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Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons
Brain Research 885 (2000) 94–101 www.elsevier.com / locate / bres
Research report
Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons George G. Somjen* Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710, USA Accepted 12 September 2000
Abstract Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the ‘Hodgkin–Huxley’ type early, fast inward surge followed by sustained outward current. Over 5–20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K 1 currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin (TTX) and it had current–voltage (I–V ) characteristics of a persistent sodium current, INa,P . In cells not filled with dye but illuminated by laser, and in cells with dye but not illuminated, INa,P remained small. There was a more than 12-fold difference in the maximal amplitude of INa,P of fluorescent compared to non-fluorescent cells. Once induced, INa,P decreased very slowly. Fluorescence increased the duration but not the amplitude of the transient Na 1 current, INa,T . With membrane potential clamped to a constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced INa,P potentiation is related to photodynamic action. 2000 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Sodium channels Keywords: Persistent sodium current; Sodium channel; Photodynamic action; Fluorescence; Dissociated neuron; Whole-cell current
1. Introduction In a previous study [19] of the effects of low sodium chloride concentration and low osmolarity, whole-cell sodium and potassium currents were recorded in patchclamped hippocampal neurons filled with the calciumsensitive indicator dyes, Fluo-3 and Fura-red. The cells were stimulated by series of depolarizing voltage pulses, and they were intermittently excited by laser light to record confocal images and to measure calcium-dependent fluorescence. In many trials in which no ion channel blocking drugs were used, a slowly inactivating, inward current appeared. This current was not usually detectable at first, but it increased in amplitude over 5–20 min. As the *Tel.: 11-919-681-8404; fax: 11-919-684-5481. E-mail address: [email protected] (G.G. Somjen).
persistent inward current grew, the presumably K 1 -mediated outward currents became reduced in amplitude. It seemed as though the slowly inactivating opposing currents interfered one with the other because, as the one waned, the other became more prominent [19]. The slow inward current appeared to be similar to the persistent voltage-dependent sodium current, INa,P , recorded in many central neurons [6,9,23], except for its growth over time and its final, unusually large amplitude. Because of the possible role of INa,P in seizures, spreading depression and hypoxia [7,10,13] it seemed important to define the conditions under which this current can attain such unusual intensity. In this article I report that internal fluorescence itself causes the growth of INa,P which, once it is induced, reverses only very slowly. An abstract of some of the findings has been published [20]. A companion paper [21]
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02947-4
G.G. Somjen / Brain Research 885 (2000) 94 – 101
reports the effect of elevated external K 1 concentration on INa,P .
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2. Materials and methods
The current records were read with Clampfit (Axon Instruments) software. After subtraction of linear leak and holding currents, the data were further processed with the Excel (Microsoft) program. Junction potentials were calculated with the JPCalc program [3].
2.1. Isolation of neurons
2.3. Fluorescence imaging
Hippocampal CA1 pyramidal cells were isolated according to the method of Kay and Wong [14]. Briefly: rats of 60–120 g body weight were decapitated under deep ether anesthesia. Brains were removed and 500-mm-thick slices were cut from hippocampus. The CA1 region was cut into smaller pieces and these tissue fragments were digested for 75 min. The digestion medium contained (in mM l 21 ): NaCl 125, KCl 5, CaCl 2 1, MgCl 2 2, D-glucose 25, [2-hydoxyethyl]piperazine-[2-ethanesulfonic acid] (HEPES) 10, pH 7.0, with trypsin 0.75 mg / ml, at room temperature (20–228C). After digestion the tissue pieces were washed and then incubated in trypsin-free oxygenated medium at room temperature. Tissue fragments were dispersed by trituration with a graded series of Pasteur pipettes.
The non-permeant forms of the fluorescent calcium indicator dyes Fluo-3 (10 mM) and Fura-red (30 mM) (Molecular Probes) were added to the pipette solution [11]. The 488 nm excitation light was used and emission was recorded at 520 nm (Fluo-3) as well as 640 nm (Fura-red) with COMOS (Biorad) software. Fluorescence intensities were recorded from two intersecting elongated rectangular areas of interest at 10 or 20 s intervals; whole images were recorded at 60 s intervals. Background-corrected fluorescence ratios (Fluo-3 / Fura-red) were computed subsequently using Excel (Microsoft) software. Fluo-3 fluorescence increases while Fura-red fluorescence decreases with rising [Ca 21 ] i .
2.2. Recording of voltage-dependent currents Cell suspensions were placed in a chamber of about 0.7 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 composition: (in mM l 21 ): NaCl 130, KCl 3.5, CaCl 2 1.2, MgCl 2 1.0, glucose 25, HEPES 10, pH 7.3 or 7.35, at 20–228C. Cells were approached under the microscope objective with patch pipettes; tight seal was established, and the whole-cell recording condition created by suction. To record Na and K currents the pipettes were filled with a solution containing (in mM l 21 ): KF 129, NaCl 4, EGTA 10, CaCl 2 0.5, MgCl 2 2, HEPES 10, Na 2 ATP 4, pH 7.1 or 7.3, tip resistance 2.5–4.5 MV. To block K 1 currents, KF was substituted by 109 mM CsF and 20 mM tetraethylammonium–Cl (TEA–Cl). An Axopatch 1D amplifier in voltage-clamp configuration and the pClamp-6 (Axon Instruments) suite of programs was used to record whole-cell currents. Pipette and cell capacitances were compensated in the customary manner. Series resistance was compensated to 70%. The holding potential was 270 mV pipette voltage. Current– voltage (I–V ) curves were recorded usually at 1-, sometimes at 2-min intervals. Two different protocols were used: Either eight sweeps, each beginning with a pre-pulse of 100 ms to 290 mV to remove inactivation, followed by 200 ms depolarizing steps at 2 s intervals of 15 mV increments, taking the pipette voltage from 270 to 135 mV; or 12 sweeps of a 200 ms hyperpolarizing pre-pulse followed by 400 ms depolarizing steps in 10 mV increments, taking the pipette from 270 to 140 mV.
2.4. Statistics Except when otherwise noted, numerical data are given as the mean6S.E.M. Significance of differences was calculated by paired two-tailed t-test.
3. Results Whole-cell currents evoked by voltage steps were recorded from patch-clamped freshly isolated hippocampal CA1 neurons. The recording pipette was filled either with a KF or with a CsF based solution, that also contained the fluorescent dyes Fura-red and Fluo-3. At 1-min intervals the cells were stimulated by a series of voltage steps to construct current–voltage (I–V ) curves, and they were also excited at 10 or 20 s intervals by 1 s pulses of laser light to determine changes in internal Ca 21 activity. When KF was the main electrolyte, all the voltage-gated channels were available for activation and the records were the sums of several individual currents. Fig. 1A illustrates current–voltage (I–V ) curves of the slowly inactivating currents recorded with a KF-based pipette from one cell over an 18-min period. The graph shows the average currents measured during the last 15 ms of each depolarizing step after subtracting linear leak and holding current and correcting pipette potentials for junction potential. Fig. 1B shows sample currents evoked by a depolarizing pulse to 225 mV pipette potential (VP ) (equaling 232.6 mV membrane potential (Vm ), after correction for junction potential). In most cases, as in Fig. 1A, during the first few minutes of observation the slowly inactivating component flowed outward at all potentials and the I–V curve appeared to be dominated by the delayed
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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 (Vm ) corrected for junction potential. (B) Sample currents recorded 1 min and 18 min after establishing whole-cells’ condition, evoked by depolarization to 225 mV pipette voltage, corresponding to Vm of 232.6 mV after correction of junction potential.
rectifier K 1 current, IK,Dr . After 5–10 min, the persistent inward current appeared in most fluorescent laser illuminated cells, and then it grew in amplitude. As the persistent inward current increased, outward currents became smaller and the reversal potential (zero-current potential) of the compound current shifted to more positive levels. The persistent inward current was blocked by 0.5 mM tetrodotoxin (TTX). In the cell illustrated in Fig. 2A, a trace of a persistent inward current was recorded immediately after establishing whole-cell condition. After 24 min of stimulation by voltage pulses alternated with laser
pulses, a sizeable inward current appeared. Adding TTX to the bathing solution completely blocked both transient and persistent inward currents within 3–5 min, but after washing for 16 min with normal solution both components of the inward currents reappeared. By subtracting the current amplitudes recorded under the influence of TTX from those recorded at the end of the 24 min of observation, the TTX-sensitive component of the persistent current is revealed (Fig. 2B). Fig. 2C shows conductances computed by dividing the currents of Fig. 2B by the driving voltage, the latter being defined as the difference between
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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.
the membrane potential and the extrapolated reversal potential of the TTX-sensitive current. The TTX-sensitive persistent component activated at a level more negative than 260 mV, it was maximal at 220 mV and it reversed around 130 mV. TTX sensitivity, activation and reversal potentials, and the shape of its I–V curve identify it as a persistent sodium current, INa,P [6,9]. The fact that outward currents became smaller while INa,P grew, suggested the possibility that the ‘rundown’ of 1 K currents had unmasked the previously hidden, slow inward current [19]. To test this, cells were tested in which most of the K 1 currents were suppressed by using pipettes filled with a solution containing CsF instead of KF, with 20 mM tetraethylammonium (TEA1 ) added. Even after replacing K 1 with Cs 1 and TEA1 , a small non-inactivating outward current often remained visible at strongly depolarized voltages (Fig. 5B). This may be due to Cs 1 flowing through K 1 channels, or it may represent the non-specific cation current described by Alzheimer [1]. As
expected, when most K 1 currents were blocked, INa,P could readily be evoked right away in almost all cells by prolonged depolarizing pulses. INa,P did, however, grow with time and with repeated fluorescent excitation, even when the K 1 currents were eliminated, demonstrating that rundown of K 1 current cannot account for its growth. As the persistent inward current increased in amplitude, its reversal shifted in the positive direction, regardless of whether K 1 currents were blocked or not (Fig. 1A, 5A, B). To test whether repeated stimulation by depolarizing voltage steps causes the gradual increase of the INa,P (use-dependent facilitation), recordings were made from cells without fluorescent dyes and without laser illumination (three cells with KF and 14 cells with CsF in the pipettes). In these cells the INa,P was small and it did not grow during repeated stimulation. The question then was, whether the dyes themselves potentiate INa,P , and, conversely, whether laser light in the absence of internal fluorescence has this effect. Laser illumination of cells not
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containing dye (three with KF, three with CsF), and filling cells with dye but not using the laser (n511, all CsF) had the same negative result: in the absence of fluorescence, the INa,P was, and remained, small. Fig. 3 shows a statistical summary of the maximal persistent inward current amplitudes normalized to cell capacitance, recorded at different times from groups of cells with and without fluorescence, in the presence and in the absence of K 1 mediated outward current. The difference between the fluorescent and the non-fluorescent cells was striking. In the fluorescent cells that were observed for more than 10 min with CsF-filled pipettes, the mean maximal INa,P amplitude was 280.1619.7 pA / pF (n56), which is 12.9 times larger than the 26.261.2 (n57)
Fig. 3. Statistical summary of the potentiation of the persistent inward current by repeated pulses of fluorescence. The columns show mean6S.E.M. of the maximal slowly inactivating inward current, normalized to cell capacitance. The numbers above the horizontal axis show the time in minutes elapsed from establishing whole-cell conditions until the recording. ‘0–5 min’ show the largest persistent inward current measured 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 of cells in each category are shown below the columns. Pipette solutions based on CsF also contained 20 mM tetraethylammonium (TEA1 ) to block most K 1 currents. (A) Non-fluorescent cells include those filled with dye but not illuminated, not filled with dye but illuminated, and free of dye and not illuminated. (B) Fluorescent cells were filled with Fluo-3 and Fura-red dyes and were intermittently illuminated by laser light.
pA / pF recorded from cells under similar conditions but in the absence of fluorescence. Without exception, in all fluorescent cells the INa,P increased in amplitude with time but the magnitude of the effect was variable. In the fluorescent, CsF group there were 18 cells from which the current was recorded for 5–10 min after establishing whole-cell condition; the largest current in a cell in this group was 21700 pA (185 pA / pF) and the smallest 2116 pA (12.2 pA / pF). The mean amplitude of all the cells in the 5–10 min, fluorescent, CsF group was 68.6 pA / pF with a standard deviation of 642.1 and standard error of 69.9. In the non-fluorescent group, and initially also among the fluorescent cells, the difference between the INa,P amplitude recorded with and without blocking K 1 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 of the INa,P and the cytoplasmic calcium activity, [Ca 21 ] 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 responses of [Ca 21 ] i were presumably caused by voltagedependent calcium currents. In the course of repeated stimulation they usually subsided, probably because the pipettes did not contain the ingredients required to counteract ‘rundown’ of Ca 21 currents [19]. The ‘resting’ or baseline [Ca 21 ] i showed upward or downward drift in different experiments and was not correlated with the growth of INa,P . In the great majority of the trials F 2 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 INa,P was seen, but in two trials with gluconate and in one with acetate the seal was maintained and INa,P appeared and grew as usual. At this stage of the study three questions arose. First, do the laser induced pulses of fluorescence induce a membrane current? Second, can fluorescence by itself potentiate the INa,P , or does it have to be combined with depolarization? And third, is the potentiation of the INa,P reversible, or is it an irreversibly altered state of the membrane channel? To answer these questions, I–V curves were first recorded for 6–14 min from cells that were filled with dye, but without turning on the laser. Then electrical stimulation was suspended and intermittent laser excitation was turned on for 7–10 min. In this period the holding current was continuously recorded. The voltage to which the membrane was clamped was adjusted from time to time to detect any laser-induced currents that may be voltage
G.G. Somjen / Brain Research 885 (2000) 94 – 101
dependent. Subsequently, I–V curves were again recorded without laser illumination for additional 28–32 min. Figs. 4 A and B show the maximal INa,P recorded with CsF pipettes from four cells before and after laser illumination under this protocol. Figs. 4C and D show the holding current of one of these cells recorded during intermittent laser illumination, reproduced on two different time-scales. Fig. 5 illustrates recordings obtained from individual cells before and after laser excitation. As illustrated by the examples of Fig. 4C and D, laser pulse excitation of fluorescence did not evoke a detectable change in the holding current, regardless of holding potential. The same was true in four cells from which recordings were made with CsF and two cells with KF pipettes. Recordings have been obtained at 290, 270, 250, 240 and 230 mV holding potentials (not all levels in each cells). Not surprisingly, shifting of the holding potential did evoke increases in holding current that inactivated slowly (Fig. 4C, D). Following 5–8 min of intermittent laser illumination,
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there was a striking increase in amplitude of the INa,P in all 6 trials. The mean maximal amplitude immediately before laser treatment was 243.7613.2 pA and immediately after the series of illuminations 29136172 pA (mean6S.E.M.). One of the six cells was lost early in the post-laser period, the persistent current of the five other cells at the end of 28–32 min following laser treatment was still 28076162 pA. The decay was slow but consistent and statistically significant (P,0.009, paired two-tailed t-test). This slow decrease could perhaps be caused by ‘rundown’ of INa,P , but the transient Na 1 current, INa,T did not show a similar decay. INa,T amplitude was on average 214,60761591 pA in these six cells before laser treatment, 212,14661406 pA immediately after laser and 212,37962425 pA half an hour later. Fig. 5 illustrates examples of I–V curves and current traces obtained from two of the six cells before and after laser-induced fluorescence. Fig. 5A, C and D were recorded with KF filled pipette from one cell, and Fig. 5B from another cell, using CsF. With KF, INa,P was not
Fig. 4. Intermittent pulses of fluorescence do not evoke a membrane current, but they cause long-lasting potentiation of the persistent inward current, INa,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 14 min. The recording pipettes were filled with CsF based solution containing 20 mM TEA1 . (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.
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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, but recorded with K 1 currents mostly blocked, with CsF and TEA1 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 INa,P . (C) Transient sodium current, INa,T evoked by depolarizing pulse of 230 mV pipette voltage (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 thereafter (Fig. 5A, D). With CsF the originally small INa,P increased manifold following laser pulses (Fig. 5B). As shown in Fig. 5C, while the amplitude of INa,T did not change, its duration increased. Such a widening of the INa,T trace was consistently seen in conjunction with the growth of the INa,P . The amplitude of INa,T usually showed slow drift up or down, unrelated to laser treatment and not linked to the changes in other ion currents.
4. Discussion Two conclusions emerge. First, that depression of outward K 1 currents reveals the voltage-gated persistent Na 1 current, INa,P , which otherwise can be concealed by IK . Second, that laser-induced internal fluorescence powerfully potentiates INa,P . This potentiation accumulates with repeated laser pulse excitation and it reverses only very
slowly after laser excitation has ceased. The first of these two findings could be expected, the second was a surprise. Since laser illumination and intracellular fluorescence are not usual hazards for hippocampus in situ, the effect is not directly relevant to human pathophysiology. Nonetheless it is of interest for the understanding of sodium currents in mammalian central neurons. The fact that the persistent current can grow more than 10-fold under the relatively mild influence of intracellular fluorescence suggests that the maximal current carrying capacity of slowly inactivating Na 1 channels is much greater than is normally manifested. It is not known whether INa,P arises because of incapacitated inactivation of the channel that generates the common, transient Na 1 current, INa,T , or whether it represents current flowing through a distinct channel type. There is evidence favoring the former interpretations [2,4,5]. Our data do not distinguish between these two alternatives, but the fact that INa,T duration increased whenever INa,P amplitude increased does suggest a link between these two.
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In the data there is also a message of caution. Fluorescent dyes have many uses in cell biology. One must be mindful of the possibility that the fluorescence might alter the very function one is about to study. The very slow reversibility of the fluorescence-induced potentiation of INa,P raises the question whether it is an example of phototoxicity or of photodynamic action. Photodynamic cell injury has been attributed to the formation of reactive oxygen species [22], and as such it has been successfully prevented by replacing oxygen by argon in the solution bathing Aplysia neurons [17]. Unlike the specific effect of fluorescence in hippocampal neurons described here, photodynamic cell damage is typically associated with non-specific increase of membrane ion permeability and consequently with depolarization [16,22]. Other, more subtle photodynamic effects, which do not necessarily destroy cells have, however, also been described. Among them are inactivation of the mitochondrial permeability transition pore [18]; the triggering of calcium oscillations in pancreatic cells [8]; and light-induced potentiation of NMDA currents in cultured neurons [15]. Each of these effects differs from the one described here, yet each exemplifies the novel fact, that light energy can change channel function. Gage and associates [10,12] reported enhancement of INa,P by hypoxia and cyanide poisoning in neurons and heart muscle cells. We have found a similar effect in hippocampal pyramidal cells by elevated [K 1 ] o [20,21]. The effects of hypoxia and high [K 1 ] o were weaker and more readily reversible than those of fluorescence. This quantitative difference does not, however, exclude a ‘final common path’ of the enhancement of INa,P by these three agents.
Acknowledgements Supported by NIH grant NS 18670.
References [1] C. Alzheimer, A novel voltage-dependent cation current in rat neocortical neurones, J. Physiol. 479 (1994) 199–205. [2] C. Alzheimer, P.C. Schwindt, W.E. Crill, Modal gating of Na 1 channels as a mechanism of persistent Na 1 current in pyramidal neurons from rat and cat sensorimotor cortex, J. Neurosci. 13 (1993) 660–673. [3] P.H. Barry, JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements, J. Neurosci. Meth. 51 (1994) 107–116.
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[4] W.A. Catterall, Structure and function of voltage sensitive ion channels, Science 242 (1988) 50–61. [5] W.A. Catterall, Modulation of sodium and calcium channels by protein phosphorylation and G proteins. [Review] [136 refs], Adv. Second Messeng. Phosphoprot. Res. 31 (1997) 159–181. [6] W.E. Crill, Persistent sodium current in mammalian central neurons, Ann. Rev. Physiol. 58 (1996) 349–362. [7] W.E. Crill, P.C. Schwindt, Role of persistent inward and outward membrane currents in epileptiform bursting in mammalian neurons, in: A.V. Delgado-Escueta, A.A. Ward, D.M. Woodbury, R.J. Porter (Eds.), Basic Mechanisms of the Epilepsies, Raven Press, New York, 1986, pp. 225–233. [8] Z.J. Cui, T. Kanno, Photodynamic triggering of calcium oscillation in the isolated rat pancreatic acini, J. Physiol. 504 (1997) 47–55. [9] C.R. French, P. Sah, K.J. Buckett, P.W. Gage, A voltage-dependent persistent sodium current in mammalian hippocampal neurons, J. Gen. Physiol. 95 (1990) 1139–1157. ¨ [10] A.K.M. Hammarstrom, P.W. Gage, Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons, J. Physiol. 510 (1998) 735–741. [11] R.P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, 1996. [12] Y.-K. Ju, D.A. Saint, P.W. Gage, Hypoxia increases persistent sodium current in rat ventricular myocytes, J. Physiol. 497 (1999) 337–347. [13] H. Kager, W.J. Wadman, G.G. Somjen, G.G., Simulated seizure discharges and spreading depression-like depolarization in a neuron model incorporating interstitial space and ion concentration changes, J. Neurophysiol. (2000) (in press). [14] A.R. Kay, R.K.S. Wong, Isolation of neurons suitable for patch clamping from adult mammalian central nervous system, J. Neurosci. Meth. 16 (1986) 227–238. [15] D.N. Leskiewicz, W.K. Potthoff, K.K. Kandler, E. Aizenman, Selective enhancement of NMDA receptor mediated currents by light, Soc. Neurosci. Abstr. 25 (1999) 1978. [16] F. Mendez, R. Penner, Near-visible ultraviolet light induces a novel ubiquitous calcium-permeable cation current in mammalian cell lines, J. Physiol. 507 (1999) 365–377. [17] T.D. Parsons, D. Kleinfeld, F. Raccuia-Behling, B.M. Salzberg, Optical recording of the electrical activity of synaptically interacting Aplysia neurons in culture using potentiometric probes, Biophys. J. 56 (1989) 213–221. [18] C. Salet, G. Moreno, F. Ricchelli, P. Bernardi, Singlet oxygen produced by photodynamic action causes inactivation of the mitochondrial permeability transition pore, J. Biol. Chem. 272 (1997) 21938–21943. [19] G.G. Somjen, Low external NaCl concentration and low osmolarity enhance voltage gated Ca currents but depress K currents in freshly isolated rat hippocampal neurons, Brain Res. 851 (1999) 189–197. [20] G.G. Somjen, Intracellular fluorescence and elevated [K 1 ] o potentiate persistent sodium current in isolated hippocampal neurons, FASEB J. 14 (2000) A107. ¨ [21] G.G. Somjen, M. Muller, Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices, Brain Res. 885 (2000) 102–110. [22] K.G. Specht, M.A.J. Rodgers, Plasma membrane depolarization and calcium influx during cell injury by photodynamic action, Biochim. Biophys. Acta 1070 (1991) 60–68. [23] C.P. Taylor, Na 1 currents that fail to inactivate, Trends Neurosci. 16 (1993) 455–460.
Research report
Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons George G. Somjen* Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710, USA Accepted 12 September 2000
Abstract Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the ‘Hodgkin–Huxley’ type early, fast inward surge followed by sustained outward current. Over 5–20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K 1 currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin (TTX) and it had current–voltage (I–V ) characteristics of a persistent sodium current, INa,P . In cells not filled with dye but illuminated by laser, and in cells with dye but not illuminated, INa,P remained small. There was a more than 12-fold difference in the maximal amplitude of INa,P of fluorescent compared to non-fluorescent cells. Once induced, INa,P decreased very slowly. Fluorescence increased the duration but not the amplitude of the transient Na 1 current, INa,T . With membrane potential clamped to a constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced INa,P potentiation is related to photodynamic action. 2000 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Sodium channels Keywords: Persistent sodium current; Sodium channel; Photodynamic action; Fluorescence; Dissociated neuron; Whole-cell current
1. Introduction In a previous study [19] of the effects of low sodium chloride concentration and low osmolarity, whole-cell sodium and potassium currents were recorded in patchclamped hippocampal neurons filled with the calciumsensitive indicator dyes, Fluo-3 and Fura-red. The cells were stimulated by series of depolarizing voltage pulses, and they were intermittently excited by laser light to record confocal images and to measure calcium-dependent fluorescence. In many trials in which no ion channel blocking drugs were used, a slowly inactivating, inward current appeared. This current was not usually detectable at first, but it increased in amplitude over 5–20 min. As the *Tel.: 11-919-681-8404; fax: 11-919-684-5481. E-mail address: [email protected] (G.G. Somjen).
persistent inward current grew, the presumably K 1 -mediated outward currents became reduced in amplitude. It seemed as though the slowly inactivating opposing currents interfered one with the other because, as the one waned, the other became more prominent [19]. The slow inward current appeared to be similar to the persistent voltage-dependent sodium current, INa,P , recorded in many central neurons [6,9,23], except for its growth over time and its final, unusually large amplitude. Because of the possible role of INa,P in seizures, spreading depression and hypoxia [7,10,13] it seemed important to define the conditions under which this current can attain such unusual intensity. In this article I report that internal fluorescence itself causes the growth of INa,P which, once it is induced, reverses only very slowly. An abstract of some of the findings has been published [20]. A companion paper [21]
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02947-4
G.G. Somjen / Brain Research 885 (2000) 94 – 101
reports the effect of elevated external K 1 concentration on INa,P .
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2. Materials and methods
The current records were read with Clampfit (Axon Instruments) software. After subtraction of linear leak and holding currents, the data were further processed with the Excel (Microsoft) program. Junction potentials were calculated with the JPCalc program [3].
2.1. Isolation of neurons
2.3. Fluorescence imaging
Hippocampal CA1 pyramidal cells were isolated according to the method of Kay and Wong [14]. Briefly: rats of 60–120 g body weight were decapitated under deep ether anesthesia. Brains were removed and 500-mm-thick slices were cut from hippocampus. The CA1 region was cut into smaller pieces and these tissue fragments were digested for 75 min. The digestion medium contained (in mM l 21 ): NaCl 125, KCl 5, CaCl 2 1, MgCl 2 2, D-glucose 25, [2-hydoxyethyl]piperazine-[2-ethanesulfonic acid] (HEPES) 10, pH 7.0, with trypsin 0.75 mg / ml, at room temperature (20–228C). After digestion the tissue pieces were washed and then incubated in trypsin-free oxygenated medium at room temperature. Tissue fragments were dispersed by trituration with a graded series of Pasteur pipettes.
The non-permeant forms of the fluorescent calcium indicator dyes Fluo-3 (10 mM) and Fura-red (30 mM) (Molecular Probes) were added to the pipette solution [11]. The 488 nm excitation light was used and emission was recorded at 520 nm (Fluo-3) as well as 640 nm (Fura-red) with COMOS (Biorad) software. Fluorescence intensities were recorded from two intersecting elongated rectangular areas of interest at 10 or 20 s intervals; whole images were recorded at 60 s intervals. Background-corrected fluorescence ratios (Fluo-3 / Fura-red) were computed subsequently using Excel (Microsoft) software. Fluo-3 fluorescence increases while Fura-red fluorescence decreases with rising [Ca 21 ] i .
2.2. Recording of voltage-dependent currents Cell suspensions were placed in a chamber of about 0.7 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 composition: (in mM l 21 ): NaCl 130, KCl 3.5, CaCl 2 1.2, MgCl 2 1.0, glucose 25, HEPES 10, pH 7.3 or 7.35, at 20–228C. Cells were approached under the microscope objective with patch pipettes; tight seal was established, and the whole-cell recording condition created by suction. To record Na and K currents the pipettes were filled with a solution containing (in mM l 21 ): KF 129, NaCl 4, EGTA 10, CaCl 2 0.5, MgCl 2 2, HEPES 10, Na 2 ATP 4, pH 7.1 or 7.3, tip resistance 2.5–4.5 MV. To block K 1 currents, KF was substituted by 109 mM CsF and 20 mM tetraethylammonium–Cl (TEA–Cl). An Axopatch 1D amplifier in voltage-clamp configuration and the pClamp-6 (Axon Instruments) suite of programs was used to record whole-cell currents. Pipette and cell capacitances were compensated in the customary manner. Series resistance was compensated to 70%. The holding potential was 270 mV pipette voltage. Current– voltage (I–V ) curves were recorded usually at 1-, sometimes at 2-min intervals. Two different protocols were used: Either eight sweeps, each beginning with a pre-pulse of 100 ms to 290 mV to remove inactivation, followed by 200 ms depolarizing steps at 2 s intervals of 15 mV increments, taking the pipette voltage from 270 to 135 mV; or 12 sweeps of a 200 ms hyperpolarizing pre-pulse followed by 400 ms depolarizing steps in 10 mV increments, taking the pipette from 270 to 140 mV.
2.4. Statistics Except when otherwise noted, numerical data are given as the mean6S.E.M. Significance of differences was calculated by paired two-tailed t-test.
3. Results Whole-cell currents evoked by voltage steps were recorded from patch-clamped freshly isolated hippocampal CA1 neurons. The recording pipette was filled either with a KF or with a CsF based solution, that also contained the fluorescent dyes Fura-red and Fluo-3. At 1-min intervals the cells were stimulated by a series of voltage steps to construct current–voltage (I–V ) curves, and they were also excited at 10 or 20 s intervals by 1 s pulses of laser light to determine changes in internal Ca 21 activity. When KF was the main electrolyte, all the voltage-gated channels were available for activation and the records were the sums of several individual currents. Fig. 1A illustrates current–voltage (I–V ) curves of the slowly inactivating currents recorded with a KF-based pipette from one cell over an 18-min period. The graph shows the average currents measured during the last 15 ms of each depolarizing step after subtracting linear leak and holding current and correcting pipette potentials for junction potential. Fig. 1B shows sample currents evoked by a depolarizing pulse to 225 mV pipette potential (VP ) (equaling 232.6 mV membrane potential (Vm ), after correction for junction potential). In most cases, as in Fig. 1A, during the first few minutes of observation the slowly inactivating component flowed outward at all potentials and the I–V curve appeared to be dominated by the delayed
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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 (Vm ) corrected for junction potential. (B) Sample currents recorded 1 min and 18 min after establishing whole-cells’ condition, evoked by depolarization to 225 mV pipette voltage, corresponding to Vm of 232.6 mV after correction of junction potential.
rectifier K 1 current, IK,Dr . After 5–10 min, the persistent inward current appeared in most fluorescent laser illuminated cells, and then it grew in amplitude. As the persistent inward current increased, outward currents became smaller and the reversal potential (zero-current potential) of the compound current shifted to more positive levels. The persistent inward current was blocked by 0.5 mM tetrodotoxin (TTX). In the cell illustrated in Fig. 2A, a trace of a persistent inward current was recorded immediately after establishing whole-cell condition. After 24 min of stimulation by voltage pulses alternated with laser
pulses, a sizeable inward current appeared. Adding TTX to the bathing solution completely blocked both transient and persistent inward currents within 3–5 min, but after washing for 16 min with normal solution both components of the inward currents reappeared. By subtracting the current amplitudes recorded under the influence of TTX from those recorded at the end of the 24 min of observation, the TTX-sensitive component of the persistent current is revealed (Fig. 2B). Fig. 2C shows conductances computed by dividing the currents of Fig. 2B by the driving voltage, the latter being defined as the difference between
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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.
the membrane potential and the extrapolated reversal potential of the TTX-sensitive current. The TTX-sensitive persistent component activated at a level more negative than 260 mV, it was maximal at 220 mV and it reversed around 130 mV. TTX sensitivity, activation and reversal potentials, and the shape of its I–V curve identify it as a persistent sodium current, INa,P [6,9]. The fact that outward currents became smaller while INa,P grew, suggested the possibility that the ‘rundown’ of 1 K currents had unmasked the previously hidden, slow inward current [19]. To test this, cells were tested in which most of the K 1 currents were suppressed by using pipettes filled with a solution containing CsF instead of KF, with 20 mM tetraethylammonium (TEA1 ) added. Even after replacing K 1 with Cs 1 and TEA1 , a small non-inactivating outward current often remained visible at strongly depolarized voltages (Fig. 5B). This may be due to Cs 1 flowing through K 1 channels, or it may represent the non-specific cation current described by Alzheimer [1]. As
expected, when most K 1 currents were blocked, INa,P could readily be evoked right away in almost all cells by prolonged depolarizing pulses. INa,P did, however, grow with time and with repeated fluorescent excitation, even when the K 1 currents were eliminated, demonstrating that rundown of K 1 current cannot account for its growth. As the persistent inward current increased in amplitude, its reversal shifted in the positive direction, regardless of whether K 1 currents were blocked or not (Fig. 1A, 5A, B). To test whether repeated stimulation by depolarizing voltage steps causes the gradual increase of the INa,P (use-dependent facilitation), recordings were made from cells without fluorescent dyes and without laser illumination (three cells with KF and 14 cells with CsF in the pipettes). In these cells the INa,P was small and it did not grow during repeated stimulation. The question then was, whether the dyes themselves potentiate INa,P , and, conversely, whether laser light in the absence of internal fluorescence has this effect. Laser illumination of cells not
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containing dye (three with KF, three with CsF), and filling cells with dye but not using the laser (n511, all CsF) had the same negative result: in the absence of fluorescence, the INa,P was, and remained, small. Fig. 3 shows a statistical summary of the maximal persistent inward current amplitudes normalized to cell capacitance, recorded at different times from groups of cells with and without fluorescence, in the presence and in the absence of K 1 mediated outward current. The difference between the fluorescent and the non-fluorescent cells was striking. In the fluorescent cells that were observed for more than 10 min with CsF-filled pipettes, the mean maximal INa,P amplitude was 280.1619.7 pA / pF (n56), which is 12.9 times larger than the 26.261.2 (n57)
Fig. 3. Statistical summary of the potentiation of the persistent inward current by repeated pulses of fluorescence. The columns show mean6S.E.M. of the maximal slowly inactivating inward current, normalized to cell capacitance. The numbers above the horizontal axis show the time in minutes elapsed from establishing whole-cell conditions until the recording. ‘0–5 min’ show the largest persistent inward current measured 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 of cells in each category are shown below the columns. Pipette solutions based on CsF also contained 20 mM tetraethylammonium (TEA1 ) to block most K 1 currents. (A) Non-fluorescent cells include those filled with dye but not illuminated, not filled with dye but illuminated, and free of dye and not illuminated. (B) Fluorescent cells were filled with Fluo-3 and Fura-red dyes and were intermittently illuminated by laser light.
pA / pF recorded from cells under similar conditions but in the absence of fluorescence. Without exception, in all fluorescent cells the INa,P increased in amplitude with time but the magnitude of the effect was variable. In the fluorescent, CsF group there were 18 cells from which the current was recorded for 5–10 min after establishing whole-cell condition; the largest current in a cell in this group was 21700 pA (185 pA / pF) and the smallest 2116 pA (12.2 pA / pF). The mean amplitude of all the cells in the 5–10 min, fluorescent, CsF group was 68.6 pA / pF with a standard deviation of 642.1 and standard error of 69.9. In the non-fluorescent group, and initially also among the fluorescent cells, the difference between the INa,P amplitude recorded with and without blocking K 1 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 of the INa,P and the cytoplasmic calcium activity, [Ca 21 ] 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 responses of [Ca 21 ] i were presumably caused by voltagedependent calcium currents. In the course of repeated stimulation they usually subsided, probably because the pipettes did not contain the ingredients required to counteract ‘rundown’ of Ca 21 currents [19]. The ‘resting’ or baseline [Ca 21 ] i showed upward or downward drift in different experiments and was not correlated with the growth of INa,P . In the great majority of the trials F 2 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 INa,P was seen, but in two trials with gluconate and in one with acetate the seal was maintained and INa,P appeared and grew as usual. At this stage of the study three questions arose. First, do the laser induced pulses of fluorescence induce a membrane current? Second, can fluorescence by itself potentiate the INa,P , or does it have to be combined with depolarization? And third, is the potentiation of the INa,P reversible, or is it an irreversibly altered state of the membrane channel? To answer these questions, I–V curves were first recorded for 6–14 min from cells that were filled with dye, but without turning on the laser. Then electrical stimulation was suspended and intermittent laser excitation was turned on for 7–10 min. In this period the holding current was continuously recorded. The voltage to which the membrane was clamped was adjusted from time to time to detect any laser-induced currents that may be voltage
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dependent. Subsequently, I–V curves were again recorded without laser illumination for additional 28–32 min. Figs. 4 A and B show the maximal INa,P recorded with CsF pipettes from four cells before and after laser illumination under this protocol. Figs. 4C and D show the holding current of one of these cells recorded during intermittent laser illumination, reproduced on two different time-scales. Fig. 5 illustrates recordings obtained from individual cells before and after laser excitation. As illustrated by the examples of Fig. 4C and D, laser pulse excitation of fluorescence did not evoke a detectable change in the holding current, regardless of holding potential. The same was true in four cells from which recordings were made with CsF and two cells with KF pipettes. Recordings have been obtained at 290, 270, 250, 240 and 230 mV holding potentials (not all levels in each cells). Not surprisingly, shifting of the holding potential did evoke increases in holding current that inactivated slowly (Fig. 4C, D). Following 5–8 min of intermittent laser illumination,
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there was a striking increase in amplitude of the INa,P in all 6 trials. The mean maximal amplitude immediately before laser treatment was 243.7613.2 pA and immediately after the series of illuminations 29136172 pA (mean6S.E.M.). One of the six cells was lost early in the post-laser period, the persistent current of the five other cells at the end of 28–32 min following laser treatment was still 28076162 pA. The decay was slow but consistent and statistically significant (P,0.009, paired two-tailed t-test). This slow decrease could perhaps be caused by ‘rundown’ of INa,P , but the transient Na 1 current, INa,T did not show a similar decay. INa,T amplitude was on average 214,60761591 pA in these six cells before laser treatment, 212,14661406 pA immediately after laser and 212,37962425 pA half an hour later. Fig. 5 illustrates examples of I–V curves and current traces obtained from two of the six cells before and after laser-induced fluorescence. Fig. 5A, C and D were recorded with KF filled pipette from one cell, and Fig. 5B from another cell, using CsF. With KF, INa,P was not
Fig. 4. Intermittent pulses of fluorescence do not evoke a membrane current, but they cause long-lasting potentiation of the persistent inward current, INa,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 14 min. The recording pipettes were filled with CsF based solution containing 20 mM TEA1 . (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.
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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, but recorded with K 1 currents mostly blocked, with CsF and TEA1 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 INa,P . (C) Transient sodium current, INa,T evoked by depolarizing pulse of 230 mV pipette voltage (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 thereafter (Fig. 5A, D). With CsF the originally small INa,P increased manifold following laser pulses (Fig. 5B). As shown in Fig. 5C, while the amplitude of INa,T did not change, its duration increased. Such a widening of the INa,T trace was consistently seen in conjunction with the growth of the INa,P . The amplitude of INa,T usually showed slow drift up or down, unrelated to laser treatment and not linked to the changes in other ion currents.
4. Discussion Two conclusions emerge. First, that depression of outward K 1 currents reveals the voltage-gated persistent Na 1 current, INa,P , which otherwise can be concealed by IK . Second, that laser-induced internal fluorescence powerfully potentiates INa,P . This potentiation accumulates with repeated laser pulse excitation and it reverses only very
slowly after laser excitation has ceased. The first of these two findings could be expected, the second was a surprise. Since laser illumination and intracellular fluorescence are not usual hazards for hippocampus in situ, the effect is not directly relevant to human pathophysiology. Nonetheless it is of interest for the understanding of sodium currents in mammalian central neurons. The fact that the persistent current can grow more than 10-fold under the relatively mild influence of intracellular fluorescence suggests that the maximal current carrying capacity of slowly inactivating Na 1 channels is much greater than is normally manifested. It is not known whether INa,P arises because of incapacitated inactivation of the channel that generates the common, transient Na 1 current, INa,T , or whether it represents current flowing through a distinct channel type. There is evidence favoring the former interpretations [2,4,5]. Our data do not distinguish between these two alternatives, but the fact that INa,T duration increased whenever INa,P amplitude increased does suggest a link between these two.
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In the data there is also a message of caution. Fluorescent dyes have many uses in cell biology. One must be mindful of the possibility that the fluorescence might alter the very function one is about to study. The very slow reversibility of the fluorescence-induced potentiation of INa,P raises the question whether it is an example of phototoxicity or of photodynamic action. Photodynamic cell injury has been attributed to the formation of reactive oxygen species [22], and as such it has been successfully prevented by replacing oxygen by argon in the solution bathing Aplysia neurons [17]. Unlike the specific effect of fluorescence in hippocampal neurons described here, photodynamic cell damage is typically associated with non-specific increase of membrane ion permeability and consequently with depolarization [16,22]. Other, more subtle photodynamic effects, which do not necessarily destroy cells have, however, also been described. Among them are inactivation of the mitochondrial permeability transition pore [18]; the triggering of calcium oscillations in pancreatic cells [8]; and light-induced potentiation of NMDA currents in cultured neurons [15]. Each of these effects differs from the one described here, yet each exemplifies the novel fact, that light energy can change channel function. Gage and associates [10,12] reported enhancement of INa,P by hypoxia and cyanide poisoning in neurons and heart muscle cells. We have found a similar effect in hippocampal pyramidal cells by elevated [K 1 ] o [20,21]. The effects of hypoxia and high [K 1 ] o were weaker and more readily reversible than those of fluorescence. This quantitative difference does not, however, exclude a ‘final common path’ of the enhancement of INa,P by these three agents.
Acknowledgements Supported by NIH grant NS 18670.
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