Materials and methods lated with the JPCalc program [3]. Results

G .G. Somjen Brain Research 885 2000 94 –101 95 1 reports the effect of elevated external K concentration on The current records were read with Clampfit Axon I . Instruments software. After subtraction of linear leak and Na,P holding currents, the data were further processed with the Excel Microsoft program. Junction potentials were calcu- 2. Materials and 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 [14]. Briefly: rats of indicator dyes Fluo-3 10 mM and Fura-red 30 mM 60–120 g body weight were decapitated under deep ether Molecular Probes were added to the pipette solution [11]. 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 21 75 min. The digestion medium contained in mM l : were recorded from two intersecting elongated rectangular NaCl 125, KCl 5, CaCl 1, MgCl 2, D -glucose 25, areas of interest at 10 or 20 s intervals; whole images were 2 2 [2-hydoxyethyl]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 subsequent- temperature 20–228C. After digestion the tissue pieces ly using Excel Microsoft software. Fluo-3 fluorescence were washed and then incubated in trypsin-free oxygenated increases while Fura-red fluorescence decreases with rising 21 medium at room temperature. Tissue fragments were [Ca ] . i dispersed by trituration with a graded series of Pasteur pipettes. 2.4. Statistics 2.2. Recording of voltage-dependent currents Except when otherwise noted, numerical data are given as the mean6S.E.M. Significance of differences was Cell suspensions were placed in a chamber of about 0.7 calculated by paired two-tailed t-test. ml capacity on the stage of a Zeiss Axioskop. When Na and K currents were recorded, the cells were maintained in flowing HEPES-buffered medium of the following com-

3. Results

21 position: in mM l : NaCl 130, KCl 3.5, CaCl 1.2, 2 MgCl 1.0, glucose 25, HEPES 10, pH 7.3 or 7.35, at Whole-cell currents evoked by voltage steps were 2 20–228C. Cells were approached under the microscope recorded from patch-clamped freshly isolated hippocampal objective with patch pipettes; tight seal was established, CA1 neurons. The recording pipette was filled either with a and the whole-cell recording condition created by suction. KF or with a CsF based solution, that also contained the To record Na and K currents the pipettes were filled with a fluorescent dyes Fura-red and Fluo-3. At 1-min intervals 21 solution containing in mM l : KF 129, NaCl 4, EGTA the cells were stimulated by a series of voltage steps to 10, CaCl 0.5, MgCl 2, HEPES 10, Na ATP 4, pH 7.1 or construct current–voltage I –V curves, and they were also 2 2 2 1 7.3, tip resistance 2.5–4.5 MV. To block K currents, KF excited at 10 or 20 s intervals by 1 s pulses of laser light to 21 was substituted by 109 mM CsF and 20 mM tetraethylam- determine changes in internal Ca activity. When KF was monium–Cl TEA–Cl. the main electrolyte, all the voltage-gated channels were An Axopatch 1D amplifier in voltage-clamp configura- available for activation and the records were the sums of tion and the pClamp-6 Axon Instruments suite of pro- several individual currents. grams was used to record whole-cell currents. Pipette and Fig. 1A illustrates current–voltage I –V curves of the cell capacitances were compensated in the customary slowly inactivating currents recorded with a KF-based manner. Series resistance was compensated to 70. The pipette from one cell over an 18-min period. The graph holding potential was 270 mV pipette voltage. Current– shows the average currents measured during the last 15 ms voltage I –V curves were recorded usually at 1-, some- of each depolarizing step after subtracting linear leak and times at 2-min intervals. Two different protocols were holding current and correcting pipette potentials for junc- used: Either eight sweeps, each beginning with a pre-pulse tion potential. Fig. 1B shows sample currents evoked by a of 100 ms to 290 mV to remove inactivation, followed by depolarizing pulse to 225 mV pipette potential V P 200 ms depolarizing steps at 2 s intervals of 15 mV equaling 232.6 mV membrane potential V , after correc- m increments, taking the pipette voltage from 270 to 135 tion for junction potential. In most cases, as in Fig. 1A, mV; or 12 sweeps of a 200 ms hyperpolarizing pre-pulse during the first few minutes of observation the slowly followed by 400 ms depolarizing steps in 10 mV incre- inactivating component flowed outward at all potentials ments, taking the pipette from 270 to 140 mV. and the I –V curve appeared to be dominated by the delayed 96 G Fig. 1. Intermittent fluorescent excitation causes the growth of a persistent inward current. A Current–voltage I –V curves of slowly inactivating current of an isolated CA1 neuron recorded at different times after establishing whole-cell recording condition, using a patch pipette filled with KF solution. After a hyperpolarizing pre-pulse, eight sweeps of 200 ms depolarizing steps at 15 mV increments were applied. The average current measured during the final 15 ms of the depolarizing pulses is plotted against the membrane potential V corrected for junction potential. B Sample currents recorded 1 min and 18 m min after establishing whole-cells’ condition, evoked by depolarization to 225 mV pipette voltage, corresponding to V of 232.6 mV after correction of m junction potential. 1 rectifier K current, I . After 5–10 min, the persistent pulses, a sizeable inward current appeared. Adding TTX to K,Dr inward current appeared in most fluorescent laser illumi- the bathing solution completely blocked both transient and nated cells, and then it grew in amplitude. As the persistent persistent inward currents within 3–5 min, but after inward current increased, outward currents became smaller washing for 16 min with normal solution both components and the reversal potential zero-current potential of the of the inward currents reappeared. By subtracting the compound current shifted to more positive levels. current amplitudes recorded under the influence of TTX The persistent inward current was blocked by 0.5 mM from those recorded at the end of the 24 min of observa- tetrodotoxin TTX. In the cell illustrated in Fig. 2A, a tion, the TTX-sensitive component of the persistent current trace of a persistent inward current was recorded immedi- is revealed Fig. 2B. Fig. 2C shows conductances com- ately after establishing whole-cell condition. After 24 min puted by dividing the currents of Fig. 2B by the driving of stimulation by voltage pulses alternated with laser voltage, the latter being defined as the difference between G .G. Somjen Brain Research 885 2000 94 –101 97 Fig. 2. The persistent inward current is tetrodotoxin TTX sensitive. A I –V curves of the slowly inactivating current recorded similarly to those of Fig. 1, at 1 min and 24 min in control solution, followed by 4 min of TTX administration, and 16 min of washing with normal solution. B The I –V relation of the TTX-sensitive persistent inward current, obtained by subtracting the I –V curve recorded under the influence from TTX Fig. 2A, triangles from the I –V curve recorded at 24 min in normal solution Fig. 2A, diamonds. C The voltage-dependent TTX-sensitive conductance in nanosiemens, plotted against membrane voltage in mV. Conductance was calculated by dividing the TTX-sensitive current Fig. 2B by the driving potential, defined as the difference between the membrane potential and the reversal potential. 1 the membrane potential and the extrapolated reversal expected, when most K currents were blocked, I Na,P potential of the TTX-sensitive current. The TTX-sensitive could readily be evoked right away in almost all cells by persistent component activated at a level more negative prolonged depolarizing pulses. I did, however, grow Na,P than 260 mV, it was maximal at 220 mV and it reversed with time and with repeated fluorescent excitation, even 1 around 130 mV. TTX sensitivity, activation and reversal when the K currents were eliminated, demonstrating that 1 potentials, and the shape of its I –V curve identify it as a rundown of K current cannot account for its growth. As persistent sodium current, I [6,9]. the persistent inward current increased in amplitude, its Na,P The fact that outward currents became smaller while reversal shifted in the positive direction, regardless of 1 I grew, suggested the possibility that the ‘rundown’ of whether K currents were blocked or not Fig. 1A, 5A, B. Na,P 1 K currents had unmasked the previously hidden, slow To test whether repeated stimulation by depolarizing inward current [19]. To test this, cells were tested in which voltage steps causes the gradual increase of the I Na,P 1 most of the K currents were suppressed by using pipettes use-dependent facilitation, recordings were made from filled with a solution containing CsF instead of KF, with cells without fluorescent dyes and without laser illumina- 1 20 mM tetraethylammonium TEA added. Even after tion three cells with KF and 14 cells with CsF in the 1 1 1 replacing K with Cs and TEA , a small non-inactivat- pipettes. In these cells the I was small and it did not Na,P ing outward current often remained visible at strongly grow during repeated stimulation. The question then was, 1 depolarized voltages Fig. 5B. This may be due to Cs whether the dyes themselves potentiate I , and, con- Na,P 1 flowing through K channels, or it may represent the versely, whether laser light in the absence of internal non-specific cation current described by Alzheimer [1]. As fluorescence has this effect. Laser illumination of cells not 98 G containing dye three with KF, three with CsF, and filling pA pF recorded from cells under similar conditions but in cells with dye but not using the laser n511, all CsF had the absence of fluorescence. Without exception, in all the same negative result: in the absence of fluorescence, fluorescent cells the I increased in amplitude with time Na,P the I was, and remained, small. but the magnitude of the effect was variable. In the Na,P Fig. 3 shows a statistical summary of the maximal fluorescent, CsF group there were 18 cells from which the persistent inward current amplitudes normalized to cell current was recorded for 5–10 min after establishing capacitance, recorded at different times from groups of whole-cell condition; the largest current in a cell in this cells with and without fluorescence, in the presence and in group was 21700 pA 185 pA pF and the smallest 2116 1 the absence of K mediated outward current. The differ- pA 12.2 pA pF. The mean amplitude of all the cells in ence between the fluorescent and the non-fluorescent cells the 5–10 min, fluorescent, CsF group was 68.6 pA pF was striking. In the fluorescent cells that were observed for with a standard deviation of 642.1 and standard error of more than 10 min with CsF-filled pipettes, the mean 69.9. In the non-fluorescent group, and initially also maximal I amplitude was 280.1619.7 pA pF n56, among the fluorescent cells, the difference between the Na,P 1 which is 12.9 times larger than the 26.261.2 n57 I amplitude recorded with and without blocking K Na,P currents was also quite marked. With time, however, the currents recorded from fluorescent cells with KF pipettes approached those recorded with CsF. In the greater-than 10 min groups the data are too few and the variability is too large for reliable statistical comparison; for the few cells tested for more than 10 min the difference between KF and CsF recording is not significant Fig. 3B. There was no detectable relationship between the growth 21 of the I and the cytoplasmic calcium activity, [Ca ] , Na,P i as indicated by the ratio of the fluorescences of Fluo-3 and Fura-red. Initially the fluorescence ratio increased sharply but transiently whenever the cell was stimulated by depolarizing currents see also [19,21]. These transient 21 responses of [Ca ] were presumably caused by voltage- i dependent calcium currents. In the course of repeated stimulation they usually subsided, probably because the pipettes did not contain the ingredients required to coun- 21 teract ‘rundown’ of Ca currents [19]. The ‘resting’ or 21 baseline [Ca ] showed upward or downward drift in i different experiments and was not correlated with the growth of I . Na,P 2 In the great majority of the trials F was the main anion in the recording pipette. K-gluconate or K-acetate replaced KF in several experiments. The seal between pipette and cell membrane often loosened within 5–7 min so that no I was seen, but in two trials with gluconate and in one Na,P with acetate the seal was maintained and I appeared Na,P and grew as usual. At this stage of the study three questions arose. First, do the laser induced pulses of fluorescence induce a mem- brane current? Second, can fluorescence by itself potentiate Fig. 3. Statistical summary of the potentiation of the persistent inward current by repeated pulses of fluorescence. The columns show the I , or does it have to be combined with depolariza- Na,P mean6S.E.M. of the maximal slowly inactivating inward current, normal- tion? And third, is the potentiation of the I reversible, Na,P ized to cell capacitance. The numbers above the horizontal axis show the or is it an irreversibly altered state of the membrane time in minutes elapsed from establishing whole-cell conditions until the channel? To answer these questions, I –V curves were first recording. ‘0–5 min’ show the largest persistent inward current measured recorded for 6–14 min from cells that were filled with dye, toward the end of the first 5 min of recording, ‘5–10 min’ show measurements usually 8–10 min after the start of recording. The numbers but without turning on the laser. Then electrical stimula- of cells in each category are shown below the columns. Pipette solutions tion was suspended and intermittent laser excitation was 1 based on CsF also contained 20 mM tetraethylammonium TEA to turned on for 7–10 min. In this period the holding current 1 block most K currents. A Non-fluorescent cells include those filled was continuously recorded. The voltage to which the with dye but not illuminated, not filled with dye but illuminated, and free membrane was clamped was adjusted from time to time to of dye and not illuminated. B Fluorescent cells were filled with Fluo-3 and Fura-red dyes and were intermittently illuminated by laser light. detect any laser-induced currents that may be voltage G .G. Somjen Brain Research 885 2000 94 –101 99 dependent. Subsequently, I –V curves were again recorded there was a striking increase in amplitude of the I in all Na,P without laser illumination for additional 28–32 min. Figs. 6 trials. The mean maximal amplitude immediately before 4 A and B show the maximal I recorded with CsF laser treatment was 243.7613.2 pA and immediately after Na,P pipettes from four cells before and after laser illumination the series of illuminations 29136172 pA mean6S.E.M.. under this protocol. Figs. 4C and D show the holding One of the six cells was lost early in the post-laser period, current of one of these cells recorded during intermittent the persistent current of the five other cells at the end of laser illumination, reproduced on two different time-scales. 28–32 min following laser treatment was still 28076162 Fig. 5 illustrates recordings obtained from individual cells pA. The decay was slow but consistent and statistically before and after laser excitation. significant P,0.009, paired two-tailed t-test. This slow As illustrated by the examples of Fig. 4C and D, laser decrease could perhaps be caused by ‘rundown’ of I , Na,P 1 pulse excitation of fluorescence did not evoke a detectable but the transient Na current, I did not show a similar Na,T change in the holding current, regardless of holding decay. I amplitude was on average 214,60761591 pA Na,T potential. The same was true in four cells from which in these six cells before laser treatment, 212,14661406 recordings were made with CsF and two cells with KF pA immediately after laser and 212,37962425 pA half an pipettes. Recordings have been obtained at 290, 270, hour later. 250, 240 and 230 mV holding potentials not all levels in Fig. 5 illustrates examples of I –V curves and current each cells. Not surprisingly, shifting of the holding traces obtained from two of the six cells before and after potential did evoke increases in holding current that laser-induced fluorescence. Fig. 5A, C and D were re- inactivated slowly Fig. 4C, D. corded with KF filled pipette from one cell, and Fig. 5B Following 5–8 min of intermittent laser illumination, from another cell, using CsF. With KF, I was not Na,P Fig. 4. Intermittent pulses of fluorescence do not evoke a membrane current, but they cause long-lasting potentiation of the persistent inward current, I . Na,P A Maximal persistent inward currents recorded from four dye-filled cells before illumination by laser. The period of pre-laser observation varied from 6 to 1 14 min. The recording pipettes were filled with CsF based solution containing 20 mM TEA . B Maximal persistent inward currents recorded from the same four cells after intermittent laser illumination. Note different ordinate scales for A and B. C Continuous recording of the holding current during intermittent laser illumination of one of the cells represented in A and B. The lower trace shows the switching of the laser. The broken vertical lines indicate changes in the holding potential. No voltage pulses were applied during intermittent laser illumination. D Part of the recording of C, on an expanded time-scale. 100 G Fig. 5. Sample I –V curves and current recordings before and after fluorescent excitation. Parts A, C and D are from the same cell. A I –V curves just before and 2 min after intermittent laser illumination, recorded with KF-based pipette solution with fluorescent dyes. B Similar to A, from another cell, 1 1 but recorded with K currents mostly blocked, with CsF and TEA in the recording pipette. The two I –V curves recorded 2 min and 30 min after the intermittent laser treatment show the slow decrease of I . C Transient sodium current, I evoked by depolarizing pulse of 230 mV pipette voltage Na,P Na,T corresponding to 237.9 mV membrane potential recorded before and 3 min after intermittent laser illumination. The current was sampled at 2000 Hz. D Currents evoked by 220 mV and 130 mV pipette voltage steps 227.9 mV and 122.1 mV membrane potential before and 3 min after intermittent laser treatment. The currents were sampled at 2000 Hz, but were filtered at 100 Hz off-line. detectable before laser treatment, but it was marked slowly after laser excitation has ceased. The first of these thereafter Fig. 5A, D. With CsF the originally small I two findings could be expected, the second was a surprise. Na,P increased manifold following laser pulses Fig. 5B. As Since laser illumination and intracellular fluorescence shown in Fig. 5C, while the amplitude of I did not are not usual hazards for hippocampus in situ, the effect is Na,T change, its duration increased. Such a widening of the I not directly relevant to human pathophysiology. Nonethe- Na,T trace was consistently seen in conjunction with the growth less it is of interest for the understanding of sodium of the I . The amplitude of I usually showed slow currents in mammalian central neurons. The fact that the Na,P Na,T drift up or down, unrelated to laser treatment and not persistent current can grow more than 10-fold under the linked to the changes in other ion currents. relatively mild influence of intracellular fluorescence sug- gests that the maximal current carrying capacity of slowly 1 inactivating Na channels is much greater than is normally manifested. It is not known whether I arises because of Na,P

4. Discussion incapacitated inactivation of the channel that generates the