic and bath samples, different calibration points were used. A five-point calibration curve was used in the range of 1 to 100 μM and only 10 μl of samples were injected for cytosolic samples. For bath samples, a seven-point calibration curve in range of 0.05 to 10 μM was used with injection volume of 30 μl. The built-in quantitation module of Analyst 1.5.1 software (Framingham, MA, USA) was used for the quantitation.
The concentrations of the analytes were finally calculated as pmol/mg wet tissue for both the cytosolic and the bath samples. Molar GABA concentrations were estimated by converting the pmol/mg values assuming 0.8 g/ml tissue density and 0.17 as the ratio of extracellular/total volume.
Unless stated otherwise data are expressed as means ± S.E.M. and were analyzed using Student's paired t-test or one-way analysis of variances with Bonferroni post hoc tests (OriginPro 8.0, OriginLab Coporation, Northampton, MA, USA). A value of P < 0.05 was considered significant.