Cell Isolation and Culture
Following the National Institutes of Health (NIH) Guide for the care and use of laboratory animals and a protocol approved by the University of Virginia Animal Research Committee, CG neurons were prepared by a modification of the method of Novelli et al. (15) using cerebella isolated from 5- to 7-day-old Sprague-Dawley rat pups. The tissue was coarsely chopped, trypsinized (Type III) for 45 minutes at 37℃, followed by addition of DNase I and trypsin inhibitor and gentle centrifugation. The supernatant was discarded, and the pellet was triturated, and after 5 minutes, MgCl2 (2.5 mM) and CaCl2 (0.1 mM) were added to solution. The neuronal suspension was filtered through 70 µm mesh and recentrifuged for 2 minutes. Neurons from the resuspended pellet (2 ×106) were plated onto poly-L-lysine-coated glass coverslips (11 mm×22 mm) cultured in basal Eagle's medium with 10% fetal calf serum, 2 mM glutamine, 100 µg/ml gentamicin and 25 mM K+. Glial cell proliferation was prevented by addition of 10 µM cytosine arabinoside 24 hours after plating. Granule neurons were maintained in 5% CO2 : 95% air at 37℃ and were used 4 to 10 days after isolation. A small series of neurons was grown in solution containing 5 mM KCl. Biochemical reagents, buffers and toxins were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Halothane was obtained from Halocarbon Laboratories (Riveredge, NJ), sevoflurane from Abbott Laboratories (North Chicago, IL), isoflurane and enflurane from Anaquest/Ohmeda (Liberty Corner, NJ).
[Ca2+]i Measurement in Cultured Granule Neurons
For measurement of cytosolic Ca2+ concentration ([Ca2+]i), neurons on coverslips were incubated at 37℃ for 20 minutes in basal medium containing 3 µM fura-2-AM, BSA 16 µM and (in mM): NaCl 153, KCl 3.5, NaHCO3 5, KH2PO4 0.4, MgSO4 1.2, CaCl2 1.3, glucose 5, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) 20 with pH adjusted to 7.4. In some experiments HEPES was substituted for TES with no alteration in behavior. After washing the neurons twice in fura-2-free solution, coverslips were inserted into a holder and placed in a 2 ml cuvette and washed twice more with 2 ml fresh medium. [Ca2+]i was determined at 37℃in a PTI (Photon Technology Incorporated, Monmouth Junction, NJ) DeltaScan luminescence spectrofluorometer equipped with a cuvette warmer and magnetic stirrer to ensure adequate mixing during each experiment. Ca2+ influx into neurons was initiated with addition of 33 µl of 3.0 M KCl, which increased [K+]o from 5 to 55 mM. Fluorescence at 510 nm was determined for alternating excitation wavelengths of 340 and 380 nm with fluorescence (340/380) ratios collected every 0.5 to 1.9 seconds for 60 to 180 seconds. Subsequent calibration was carried out by determining maximum and minimum fluorescence ratios using 10 µM ionomycin for maximum (Ca2+-saturated) values and 10 mM ethylene glycol-bis (-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) for minimum values for each coverslip. [Ca2+]i was calculated according to the standard formula using a Ca2+-fura-2 Kd of 224 nM (20). The signal amplitude and time course were extremely reproducible among cells on a given day. Addition of 100 µmoles of 1M NaCl instead of KCl elicited no response, suggesting change in tonicity by itself had minimal effect. In the presence of 1 mM EGTA and in the absence of added Ca2+, extracellular [Ca2+] ([Ca2+]o) was less than 100 nM and KCl addition elicited no [Ca2+]i transient. To further verify the assay, Mg2+ ([Mg2+]o) was adjusted to change the amount of entering Ca2+ as well as the resulting glutamate release.
Measurement of Neuronal Glutamate Release
Glutamate release was measured in CG neurons at 4-7 days using a glutamate dehydrogenase (GluDH)-coupled assay (Boehringer Mannhein GmbH, Germany). As described (21, 22), 50 U/ml GluDH was employed in the presence of 1 mM NADP+ to catalyze the formation of α-ketoglutarate and the fluorescent species NADPH from glutamate. NADPH fluorescence was excited at 340 nm and measured at 460 nm using the PTI spectrofluorometer. The coverslip of granule neurons was washed in buffer solution and then incubated at 37℃ for 5 min in a 2 ml cuvette containing (in mM): 145 NaCl, 5 KCl, 1.3 MgCl2, 1.5 CaCl2, 1.2 NaH2PO4, 10 glucose, and 20 HEPES, pH 7.4. As in the [Ca2+]i transient study, glutamate release was activated by adding KCl to achieve a final concentration of 55 mM, while monitoring the change in NADPH fluorescence for 300 seconds at a sampling rate of 1-2 Hz. The fluorescence signal in this setting increased to a value 10-20% above the baseline fluorescence, with a typically stable plateau being reached within 10 seconds.
To calibrate the fluorescent response to glutamate release, studies were performed with direct addition of NADPH or glutamate under identical conditions. Addition of NADPH in the cuvette solution to obtain a 0.2, 0.5, and 1.0 µM concentration resulted in abrupt increases in the fluorescence signal of 0.92±0.21, 1.94±0.86, and 3.1±1.31 × 105 counts per second (cps), respectively (±SD, n=5). When glutamate was added (in aliquots of 0.5 mM solution) to solutions containing 50 mg/ml GluDH enzyme solution, the fluorescence signal increased with an exponential time course time constant of ~60 seconds at 37℃. When the added (glutamate) was 0.2, 0.5 and 1.0 µM (equimolar to the increases in [NADPH]), the respective steady-state increases in fluorescence signals were 0.89±0.19, 1.83±0.29, and 2.89±0.30×105 cps, or ~95 percent of the NADPH values. The close agreement of the fluorescence signal between the same quantity of NADPH and glutamate suggests that the glutamate reaction producing NADPH proceeded to completion. In control experiments, when [glutamate] was abruptly increased to 0.2, 0.5 and 1.0 µM in the NADPH buffer and GDH mixture in the presence of halothane or isoflurane, and no difference was observed in the rate or extent of fluorescence increase seen in their absence.
Upon addition of KCl and depolarization of the neurons, there was a sudden increase in the NADPH fluorescence signal, followed by a much smaller and slower increase, which typically stabilized by 5 to 15 seconds (Figure 1b, 2b, 3b). The increase in the fluorescence signal was typically on the order of 0.4-1.5 × 105 cps, reflecting a final metabolism of 0.2-0.4 nmoles of released glutamate. The maximum value varied with the degree of confluence and coverage of neurons on the coverslips. Compared to the addition of glutamate in solution, the stabilization of the fluorescence signal in the presence of depolarization-induced glutamate released from neurons was far more rapid. Such rapidity suggests that there must be rapid release of a high concentration of glutamate (>40 nmoles yielding >20 µM), followed by rapid arrest (≤1 second) of glutamate release, as well as substantial local glutamate uptake into neurons which would then cause cessation of NADPH production in the first few seconds. A high concentration of glutamate (>1 mM) with rapid reuptake has been predicted to be found in synaptic clefts (23), while a high-capacity system for glutamate uptake in neurons (24) could account for the rapid stabilization of the signal.
In additional previously reported control experiments, GABAA receptors (chloride ion channels) were blocked using 100 µM bicuculline, GABAB receptors were activated by 10 µM baclofen, NMDA glutamate receptors were inhibited by D-(–)-2-amino-5-phosphonovaleric acid (AP-5), or intracellular Ca2+ was mobilized by 5 mM caffeine. None of these separate interventions had any significant action on the depolarization-evoked Ca2+ transient or glutamate release (25). In that same study, Na channel blockade by 10 µM tetrodotoxin caused an 11% decrease in the Ca2+ transient peak (a non-significant decrease in glutamate release), while intracellular Ca2+ immobilization with 10 µM ryanodine caused a 14% decrease in glutamate release (no effect on the Ca2+ transient).
Anesthetic and Drug Administration
Prior to either type of experimental study, CG neurons were incubated in the cuvette for 5 minutes in VA-equilibrated solution, which was generated by bubbling filtered VA-containing air which had passed through anesthetic vaporizers (Ohmeda, Madison, WI) calibrated to deliver the specified percent vapor in air. VA vapor concentrations were approximately 0.8 and 1.6 times the minimal alveolar concentration (MAC) value for rats (that concentration at which 50% of rats do not respond to painful stimulation) (26). As periodically verified by gas chromatography, 0.75 and 1.5% halothane yielded aqueous concentrations of 0.25 and 0.5 mM; 1.3 and 2.5% isoflurane yielded 0.23 and 0.42 mM; 2% and 4% sevoflurane yielded 0.22 and 0.43 mM; 1.7 and 3.5% enflurane yielded 0.35 and 0.70 mM enflurane. Solutions were sampled at 37℃ and aqueous concentrations typically varied by ±10%. VA-containing air continually flowed through the cuvette head-space to prevent VA loss to the atmosphere. Control solutions were bubbled with filtered air only. A five minutes incubation was sufficient to achieve a stable effect of the anesthetics, nicardipine or ω-agatoxin-IVA (Aga-IVA; Alexis Biochemical, San Diego, CA); a 20 minutes prior exposure to ω-conotoxin-GVIA (Ctx-GVIA) was found to be necessary to achieve its maximum effect on either [Ca2+]i or glutamate release.
Whole-Cell Patch-Clamp Studies
For electrophysiological studies, neurons grown on cover slips under conditions identical to those for the spectrophotometric studies were placed at the bottom of a recording chamber mounted on an inverted microscope where bathing solutions could be exchanged. Prior to establishing the whole-cell-recording configuration, the external bathing solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, adjusted to pH 7.4 with 1 N NaOH. The patch pipette solution contained (in mM): 108 CsMeSO4, 10 CsCl, 9 EGTA, 24 HEPES, 4 Mg-ATP, 0.3 GTP, adjusted to pH 7.3 with 1 N CsOH. Once whole-cell recording was achieved, the bathing solution was replaced with one that would eliminate potentially interfering K and Na currents (in mM): 160 TEA-Cl, 5 BaCl2, 10 HEPES, pH 7.3 with 1 N CsOH.
Standard whole-cell voltage-clamp methods were employed using the Axopatch 200 patch clamp amplifier (Axon Instruments, Foster City, CA). Data acquisition was performed using a pClamp system version 5.5.1 (Axon Instruments) coupled with an IBM-compatible, 386-based microcomputer. Patch electrodes were prepared from borosilicate glass 1B150F-3 (World Precision Instruments), heat polished, and had a resistance less than 5 MΩ when filled with internal solution. All experiments were conducted at room temperature (20-22℃). Four to six minutes after initiating whole-cell recording configuration, neurons were typically voltage-clamped at -80 mV to establish a stable baseline and maximize currents by reducing steady state inactivation. To define the current-voltage relation, IBa was triggered by step depolarizations 70 msec in duration from -40 to +40 mV. After control measurements the preparation was superfused for 4 to 6 minutes with solution pre-equilibrated with either halothane or isoflurane (produced by bubbling the solution at room temperature) and measurements repeated. Anesthetic solution was washed out for 5-8 minutes before recording recovery currents. Standard P/n analysis was used to estimate and subtract leakage and capacitative currents. The higher solubility of the anesthetics at room temperature produced aqueous concentrations approximately 60-70% higher than those at 37℃. In other experiments a depolarization to -10 mV was applied for 400-450 msec. A 9.5 sec depolarization to 0 mV was applied to duplicate the prolonged depolarization obtained with application of 55 mM K+.
Statistics and Analysis
For neurons at the same day in culture and growing with a similar density, KCl depolarization elicited extremely uniform control responses for either [Ca2+]i transients or glutamate release, varying by less than 8%. Results of [Ca2+]i measurements are reported and compared as absolute values and also as fraction of same day control, while glutamate measurements are only reported in the latter format. Unless otherwise indicated, results are expressed as sample mean ±sample standard error (SEM). Results were compared among anesthetics and drugs by ANOVA and the Protected Least Significant Difference (PLSD) Test.