Postnatal day 15–19 rats were deeply anesthetized with isofluorane and decapitated, as approved by the Oregon Health and Science University Institutional Animal Care and Use Committee. Hippocampi were removed, and transverse slices (300 μm) were cut using a vibroslicer (Leica, Bannockburn, IL) in ice-cold solution containing the following (in mm): 110 choline chloride, 7 MgCl2, 2.5 KCl, 1.25 KH2PO4, 0.5 CaCl2, 25 NaHCO3, 1.3 Na-ascorbate, and 10 glucose (saturated with 95% O2/5% CO2). The slices were incubated for 30–45 min at 34°C and then stored at room temperature in an external solution containing the following (in mm): 119 NaCl, 2.5 KCl, 2.0 CaCl2, 1.3 MgCl2, 1.0 NaH2PO4, 26.2 NaHCO3, and 11 glucose (saturated with 95% O2/5% CO2).
Whole-cell recordings were obtained, using an Axopatch-1B amplifier (Molecular Devices, Union City, CA), from CA1 pyramidal cells visually identified with differential interference contrast optics (Zeiss, Thornwood, NY). Slices were superfused with the above external solution with 2.5 mm CaCl2, 100 μm picrotoxin, 10 μm 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), and 10 μm d-serine, except when noted. Experiments were performed at 32–35°C, with the temperature maintained by an in-line heating device (Warner Instruments, Hamden, CT), unless otherwise noted (see ). Patch pipettes (2.0–3.5 MΩ) were filled with an internal solution containing the following (in mm): 135 Cs+ methanesulfonate, 8 NaCl, 10 HEPES, 10 Cs-BAPTA, 4 Mg-ATP, 0.4 Na-GTP, 0.2 verapamil (voltage-clamp experiments) or 135 K+ methanesulfonate, 8 NaCl, 10 HEPES, 10 K-BAPTA, 4 Mg-ATP, 0.4 Na-GTP (current-clamp experiments). Nucleated patch recordings were performed in the standard external solution with 5 mm CaCl2 and 0 mm MgCl2, using patch pipettes of ≤2.0 MΩ resistance. Electrophysiological recordings were acquired using custom software (J. S. Diamond, National Institute of Neurological Disorders and Stroke, Bethesda, MD) written in IgorPro (WaveMetrics, Lake Oswego, OR).
Figure 3 Solution exchange efficiency with flow-pipe applications. A, Currents evoked by 5 μm NMDA in control or in the presence of 70 μm DAA (Vh of +40mV; room temperature). Top bars indicate bath and bottom bars indicate flow-pipe applications. (more ...)
Agonists and antagonists were applied using a custom-built flow-pipe perfusion apparatus with a flow rate of ~ 0.1 ml/min positioned above the slice (). Flow-pipe solutions were continuously bubbled with 95% O2/5% CO2.
Figure 1 NMDAR-mediated currents in CA1 pyramidal cells. A, Schematic of flow-pipe configuration. Barrels were positioned at ~30° and ~1 mm from the recording pipette. B, Currents elicited from a CA1 pyramidal neuron by 5 μm NMDA (more ...)
Data analysis and statistics
AxoGraph X software (AxoGraph Scientific, Sydney, New South Wales, Australia) was used for analysis. Whole-cell recordings were excluded from analysis if series resistance was ≥10 MΩ or changed ≥15% during the course of an experiment. Current amplitudes were measured at peak deflection relative to baseline. The current blocked by application of 100 μm d
-(−)-2-amino-5-phosphonopentanoic acid (d
-AP-5) (Tocris Bioscience, Ellisville, MO) was normalized by the amplitude of the current evoked by 5 μm
NMDA (Tocris Bioscience) for cells recorded in the presence of TTX. Currents recorded from hippocampal astrocytes were normalized to the plateau of the test pulse, to scale for proportional rundown of the synaptic transporter current with increase in access resistance (Diamond et al., 1998
). Statistical analysis was performed using Excel (Microsoft, Seattle, WA) and Instat (GraphPad Software, San Diego, CA). Error bars on graphs correspond to the SEM. Significance was determined using ANOVA (Dunnett's or Tukey's post hoc
) or Student's t
test. Nonlinear regression analyses and EC50
estimates for dose–response data were performed with Prism (GraphPad Software).
In every whole-cell recording in which the standing NMDAR-mediated current was determined by block with d-AP-5, the current evoked by 5 μm NMDA was also measured (see Results). To estimate the concentration of NMDA that would evoke a current the size of the standing current, each current evoked by 5 μm NMDA was set to 0.073, the fraction of the maximal response (1 mm NMDA) evoked by 5 μm NMDA in nucleated patches. The standing current was scaled by the factor required to scale the 5 μm NMDA current to 0.073. Because the ratio of standing current to 5 μm NMDA current amplitude was 0.09, the standing current was 0.0065, i.e., <1% of the maximal current evoked by saturating NMDA. The concentration of NMDA necessary to evoke such a current was found by reading it off the abscissa of the dose–response curve at the point at which the current was 0.65% of maximal.