Wistar rats 10–15 days old were killed by decapitation and brains quickly removed and immediately transferred to an ice-cold, low-sodium solution, composed of (in mm): 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 4 MgCl2, 0.1 CaCl2, and gassed with 95% O2–5% CO2 to pH 7.4. Transverse brain slices, 110 to 130 μm thick, containing MNTB were obtained, using an Integraslice 7550PSDS (Campden Instruments; Loughborough, UK). Slices were placed in an incubation chamber containing artificial cerebrospinal fluid (aCSF) with composition (in mm): 125 NaCl, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2 and 2 CaCl2 which was gassed with 95% O2–5% CO2 (pH 7.4) and kept at 35°C for 1 h prior to storage at room temperature for up to 8 h.
For recording, brain slices were continually perfused with aCSF (composition as above; 31–35°C), at a rate of 1 ml min−1 and visualised with infrared differential interference contrast optics. MNTB principal cells were identified by their characteristically large (∼20 μm) spherical soma and astrocytes adjacent to principal cells were identified by their smaller soma (<10 μm). Astrocytes exhibited characteristic electrophysiological properties (membrane resistance <10 MΩ; resting membrane potential < −78 mV; lack of voltage-activated currents) and identification was confirmed in a separate series of experiments by labelling slices with 1 μm sulforhodamine 101 for 20 min at 35°C, immediately following slicing (Nimmerjahn et al. 2004; Kafitz et al. 2008). Furthermore, Lucifer yellow (<0.05%) was included in the patch pipette solution to enable fluorescent identification of cell type at the end of the experiment.
Membrane currents from MNTB principal neurones and astrocytes were obtained by whole-cell voltage clamping at −70 and −80 mV, respectively, using a HEKA EPC-10 double amplifier (HEKA Elektronik Dr Schulze GmbH; Lambrecht/Pfalz, Germany), filtered at 10 and 2.9 kHz, and digitized at 25 kHz with Patchmaster software (HEKA). Recordings were made using thick-walled glass pipettes (GC150F-7.5; Harvard Apparatus; Edenbridge, Kent, UK) with open-tip resistances of 5–8 MΩ for neurones and 6–9 MΩ for astrocytes. Whole-cell access resistances were <30 MΩ and series resistance compensation of 50% (100 μs lag time) was applied to neuronal recordings. For neuronal recordings, pipettes were filled with a solution containing (in mm): 110 caesium methanesulfonate, 40 HEPES, 10 TEA chloride, 5 Na2-phosphocreatine, 20 sucrose, 0.2 EGTA, 2 MgATP, 0.5 Na2GTP and 0.008 CaCl2 (pH 7.2 with CsOH). For astrocytes the patch pipette contained (in mm): 130 KCl, 2 NaCl, 4 glucose, 10 HEPES, 0.1 EGTA, 1 MgATP, 0.5 Na2GTP and 0.025 CaCl2 (pH 7.2 with KOH). In experiments where the effects of internal Cs+ or K+ removal were assessed, Cs+ and K+ were replaced by N-methyl-d-glucamine (NMDG).
All recordings were made in the presence of (in μm): 40 dl-2-amino-5-phospohonopentanoic acid (APV), 10 dizocilpine maleate (MK801), 10 (–)-bicuculline methochloride, 1 strychnine, 1 TTX, 20 2,3,dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7- sulfonamide (NBQX) and 10 mm TEA chloride. Transporter substrates (d-aspartate and l-glutamine) were dissolved in the external solution and applied by pressurized ejection (2–8 psi; 13.8-68.9 kPa) from a pipette (open tip resistance 4–6 MΩ) using a Picospritzer II (General Valve; Fairtrade, NJ, USA). In neurones, d-aspartate puffs were 2 s in duration and repeated every 30 s. In astrocytes, d-aspartate puffs were of 5 s duration and repeated every minute. Antagonists were applied by addition to the bathing solution and by inclusion in a second puff pipette, along with the agonist. The two puffer pipettes were of equal tip diameter (pulled from the same glass capillary), connected to the same pressure source and placed equidistant from the cells. For experiments investigating the proportional contribution of EAAT1 and EAAT2 to the glia d-aspartate response, 10 μm UCPH-101 and 450 μm dihydrokainate (DHK) were used. The percentage inhibitions were corrected for the fact that these competitive inhibitors do not block 100% of the EAAT1 or EAAT2 currents, respectively. UCPH-101 inhibition of EAAT1 was calculated to be 85%, using Ki = 0.41 μm (calculated from the data of Jensen et al. 2009) and a Km for d-aspartate of 60 μm (Arriza et al. 1994). DHK inhibition of EAAT2 was calculated to be 81%, using Ki = 23 μm and Km for d-aspartate of 54 μm (Arriza et al. 1994). Similarly 200 μm dl-threo-β-benzyloxyaspartic acid (TBOA) would be expected to inhibit EAAT1 by 52%, using Ki = 42 μm and inhibit EAAT2 by 88%, using Ki = 5.7 μm (Shimamoto et al. 1998). Using the relative proportions of EAAT1 and EAAT2 present (see Results), 200 μm TBOA would be expected to inhibit 64% of the total EAAT current in astrocytes. Furthermore, 20 μm of the higher affinity substrate inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA; Shimamoto et al. 2004) would be expected to inhibit 99.5% of the astrocytic EAAT current.
All chemicals were obtained from Sigma Aldrich (Gillingham, Dorset, UK) except TTX, bicuculline, NBQX, APV, MK801, UCPH-101 and DHK (Ascent Scientific; Bristol, UK) and TBOA and TFB-TBOA (Tocris BioScience; Bristol, UK).
For measuring sodium concentration changes in astrocytes, 0.5 mm SBFI (Minta & Tsien, 1989) was included in the patch-pipette solution, and NaCl omitted. Slices were imaged on a Nikon FN1 microscope using a 60× NA 1.0 fluorite lens (Nikon Corporation, Tokyo, Japan). Cells were illuminated at 350 and 380 nm for 100 ms at each wavelength (Optoscan monochromator, Carin Research, Faversham, UK) and emitted light separated by a 400 nm dichroic mirror and filtered through a 420 nm long-pass filter. Fluorescence was detected using a Cascade 512B electron-multiplying CCD camera (Photometrics, Tucson, AZ, USA), and 350:380 nm ratio images were analysed using Metafluor software (Molecular Devices, Sunnyvale, CA, USA). Calibration was performed by construction of a linear calibration curve using imaging with pipette solutions of known sodium concentrations. A 10% change in background-subtracted 350:380 nm ratio corresponded to a change in sodium concentration of 4.0 mm (R2 = 0.99).
Data are presented as mean ± SEM and regarded as statistically significant when P < 0.05 using one-way analysis of variance (ANOVA) with Dunnett's post hoc test (GraphPad Prism 5.01; GraphPad Software, San Diego, CA, USA), unless otherwise stated. Significance levels indicated on figures are 0.05 (*), 0.01 (**) and 0.001 (***).