Methods lated with the JPCalc program [3].

G .G. Somjen, M. Muller Brain Research 885 2000 102 –110 103 The nature of these currents was, however not known. A microscope objective with patch pipettes; tight seal was voltage-dependent persistent sodium current was demon- established, and the whole-cell recording condition created strated, amongst others, in hippocampal neurons [13,26] by suction. To record Na as well as K currents the pipettes reviewed by Crill [7] and by Taylor [43]. It is not known were filled with a solution containing in mmol l: KF 1 whether I flows through distinct classes of Na chan- 129, NaCl 4, EGTA 10, CaCl 0.5, MgCl 2, Hepes 10, Na,P 2 2 nels, or whether it represents failure of inactivation of the Na ATP 4, pH 7.1 or 7.3, tip resistance 2.5–4.5 MV. To 2 1 1 ‘conventional’, fast, transient Na current. Recently the block K currents, KF was substituted by 109 mM CsF weight of evidence seemed to be shifting toward the latter and 20 mM TEA-Cl. interpretation [2,31]. An Axopatch 1D amplifier in voltage clamp configura- The physiological role attributed to I is the regula- tion and the pClamp-6 Axon Instruments suite of pro- Na,P tion of the ‘resting’ excitability of neurons. Its usual grams was used to record whole-cell currents. Pipette and conductance is too weak to explain the depolarization seen cell capacitances were compensated in the customary in such severely pathological conditions as seizures or SD. manner. Series resistance was compensated to 70. The I greatly increases, however, under pathological con- holding potential was 270 mV pipette voltage. Current– Na,P 1 ditions. In heart and skeletal muscle a persistent Na voltage I –V curves were recorded usually at one, some- 1 current is enhanced by elevated [K ] [9] and hypoxia times at 2-min intervals. Two different protocols were o causes an increase in heart muscle [5,28] as well as in used: either eight sweeps, each beginning with a pre-pulse neurons [19]. In computer-based simulations we recently of 100 ms to 290 mV to remove inactivation, followed by 1 demonstrated that Na -mediated, voltage-dependent, slow- 200-ms depolarizing steps at 2-s intervals in 15-mV ly inactivating inward current can generate an SD-like increments, taking the pipette voltage from 270 to 135 depolarized state [29]. We have therefore asked whether mV; or 12 sweeps of a 200-ms hyperpolarizing pre-pulse 1 elevation of [K ] would also cause an increase in I in followed by 400-ms depolarizing steps in 10-mV incre- o Na,P hippocampal neurons. This indeed turned out to be the ments, taking the pipette from 270 to 140 mV. case. Some of the findings have been reported in an The current records were read with Clampfit Axon abstract [38]. Instruments software. After subtraction of linear leak and holding currents, the data were further processed with the Excel Microsoft program. Junction potentials were calcu- 2. Methods lated with the JPCalc program [3]. 2.1. Isolation of neurons 2.3. Fluorescence imaging Hippocampal CA1 pyramidal cells were isolated accord- The non-permeant forms of the fluorescent calcium ing to the method of Kay and Wong [30]. Briefly: rats of indicator dyes fluo-3 10 mM and fura-red 30 mM 60–120 g body weight were decapitated under ether Molecular Probes were added to the pipette solution [22]. anesthesia. Brains were removed and 500-mm thick slices The 488 nm excitation light was used and emission was were cut from hippocampus. The CA1 region was cut into recorded at 520 nm fluo-3 as well as 640 nm fura-red smaller pieces and these tissue fragments were digested for with COMOS Biorad software. Fluorescence intensities 75 min. The digestion medium contained in mmol l: were recorded from two intersecting elongated rectangular NaCl 125, KCl 5, CaCl 1, MgCl 2, D -glucose 25, 2 2 areas of interest at 10- or 20-s intervals; images were [2-hydroxyethyl]piperazine-[2-ethanesulfonic acid] recorded at 60-s intervals. Background-corrected fluores- Hepes 10, pH 7.0, with trypsin 0.75 mg ml, at room cence ratios fluo-3 fura-red were computed subsequently temperature. After digestion the tissue pieces were washed using Excel Microsoft software. Fluo-3 fluorescence and then incubated in trypsin-free oxygenated medium at increases while fura-red fluorescence decreases with rising room temperature. Tissue fragments were dispersed by 21 [Ca ] . i trituration with a graded series of fire polished Pasteur pipettes. 2.4. Hippocampal slices 2.2. Recording of voltage-dependent currents Tissue slices of 400 mm thickness were prepared as Cell suspensions were placed in a chamber of about 0.7 described above, and placed in an ‘Oslo’ style interface ml capacity on the stage of a Zeiss Axioskop and main- chamber in flowing artificial cerebrospinal fluid ACSF of tained in flowing Hepes-buffered medium of the following the following composition in mmol l: 130 NaCl, 3.5 composition: in mmol l: NaCl 130, KCl 3.5, CaCl 1.2, KCl, 1.25 NaH PO , 24 NaHCO , 1.2 CaCl , 1.2 MgSO , 2 2 4 3 2 4 MgCl 1.0, glucose 25, Hepes 10, pH 7.3 or 7.35, at 10 glucose, pH 7.4, saturated with 95 O , 5 CO , 2 2 2 1 1 1 1 22–268C. When K concentration was raised, Na was temperature 368C. When K concentration was raised, Na equivalently reduced. Cells were approached under the was equivalently reduced. 104 G Slices were left undisturbed for 90 min before recording slowly inactivating inward current was detectable in all but began. two of a population of 42 isolated CA1 hippocampal neurons. The mean amplitude normalized to cell capaci- 2.5. Whole-cell recording in tissue slices tance was 27.166.5 pA pF mean6st.d., with a range from 0 to 224.3 pA pF in cells with average capacitance 1 Patch pipettes were pulled from thick-walled glass tubes of 10.762.2 pF. In 10 trials the bath K concentration was and filled with the following solution in mmol l: 120 raised from 3 to 20 mM; the mean persistent inward K-gluconate, 8 KCl, 10 Hepes, 11 EGTA, 0.5 CaCl , 2 current in this sample in control solution was 24.860.9 2 MgCl , 4 ATP-Na , pH 7.35, osmolarity 290, pipette tip pA pF mean6S.E.M. and it increased to 28.461.6 pA 2 2 resistance 3–5 MV. Cells in CA1 stratum pyramidale were pF P ,0.03 by paired t-test. Fig. 1 illustrates an example found by ‘blind’ search. Seal resistances of 0.5 GV, of the reversible enhancement of the persistent inward more usually 1.0 GV were accepted. Pipette capacitance current in one of these cells. In this case the maximal was compensated. Whole-cell recording condition was persistent inward current increased during elevation of 1 established by gentle suction. Cell capacitance and series [K ] by a factor of almost 4 Fig. 1A,C while the o resistance were not compensated. Holding potential was calculated maximal conductance doubled Fig. 1D. The 265 mV. Series of test pulses were delivered at 90-s sample currents of Fig. 1B show an increase not only of intervals. Each protocol consisted of a pre-pulse of 400 ms the slowly inactivating inward current, but also of an to 290 mV followed by a series of either eight or 10 inward tail current that follows repolarization. As long as depolarizing steps of 600 ms in 10-mV increments, either the persistent inward current was small, tail currents were from 270 to 0, or from 270 to 120 mV. Data processing usually small or absent in recordings made with CsF was similar to that for isolated cells. pipettes. Marked tail currents were seen whenever the Each cell was examined first for 15 min in normal persistent inward current grew large, but there was no 1 solution, then for 15 min in elevated K 10 mmol l simple correlation between the amplitudes of the two. solution. If the seal held, then either recovery was ob- In the absence of channel blocking agents, when KF- served during washing with normal ACSF for 15–30 min, filled pipettes were used, the persistent current was a 1 1 or the cell was exposed to high K solution with tet- mixture mainly composed of the delayed rectifier K rodotoxin TTX 1.0 mM added. Slices were exposed to current, I [36] and I . As long as the cells were K,DR Na,P 1 high K only once. bathed in normal solution, a persistent inward current was 1 detected in only two out of 11 cells. When the K 2.6. Statistics concentration in the bath was raised, such an inward current appeared in nine of the 11 cells. Its amplitude was Except when otherwise noted, numerical data are given 21.1 pA pF n53 at 10 mM, 23.6 pA pF n57 at 20 1 as the mean6S.E.M. Significance was calculated by paired mM and 216.9 pA pF n 53 at 40 mM [K ] . Apparent- o two-tailed t-test. ly in control solution the powerful I masked the weak K,DR 1 I , but as [K ] increased, I strengthened sufficiently Na,P o Na,P to compete. Fig. 2B illustrates the emergence of I in Na,P 1 3. Results high [K ] solution.