These studies were performed on juvenile rats (Sprague-Dawley, P28–35). All procedures were approved by the Animal Care and Use Committee, University of Tennessee, Health Science Center. The animals were anaesthetized with isofluorane until the animal was areflexive. Briefly, the animal was placed into a sealed plastic container into which gauze soaked with isofluorane was placed under a fibreglass screen floor. After anaesthesia with isoflurane, the animals were decapitated, and the brain was removed and dropped into ice cold cutting solution for 30–60 s. The cutting solution contained (mm): 250 sucrose, 25 KCl, 1 NaH2PO4, 11 glucose, 4 MgSO4, 0.1 CaCl2, 15 Hepes (2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (pH = 7.3–7.4; 300 mosmol l−1).
We made 400 μm coronal slices of the fronto-parietal regions of the brain using a vibrating tissue slicer (World Precision Instruments, Sarasota, FL, USA). The slices were then transferred to a mesh surface in a chamber containing artificial cerebrospinal fluid (aCSF) at room temperature. The aCSF contained (mm): 125 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose (pH 7.4, 310 mosmol l−1), and was bubbled with a 95% O2–5% CO2 (carbogen) mixture.
Acute isolation of neurones
Just prior to enzyme treatment, the combined primary motor and primary somatosensory cortices were dissected from brain slices under a stereomicroscope. The cortex was further cut to restrict enzyme treatment to the supragranular layers (I/III). Two to three cortex pieces at a time were transferred to oxygenated aCSF (35°C) with added enzyme (Sigma Protease type XIV, 1.2 mg ml−1: Sigma Chemicals, St Louis, MO, USA). After 20–30 min of incubation in enzyme, the tissue was washed with sodium isethionate solution, which consisted of (mm): 140 sodium isethionate, 2 KCl, 4 MgCl2, 23 glucose, 15 Hepes, pH 7.3 (adjusted with 1 m NaOH).
This solution and enzyme-treated tissue were triturated using three successively smaller fire-polished pipettes to release individual neuronal somata. The supernatant from each trituration step (containing dissociated neurones) was transferred to a fresh container, plated onto a plastic Petri dish (Nunc, Rochester, NY, USA) on an inverted microscope stage, and allowed to settle for approximately 5 min. A background flow of ∼1 ml min−1 of Hepes-buffered saline solution (HBSS) was then established. HBSS consisted of (mm): 138 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 10 Hepes, 10–20 dextrose, pH 7.3 (adjusted with 1 n NaOH), and osmolarity = 300–305 mosmol l−1. The external recording solution was HBSS, plus tetrodotoxin (TTX: 1 μm) and CdCl2 (400 μm) to block Na+ and Ca2+ channels, respectively.
Corning 7052 capillary glass (Garner Glass: Claremont, CA, USA) was used to create electrodes on a Sutter Instruments (Novato, CA, USA) Model P-87 Flaming/Brown micropipette puller. Electrodes were fire-polished and filled with internal solution. Two internal solutions were used (no differences were observed in the recorded currents between the two internals). The first consisted of (mm): 120 KMeSO4, 15 KOH, 2 MgCl2, 7.5 NaCl, 30 Hepes, 2 adenosine 5′-triphosphate (ATP), 0.2 guanosine 5′-triphosphate (GTP), 0.1 leupeptin, 1–10 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The second internal solution contained 75 KMeSO4, 60 KOH, 2 MgCl2, 7.5 NaCl, 40 Hepes, 2 ATP (Na+ salt), 0.2 GTP, 0.1 leupeptin, 10 BAPTA. Both internals were ∼270 mosmol l−1 and adjusted to pH 7.2 with KOH.
A multibarrel array of glass capillaries (∼500 μm outer diameter) in ‘sewer pipe’ configuration was used to apply solutions. Six capillaries were glued side to side and attached to a micromanipulator. Solutions were changed by moving the active barrel so that the flow surrounded the recorded cell. Care was taken to regulate flow through the array to prevent flow artifacts. The following channel blockers (Alomone Laboratories, Ltd, Jerusalem, Israel) were used: TTX (1 μm), CdCl2 (400 μm), α-dendrotoxin (0.1–1000 nm), δ-dendrotoxin (10 nm), dendrotoxin-K (10–100 nm), r-tityustotoxin-Kα (100 nm), r-margatoxin (1–30 nm).
Recordings were made with a Dagan 8900 (Minneapolis, MN, USA) amplifier at room temperature (21–23°C). Electrode resistances were 1.4–2.2 MΩ after polishing, and series resistance compensation was 70–90%. For experiments detailing voltage dependence and kinetics of currents, cells with calculated series resistance errors of ≥ 5 mV were discarded ((V = IR): series resistance error (V) = remaining series resistance after compensation (R) multiplied by peak current (I)). Data were acquired using pCLAMP 8. Reported membrane potentials were corrected off-line for the measured liquid junctional potential (∼8 mV). Data acquisition (20 kHz sampling, filtered at 5 kHz) and analysis were done using pCLAMP and Axograph software (Axon Instruments, Union City, CA, USA). Linear leak current and the capacitative artifact were digitally subtracted before analysis, using a P/4 protocol.
Prism (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical tests of significance. Student's paired or unpaired t test was used to compare sample population data throughout, and summary data are presented as means ± standard deviation, unless noted otherwise. P= 0.05 was taken as the level of significance.
Sample population data are represented as scatter plots or as box plots (Tukey, 1977). Box plots indicate the upper and lower quartiles as edges of the box, with the median represented as a line crossing the box. The stems indicate the smallest and lowest non-outlying values, and outliers are indicated by open circles. Outlying values are greater than 1.5 times the quartile boundaries.