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Lithium ions increase action potential duration of mammalian neurons
Brain Research, 293 (1984) 173-177 Elsevier
173
BRE 20023
Lithium ions increase action potential duration of mammalian neurons MARK L. MAYER l, VINCENZO CRUNELLP and JOHN A. KEMP2 1Department of Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, London SWI 7 ORE and 2Neurochemical Pharmacology Unit, MRC Centre, Hills Road, Cambridge CB2 2QD (U.K.)
(Accepted October 11th, 1983) Key words: lithium - - action potential - - intracellular recording - - calcium current
During Lucifer Yellow staining of mammalian neurons, intracellular recording revealed a prolongation of the action potential that is probably the result of leakage of lithium ions into the intracellular fluid. In experiments on dorsal root ganglion neurons intracellular iontophoresis of lithium ions also broadened the action potential. Cadmium, a calcium channel blocker, shortens the lithium-evoked wide actions potentials. The present experiments do not reveal whether lithium directly enhances inward calcium current, or whether a block of outward rectification allows calcium currents to increase the action potential duration. The 4-aminonapthalimide dye, Lucifer Yellow CH, is a useful fluorescent marker when used in conjunction with intracellular microelectrodes to stain individual neurons from which electrical activity is being recorded19,20. The low solubility of the potassium salt of Lucifer Yellow limits the concentration of dye that can be used to fill microelectrodes and this reduces the intensity of staining achievable under experimental conditions. Thus Lucifer Yellow is prepared as the highly soluble lithium salt 19. To further improve the electrical characteristics of fine tip microelectrodes required for recording from small mammalian neurons, Lucifer Yellow has been dissolved in 1-2 M lithium acetate or lithium chloride 14,19,21. When using such electrodes for combined morphological and electrophysiological study of neurons in cultures and brain slices prepared from several regions of the mammalian nervous system we recorded marked changes in action potential duration during prolonged stable intracellular recording. This seriously compromised characterization of the electrophysiological properties of the neurons to be studied morphologically. O u r results suggest that leakage of lithium ions into the cytosol is responsible. The effects we describe are quite different from the photo-
inactivition of neurons that occurs on irradiation of dye-labeled cells 15. Cultures of rat dorsal root ganglion ( D R G ) neurons were prepared from 14-16-day-old rat embryos and grown in dissociated culture for up to 12 weeks using methods similar to those described previously 18. The recording medium contained (raM) NaCI 142, KC1 4.7, CaCI2 3, MgC12 2, glucose 9.5, phenol red 0.01 mg/ml and was buffered to p H 7.3 with 5 mM HEPES. Intracellular recording was performed at room temperature on the stage of a phase contrast microscope. Brain slices from the dorsal raphe region of the brainstem and the visual cortex were prepared using a Mcllwain tissue chopper and maintained in vitro in continually gassed (95% 0 2, 5% CO2) medium containing (mM) NaCI 134, KC1 5, KH2PO 4 1.25, MgSO4 2, CaCI 2 2, N a H C O 3 16 and glucose 10. Intracellular recording was performed at 37 °C. Microelectrodes were filled with a 5% solution of Lucifer Yellow C H dissolved in 1 M lithium acetate titrated to pH 7.0 with acetic acid. In each of the 3 preparations examined neurons impaled with Lucifer Yellow/lithium acetate microelectrodes generated action potentials that were unusually long compared to those recorded in control
Correspondence: M. L. Mayer, Department of Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, London SWl7 ORE, U.K.
174
VISUAL CORTEX
BRAINSTEM
GANGL I0 N
50 mV -G
] I
I
I
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.
.
.
.
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.
20ms
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Fig. 1. Action potential duration increases during intracellular recording with Lucifer Yellow filled electrodes. Actions potentials were recorded from neurons in preparations from 3 areas of the rat nervous system using microelectrodes filled with 5% Lucifer Yellow dissolved in ] M lithium acetate. In each case the upper record shows a response evoked immediately after a stable recording was obtained. The lower records show responses recorded from the s a m e neurons later in the experiment. The resting potential of the dorsal root ganglion neuron was initally--60 mV, and dechned to ~ 4 0 m V after 38 rain, at which time the lower record was obtained; the
current calibration represents I nA. The resting potential of the brainstem neuron was initially---63 mV and declined to --46 mV after injection of 214 nC of depolarizing current over a period of 16 min; the membrane potential was returned to ---63 mV while recording the action potential illustrated in the lower record. The current calibration is 0.5 nA. The resting potential of the visual cortex neuron was initially--74 mV and increased to --76 mV after 2.5 min at which time the lower record was obtained; the current calibration is 0.5 hA. All data was stored on tape, digitized at 2-5 KHz by a PDP 11/23 computer and plotted on Hewlet Packard 77OT digital plotter. experiments p e r f o r m e d with potassium acetate filled microelectrodes (Fig. 1). In many neurons the width of action potentials increased gradually over a p e r i o d of 5-20 min after the neuron was first impaled. This slow increase in action potential duration was particularly obvious when recording with fine tip (high resistance) microelectrodes. I n d e e d when thin wall glass was used to p r e p a r e lower resistance microelectrodes the increase in action potential duration occurred m o r e rapidly. This suggests that the intracellular accumulation of Lucifer Yellow/lithium acetate electrolyte leaking from the m i c r o e l e c t r o d e was a possible cause. F u r t h e r experiments to investigate the mechanism of this effect were p e r f o r m e d on D R G neurons in culture. A c t i o n potential duration also increased when intracellular recording was p e r f o r m e d using microelectrodes filled with 1 M lithium acetate alone, suggesting that Lucifer Yellow was not responsible. Mi-
croiontophoretic injections of lithium from high resistance electrodes (greater than 100 M Q ) were used to increase the intracellular lithium concentration in a m o r e controlled manner, such that the effect of lithium injection on resting potential, m e m b r a n e resistance (chord conductance), and action potential duration could be m e a s u r e d after each of several lithium injections into a single neuron (Fig. 2A). Such experiments were p e r f o r m e d successfully on 6 D R G neurons. Lithium was injected with a current of 0.5 n A for periods of 30--60 s, and b e t w e e n injections the m e m b r a n e potential was allowed to settle for a further 60-70 s before m e a s u r e m e n t s of m e m b r a n e p a r a m e t e r s were made. In view of uncertainty concerning the transport n u m b e r for lithium i o n o p h o r e sis, and values for diffusional and hydrostatic efflux of lithium, calculation of the intracellular lithium concentration achieved after each injection seems unwarranted. Some microelectrodes a p p a r e n t l y re-
175 leased little lithium in that no physiological effect was recorded (such microelectrodes had poor electrical characteristics, and blocked during current injection), while others leaked lithium so rapidly that further injection by microionophoresis was pointless. In successful experiments initial injections of lithium '15-30 nC) had little action on resting potential but increased input chord conductance (measured
from hyperpolarizing electrotonic potentials evoked by constant current pulses) by 6--64%; action potential duration measured at 1/2 amplitude increased by 54-65% (see Fig. 2A). Further injections of lithium (60-120 nC total dose) depolarized the membrane potential by 2-23 mV, increased the input chord conductance to 114-234% of resting values and evoked further increases in action potential duration to val-
A
0 nC LITHIUM Vm-62
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~
60nC LITHIUH
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120 nC LITHIUM
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~.~ 1-62 --,
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Fig. 2. Intracellular iontophoresis of lithium ions increases action potential duration. In A the dose-dependent action of lithium, injected ionophoretically with a current of 0.5 nA, was recorded in a dorsal root ganglion neuron impaled with a microelectrode containing 1 M lithium acetate (resistance 120 MQ). Each record shows an action potential evoked by a brief depolarizing current pulse, and a hyperpolarizing electrotonic potential evoked by a 0.2 nA current pulse. Before injection of lithium the resting potential (rmp) was ---62 mV, the input resistance 156 M ~ , the action potential duration at l/z amplitude 5.8 ms and the action potential amplitude 89 mV. These values changed with the cumulative dose of current used to inject lithium as follows: 30 nC lithium - - rmp ----61 mV, input resistance 155 Mff~, duration 9.6 ms, amplitude 79 mV; 60 nC lithium - - rmp ---60 mV, input resistance 151 Mr2, duration 13.3 ms, amplitude 72 mV; 90 nC lithium - - rmp --59 mV, input resistance 139 MQ, duration 17.5 ms, amplitude 67 mV; 120 nC lithium - - rmp - 49 mV, input resistance 113 Mf~, duration 52 ms, amplitude 58 mV. When DC current injection w~tsused to restore the membrane potential to -----62mV after injection of 120 nC lithium the action potential duration was reduced to 41 ms but did not return to control values. B1 shows an action potential recorded from a dorsal root ganglion neurone impaled with a microelectrode filled with 1 M cesium chloride; note the initial overshoot preceding the plateau phase. In contrast B2 and B3 show responses recorded from a neuron impaled with a microelectrode containing 1 M lithium acetate; B3 was obtained during perfusion with the calcium channel blocker cadmium chloride (0.2 mM). Note that cadmium shortens the action potential and also reduces the amplitude and duration of the depolarizing after potential. Data was digitized at 5 kHz by a PDP 11/23 computer.
176 ues of 28-52 ms (an increase of 552-796% over control values). These changes were maintained for as long as intracellular recording could be continued. Hyperpolarizing the membrane potential to control values recorded before injection of lithium reduced action potential widening (see Fig. 2A), but only to durations of 26-41 ms, which are considerably in excess of control values. This voltage sensitivity of the action potential duration is probably due to relief of inactivition of both transient outward current 13 and the delayed rectifier 1,5 both of which may contribute to action potential repolarization. However, the major action of lithium occurs via some mechanism other than a change in membrane potential alone. In addition to the above-described effects lithium injection also reduced the overshoot and rate of rise of D R G neuron action potentials, producing characteristically rounded spikes. In contrast intracellular injection of cesium, a potassium channel blocker 7, produces wide action potentials with an initial rapid upstroke and overshoot preceding a fast repolarizing phase followed by a cardiac-like action potential plateau (see Fig. 2B, 1). The spike afterhyperpolarization is also characteristically altered after injection of lithium, being initially reduced in amplitude, and after further injection of lithium inverted to a depolarizing after potential (see Fig. 2A). Since the action potential of D R G neurons normally represents a finely tuned balance between inward sodium and calcium and outward potassium currentsS,17, an action of lithium on any of these parameters could prolong the action potential duration. In 7 tests on 5 neurons previously injected with lithium, and held at a membrane potential o f - - 8 0 mV so that the depolarizing spike after potential was also clearly recorded, application of the calcium channel blocker cadmium3,12 reversibly reduced both the action potential duration and the amplitude and duration of the spike afterdepolarization (Fig. 2B, 3). The result of this experiment suggests that lithium alters the balance between calcium and potassium currents. Spike broadening does not appear to result from an increased sodium current. Indeed reduction of the overshoot and rate of rise of the initial component of the action potential that occurs following lithium injection suggests a reduced sodium current, as would occur if lithium was permeable through sodium chan-
nels lo and sufficient intracellular lithium accumulation occurred to reduce the driving force for inward sodium current. Several hypothesis may be put forward to explain the action of lithium. Reversal of the spike after potential, from hyperpolarizing to depolarizing, suggests a change of the potassium equilibrium potential. Since the sodium pump does not transport lithium11, injection of this ion results in an exchange of intracellular cations, principally potassium, for lithium. This reduces the driving force for outward potassium current and hence delayed rectification. This alone would tend to increase the width of action potentials. In addition in squid axons sodium and lithium ions can enter and block potassium channels 7, this also reduces outward rectification. It is plausible that lithium may also directly enhance inward calcium current. However experiments on invertebrate neurons 4 and the mammalian neuromuscular junction6 do not support this, but suggest that lithium impairs regulation of intracellular calcium activity such that cells become calcium loaded. Since inactivation of calcium channels occurs when the cytosolic calcium concentration rises above physiological values 9, calcium loading would be expected to reduce inward calcium current, and hence decrease the duration of action potentials with a major calcium component. In addition the rise of intracellular calcium activity caused by injection of lithium may be the cause of the increase in membrane potassium permeability produced by lithium in snail neurons 16. The complexity of action of lithium is clearly beyond the resolution of the simple current clamp experiments described herein, but our results suggest that leakage of lithium ions from Lucifer Yellow microelectrodes has serious implications for electrophysiological experiments on small mammalian neurons. Previous studies with Lucifer Yellow failed to report effects on the electrophysiological behavior of larger invertebrate neurons. It is probable that the larger cytoplasmic volume of invertebrate cells more effectively buffers microelectrode electrolyte leakage during intracellular recording. The alteration in electrophysiological activity that we describe may occur less frequently when microelectrodes are filled with a low concentration (5%) aqueous solution of Lucifer Yellow. However the electrical properties of such
177 e l e c t r o d e s c o m p r o m i s e s e l e c t r o p h y s i o l o g i c a l experi-
m i d e , a dye which binds to Nissl s u b s t a n c e , and sul-
m e n t s b e c a u s e t h e s e e l e c t r o d e s h a v e high resistance,
f o r h o d a m i n e 101 h a v e r e c e n t l y b e e n u s e d as fluores-
p o o r c u r r e n t passing capability and are electrically
cent m a r k e r s
noisy. L u c i f e r Y e l l o w m i c r o g r a p h s with e x t e n s i v e d e n -
(ref. 2 and P. B. G u t h r i e , p e r s o n a l c o m m u n i c a t i o n ) . B o t h are soluble in p o t a s s i u m a c e t a t e and m a y p r o v e
dritic and a x o n a l de'tail m a y b e o b t a i n e d f r o m n e u -
to be useful a l t e r n a t i v e s to L u c i f e r Yellow.
in e l e c t r o p h y s i o l o g i c a l
experiments
rons with p o o r resting p o t e n t i a l s i n d i c a t i v e of i m p a l e m e n t d a m a g e ( u n p u b l i s h e d o b s e r v a t i o n s ) . Such cells w o u l d not n o r m a l l y be u s e d for e l e c t r o p h y s i o l o g i c a l analysis, but p r o v i d e useful a n a t o m i c a l data. O u r observations a r o s e w h e n we t r i e d to c o m b i n e intracellular e l e c t r o p h y s i o l o g i c a l e x p e r i m e n t s with m o r p h o -
M . L . M . is a B e i t M e m o r i a l Fellow. W e t h a n k J. A . Wilson for p r e p a r i n g the cultures. S u p p o r t e d M R C G r a n t s G 8217750N ( M . L . M . ) and
by G
8219655N ( V . C . ) . J . A . K . thanks Smith, Kline and F r e n c h for s u p p o r t .
logical study of the s a m e n e u r o n s . E t h i d i u m bro-
1 Adams, P. R., Brown, D. A. and Constanti, A., M-Currents and other potassium currents in bullfrog sympathetic neurones, J. Physiol. (Lond.), 330 (1982) 537-572. 2 Aghajanian, G. K. and Vandermaelen, C. P., Intracellular identification of central noradrenergic and serotoninergic neurons by anew double labelling technique, J. Neurosci., 2 (1982) 1786--1792. 3 Akaike, N., Lee, K. S. and Brown, A. M., The calcium current of Helix neuron, J. gen. Physiol., 71 (1978) 509-531. 4 Aldenhoff, B. and Lux, H. D., Effects of lithium on calcium-dependent membrane properties and on intracellular calcium-concentration in Helix neurones. In H. M. Emerich, J. B. Aldenhoff and H. D. Lux (Eds.), Basic Mechanisms in the Action of Lithium, Excepta Medica, Amsterdam, 1982, pp. 50-63. 5 Aldrich, R. W., Getting, P. A. and Thompson, S. H., Inactivation of delayed outward current in molluscan neurone somata, J. Physiol. (Lond.), 291 (1979) 507-530. 6 Balnave, R..J. and Gage, P. W., On facilitation of transmitter release at the toad neuromuscular junction, J. Physiol. (Lond.), 239 (1974) 657-675. 7 Benzanilla, F. and Armstrong, C. M., Negative conductance caused by entry of sodium and cesium into the potassium channels of squid axons, J. gen. Physiol., 60 (1972) 588-608. 8 Dunlap, K. and Fischbach, G. D., Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones, J. Physiol. (Lond.), 317 (1981) 519-535. 9 Eckert, R. and Tillotson, D. L., Calcium-mediated inactivation of the calcium conductance in caesium loaded giant neurons of aplysia californica, J. Physiol. (Lond.), 314 (1981) 265-280. 10 Hodgkin, A. L. and Katz, B., The effect of sodium ions on the electrical activity of the giant axon of the squid, J. Physiol. (Lond.), 108 (1949) 37-77.
11 Keynes, R. D. and Swan, R. C., The effect of external sodium concentration on the sodium fluxes in frog skeletal muscle, J. Physiol. (Lond.), 147 (1959) 591-625. 12 Khrishtal, O. A., Blocking action of cadmium ions on calcium channels in the membrane of nerve cells, Dokl. Akad. Nauk. S.S.S.R., 231 (1976) 1003-1005. 13 Kostyuk, P. G., Veselovsky, N. S., Fedulova, S. A. and Tsyndrenko, A. V., Ionic currents in the somatic membrane of rat dorsal root ganglion neurones. III. Potassium currents, Neuroscience, 6 (1981) 2439-2444. 14 MacVicar, B. A. and Dudek, F. E., Dye-coupling between CA 3 pyramidal cells in slices of rat hippocampus, Brain Research, 196 (1980) 494-497. 15 Miller, J. P. and Selverston, A. I., Rapid killing of single neurons by irradiation of intracellularly injected dye, Science, 206 (1979) 702-704. 16 Partridge, L. D. and Thomas, R. C., The effects of lithium and sodium on the potassium conductance of snail neurones, J. Physiol. (Lond.), 254 (1976) 551-563. 17 Ransom, B. R. and Holz, R. W., Ionic determinants of excitability in cultured mouse dorsal root ganglion and spinal cord cells, Brain Research, 136 (1977) 445-453. 18 Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N. and Nelson, P. G., Mouse spinal cord in cell culture. 1. Morphology and intrinsic neuronal electrophysiological properties, J. Neurophysiol., 43 (1977) 1132-1150. 19 Stewart, W. W., Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer, Cell, 14 (1978) 741-759. 20 Stewart, W. W., Lucifer dyes - - highly fluorescent dyes for biological tracing, Nature (Lond.), 292 (1981) 17-21. 21 Shafer, M. R. and Calabrese, R. L., Similarities and differences in the structure of segmentally homologous neurones that control the heart in the leech, hirudo medicinalis, Cell. Tissue Res., 214 (1981) 137-153.
173
BRE 20023
Lithium ions increase action potential duration of mammalian neurons MARK L. MAYER l, VINCENZO CRUNELLP and JOHN A. KEMP2 1Department of Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, London SWI 7 ORE and 2Neurochemical Pharmacology Unit, MRC Centre, Hills Road, Cambridge CB2 2QD (U.K.)
(Accepted October 11th, 1983) Key words: lithium - - action potential - - intracellular recording - - calcium current
During Lucifer Yellow staining of mammalian neurons, intracellular recording revealed a prolongation of the action potential that is probably the result of leakage of lithium ions into the intracellular fluid. In experiments on dorsal root ganglion neurons intracellular iontophoresis of lithium ions also broadened the action potential. Cadmium, a calcium channel blocker, shortens the lithium-evoked wide actions potentials. The present experiments do not reveal whether lithium directly enhances inward calcium current, or whether a block of outward rectification allows calcium currents to increase the action potential duration. The 4-aminonapthalimide dye, Lucifer Yellow CH, is a useful fluorescent marker when used in conjunction with intracellular microelectrodes to stain individual neurons from which electrical activity is being recorded19,20. The low solubility of the potassium salt of Lucifer Yellow limits the concentration of dye that can be used to fill microelectrodes and this reduces the intensity of staining achievable under experimental conditions. Thus Lucifer Yellow is prepared as the highly soluble lithium salt 19. To further improve the electrical characteristics of fine tip microelectrodes required for recording from small mammalian neurons, Lucifer Yellow has been dissolved in 1-2 M lithium acetate or lithium chloride 14,19,21. When using such electrodes for combined morphological and electrophysiological study of neurons in cultures and brain slices prepared from several regions of the mammalian nervous system we recorded marked changes in action potential duration during prolonged stable intracellular recording. This seriously compromised characterization of the electrophysiological properties of the neurons to be studied morphologically. O u r results suggest that leakage of lithium ions into the cytosol is responsible. The effects we describe are quite different from the photo-
inactivition of neurons that occurs on irradiation of dye-labeled cells 15. Cultures of rat dorsal root ganglion ( D R G ) neurons were prepared from 14-16-day-old rat embryos and grown in dissociated culture for up to 12 weeks using methods similar to those described previously 18. The recording medium contained (raM) NaCI 142, KC1 4.7, CaCI2 3, MgC12 2, glucose 9.5, phenol red 0.01 mg/ml and was buffered to p H 7.3 with 5 mM HEPES. Intracellular recording was performed at room temperature on the stage of a phase contrast microscope. Brain slices from the dorsal raphe region of the brainstem and the visual cortex were prepared using a Mcllwain tissue chopper and maintained in vitro in continually gassed (95% 0 2, 5% CO2) medium containing (mM) NaCI 134, KC1 5, KH2PO 4 1.25, MgSO4 2, CaCI 2 2, N a H C O 3 16 and glucose 10. Intracellular recording was performed at 37 °C. Microelectrodes were filled with a 5% solution of Lucifer Yellow C H dissolved in 1 M lithium acetate titrated to pH 7.0 with acetic acid. In each of the 3 preparations examined neurons impaled with Lucifer Yellow/lithium acetate microelectrodes generated action potentials that were unusually long compared to those recorded in control
Correspondence: M. L. Mayer, Department of Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, London SWl7 ORE, U.K.
174
VISUAL CORTEX
BRAINSTEM
GANGL I0 N
50 mV -G
] I
I
I
I
5L_
.
.
.
.
.
I
I
I
, m
.
20ms
]
50ms
I
__
I
._3 |
J
25rns
Fig. 1. Action potential duration increases during intracellular recording with Lucifer Yellow filled electrodes. Actions potentials were recorded from neurons in preparations from 3 areas of the rat nervous system using microelectrodes filled with 5% Lucifer Yellow dissolved in ] M lithium acetate. In each case the upper record shows a response evoked immediately after a stable recording was obtained. The lower records show responses recorded from the s a m e neurons later in the experiment. The resting potential of the dorsal root ganglion neuron was initally--60 mV, and dechned to ~ 4 0 m V after 38 rain, at which time the lower record was obtained; the
current calibration represents I nA. The resting potential of the brainstem neuron was initially---63 mV and declined to --46 mV after injection of 214 nC of depolarizing current over a period of 16 min; the membrane potential was returned to ---63 mV while recording the action potential illustrated in the lower record. The current calibration is 0.5 nA. The resting potential of the visual cortex neuron was initially--74 mV and increased to --76 mV after 2.5 min at which time the lower record was obtained; the current calibration is 0.5 hA. All data was stored on tape, digitized at 2-5 KHz by a PDP 11/23 computer and plotted on Hewlet Packard 77OT digital plotter. experiments p e r f o r m e d with potassium acetate filled microelectrodes (Fig. 1). In many neurons the width of action potentials increased gradually over a p e r i o d of 5-20 min after the neuron was first impaled. This slow increase in action potential duration was particularly obvious when recording with fine tip (high resistance) microelectrodes. I n d e e d when thin wall glass was used to p r e p a r e lower resistance microelectrodes the increase in action potential duration occurred m o r e rapidly. This suggests that the intracellular accumulation of Lucifer Yellow/lithium acetate electrolyte leaking from the m i c r o e l e c t r o d e was a possible cause. F u r t h e r experiments to investigate the mechanism of this effect were p e r f o r m e d on D R G neurons in culture. A c t i o n potential duration also increased when intracellular recording was p e r f o r m e d using microelectrodes filled with 1 M lithium acetate alone, suggesting that Lucifer Yellow was not responsible. Mi-
croiontophoretic injections of lithium from high resistance electrodes (greater than 100 M Q ) were used to increase the intracellular lithium concentration in a m o r e controlled manner, such that the effect of lithium injection on resting potential, m e m b r a n e resistance (chord conductance), and action potential duration could be m e a s u r e d after each of several lithium injections into a single neuron (Fig. 2A). Such experiments were p e r f o r m e d successfully on 6 D R G neurons. Lithium was injected with a current of 0.5 n A for periods of 30--60 s, and b e t w e e n injections the m e m b r a n e potential was allowed to settle for a further 60-70 s before m e a s u r e m e n t s of m e m b r a n e p a r a m e t e r s were made. In view of uncertainty concerning the transport n u m b e r for lithium i o n o p h o r e sis, and values for diffusional and hydrostatic efflux of lithium, calculation of the intracellular lithium concentration achieved after each injection seems unwarranted. Some microelectrodes a p p a r e n t l y re-
175 leased little lithium in that no physiological effect was recorded (such microelectrodes had poor electrical characteristics, and blocked during current injection), while others leaked lithium so rapidly that further injection by microionophoresis was pointless. In successful experiments initial injections of lithium '15-30 nC) had little action on resting potential but increased input chord conductance (measured
from hyperpolarizing electrotonic potentials evoked by constant current pulses) by 6--64%; action potential duration measured at 1/2 amplitude increased by 54-65% (see Fig. 2A). Further injections of lithium (60-120 nC total dose) depolarized the membrane potential by 2-23 mV, increased the input chord conductance to 114-234% of resting values and evoked further increases in action potential duration to val-
A
0 nC LITHIUM Vm-62
~
30 nC LITHIUH -
~
60nC LITHIUH
0
-
- 60mV I
_ -
90nC LITHI?H
120 nC LITHIUM
[ O.SoA
_ 120nC LITHIUH
~...m~ ~
~.~ 1-62 --,
gl
B2
- 60
mV
-~ [ 0.SnA
B3
_
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0
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-,.
]0-5 nA
20ms
A
!
Fig. 2. Intracellular iontophoresis of lithium ions increases action potential duration. In A the dose-dependent action of lithium, injected ionophoretically with a current of 0.5 nA, was recorded in a dorsal root ganglion neuron impaled with a microelectrode containing 1 M lithium acetate (resistance 120 MQ). Each record shows an action potential evoked by a brief depolarizing current pulse, and a hyperpolarizing electrotonic potential evoked by a 0.2 nA current pulse. Before injection of lithium the resting potential (rmp) was ---62 mV, the input resistance 156 M ~ , the action potential duration at l/z amplitude 5.8 ms and the action potential amplitude 89 mV. These values changed with the cumulative dose of current used to inject lithium as follows: 30 nC lithium - - rmp ----61 mV, input resistance 155 Mff~, duration 9.6 ms, amplitude 79 mV; 60 nC lithium - - rmp ---60 mV, input resistance 151 Mr2, duration 13.3 ms, amplitude 72 mV; 90 nC lithium - - rmp --59 mV, input resistance 139 MQ, duration 17.5 ms, amplitude 67 mV; 120 nC lithium - - rmp - 49 mV, input resistance 113 Mf~, duration 52 ms, amplitude 58 mV. When DC current injection w~tsused to restore the membrane potential to -----62mV after injection of 120 nC lithium the action potential duration was reduced to 41 ms but did not return to control values. B1 shows an action potential recorded from a dorsal root ganglion neurone impaled with a microelectrode filled with 1 M cesium chloride; note the initial overshoot preceding the plateau phase. In contrast B2 and B3 show responses recorded from a neuron impaled with a microelectrode containing 1 M lithium acetate; B3 was obtained during perfusion with the calcium channel blocker cadmium chloride (0.2 mM). Note that cadmium shortens the action potential and also reduces the amplitude and duration of the depolarizing after potential. Data was digitized at 5 kHz by a PDP 11/23 computer.
176 ues of 28-52 ms (an increase of 552-796% over control values). These changes were maintained for as long as intracellular recording could be continued. Hyperpolarizing the membrane potential to control values recorded before injection of lithium reduced action potential widening (see Fig. 2A), but only to durations of 26-41 ms, which are considerably in excess of control values. This voltage sensitivity of the action potential duration is probably due to relief of inactivition of both transient outward current 13 and the delayed rectifier 1,5 both of which may contribute to action potential repolarization. However, the major action of lithium occurs via some mechanism other than a change in membrane potential alone. In addition to the above-described effects lithium injection also reduced the overshoot and rate of rise of D R G neuron action potentials, producing characteristically rounded spikes. In contrast intracellular injection of cesium, a potassium channel blocker 7, produces wide action potentials with an initial rapid upstroke and overshoot preceding a fast repolarizing phase followed by a cardiac-like action potential plateau (see Fig. 2B, 1). The spike afterhyperpolarization is also characteristically altered after injection of lithium, being initially reduced in amplitude, and after further injection of lithium inverted to a depolarizing after potential (see Fig. 2A). Since the action potential of D R G neurons normally represents a finely tuned balance between inward sodium and calcium and outward potassium currentsS,17, an action of lithium on any of these parameters could prolong the action potential duration. In 7 tests on 5 neurons previously injected with lithium, and held at a membrane potential o f - - 8 0 mV so that the depolarizing spike after potential was also clearly recorded, application of the calcium channel blocker cadmium3,12 reversibly reduced both the action potential duration and the amplitude and duration of the spike afterdepolarization (Fig. 2B, 3). The result of this experiment suggests that lithium alters the balance between calcium and potassium currents. Spike broadening does not appear to result from an increased sodium current. Indeed reduction of the overshoot and rate of rise of the initial component of the action potential that occurs following lithium injection suggests a reduced sodium current, as would occur if lithium was permeable through sodium chan-
nels lo and sufficient intracellular lithium accumulation occurred to reduce the driving force for inward sodium current. Several hypothesis may be put forward to explain the action of lithium. Reversal of the spike after potential, from hyperpolarizing to depolarizing, suggests a change of the potassium equilibrium potential. Since the sodium pump does not transport lithium11, injection of this ion results in an exchange of intracellular cations, principally potassium, for lithium. This reduces the driving force for outward potassium current and hence delayed rectification. This alone would tend to increase the width of action potentials. In addition in squid axons sodium and lithium ions can enter and block potassium channels 7, this also reduces outward rectification. It is plausible that lithium may also directly enhance inward calcium current. However experiments on invertebrate neurons 4 and the mammalian neuromuscular junction6 do not support this, but suggest that lithium impairs regulation of intracellular calcium activity such that cells become calcium loaded. Since inactivation of calcium channels occurs when the cytosolic calcium concentration rises above physiological values 9, calcium loading would be expected to reduce inward calcium current, and hence decrease the duration of action potentials with a major calcium component. In addition the rise of intracellular calcium activity caused by injection of lithium may be the cause of the increase in membrane potassium permeability produced by lithium in snail neurons 16. The complexity of action of lithium is clearly beyond the resolution of the simple current clamp experiments described herein, but our results suggest that leakage of lithium ions from Lucifer Yellow microelectrodes has serious implications for electrophysiological experiments on small mammalian neurons. Previous studies with Lucifer Yellow failed to report effects on the electrophysiological behavior of larger invertebrate neurons. It is probable that the larger cytoplasmic volume of invertebrate cells more effectively buffers microelectrode electrolyte leakage during intracellular recording. The alteration in electrophysiological activity that we describe may occur less frequently when microelectrodes are filled with a low concentration (5%) aqueous solution of Lucifer Yellow. However the electrical properties of such
177 e l e c t r o d e s c o m p r o m i s e s e l e c t r o p h y s i o l o g i c a l experi-
m i d e , a dye which binds to Nissl s u b s t a n c e , and sul-
m e n t s b e c a u s e t h e s e e l e c t r o d e s h a v e high resistance,
f o r h o d a m i n e 101 h a v e r e c e n t l y b e e n u s e d as fluores-
p o o r c u r r e n t passing capability and are electrically
cent m a r k e r s
noisy. L u c i f e r Y e l l o w m i c r o g r a p h s with e x t e n s i v e d e n -
(ref. 2 and P. B. G u t h r i e , p e r s o n a l c o m m u n i c a t i o n ) . B o t h are soluble in p o t a s s i u m a c e t a t e and m a y p r o v e
dritic and a x o n a l de'tail m a y b e o b t a i n e d f r o m n e u -
to be useful a l t e r n a t i v e s to L u c i f e r Yellow.
in e l e c t r o p h y s i o l o g i c a l
experiments
rons with p o o r resting p o t e n t i a l s i n d i c a t i v e of i m p a l e m e n t d a m a g e ( u n p u b l i s h e d o b s e r v a t i o n s ) . Such cells w o u l d not n o r m a l l y be u s e d for e l e c t r o p h y s i o l o g i c a l analysis, but p r o v i d e useful a n a t o m i c a l data. O u r observations a r o s e w h e n we t r i e d to c o m b i n e intracellular e l e c t r o p h y s i o l o g i c a l e x p e r i m e n t s with m o r p h o -
M . L . M . is a B e i t M e m o r i a l Fellow. W e t h a n k J. A . Wilson for p r e p a r i n g the cultures. S u p p o r t e d M R C G r a n t s G 8217750N ( M . L . M . ) and
by G
8219655N ( V . C . ) . J . A . K . thanks Smith, Kline and F r e n c h for s u p p o r t .
logical study of the s a m e n e u r o n s . E t h i d i u m bro-
1 Adams, P. R., Brown, D. A. and Constanti, A., M-Currents and other potassium currents in bullfrog sympathetic neurones, J. Physiol. (Lond.), 330 (1982) 537-572. 2 Aghajanian, G. K. and Vandermaelen, C. P., Intracellular identification of central noradrenergic and serotoninergic neurons by anew double labelling technique, J. Neurosci., 2 (1982) 1786--1792. 3 Akaike, N., Lee, K. S. and Brown, A. M., The calcium current of Helix neuron, J. gen. Physiol., 71 (1978) 509-531. 4 Aldenhoff, B. and Lux, H. D., Effects of lithium on calcium-dependent membrane properties and on intracellular calcium-concentration in Helix neurones. In H. M. Emerich, J. B. Aldenhoff and H. D. Lux (Eds.), Basic Mechanisms in the Action of Lithium, Excepta Medica, Amsterdam, 1982, pp. 50-63. 5 Aldrich, R. W., Getting, P. A. and Thompson, S. H., Inactivation of delayed outward current in molluscan neurone somata, J. Physiol. (Lond.), 291 (1979) 507-530. 6 Balnave, R..J. and Gage, P. W., On facilitation of transmitter release at the toad neuromuscular junction, J. Physiol. (Lond.), 239 (1974) 657-675. 7 Benzanilla, F. and Armstrong, C. M., Negative conductance caused by entry of sodium and cesium into the potassium channels of squid axons, J. gen. Physiol., 60 (1972) 588-608. 8 Dunlap, K. and Fischbach, G. D., Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones, J. Physiol. (Lond.), 317 (1981) 519-535. 9 Eckert, R. and Tillotson, D. L., Calcium-mediated inactivation of the calcium conductance in caesium loaded giant neurons of aplysia californica, J. Physiol. (Lond.), 314 (1981) 265-280. 10 Hodgkin, A. L. and Katz, B., The effect of sodium ions on the electrical activity of the giant axon of the squid, J. Physiol. (Lond.), 108 (1949) 37-77.
11 Keynes, R. D. and Swan, R. C., The effect of external sodium concentration on the sodium fluxes in frog skeletal muscle, J. Physiol. (Lond.), 147 (1959) 591-625. 12 Khrishtal, O. A., Blocking action of cadmium ions on calcium channels in the membrane of nerve cells, Dokl. Akad. Nauk. S.S.S.R., 231 (1976) 1003-1005. 13 Kostyuk, P. G., Veselovsky, N. S., Fedulova, S. A. and Tsyndrenko, A. V., Ionic currents in the somatic membrane of rat dorsal root ganglion neurones. III. Potassium currents, Neuroscience, 6 (1981) 2439-2444. 14 MacVicar, B. A. and Dudek, F. E., Dye-coupling between CA 3 pyramidal cells in slices of rat hippocampus, Brain Research, 196 (1980) 494-497. 15 Miller, J. P. and Selverston, A. I., Rapid killing of single neurons by irradiation of intracellularly injected dye, Science, 206 (1979) 702-704. 16 Partridge, L. D. and Thomas, R. C., The effects of lithium and sodium on the potassium conductance of snail neurones, J. Physiol. (Lond.), 254 (1976) 551-563. 17 Ransom, B. R. and Holz, R. W., Ionic determinants of excitability in cultured mouse dorsal root ganglion and spinal cord cells, Brain Research, 136 (1977) 445-453. 18 Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N. and Nelson, P. G., Mouse spinal cord in cell culture. 1. Morphology and intrinsic neuronal electrophysiological properties, J. Neurophysiol., 43 (1977) 1132-1150. 19 Stewart, W. W., Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer, Cell, 14 (1978) 741-759. 20 Stewart, W. W., Lucifer dyes - - highly fluorescent dyes for biological tracing, Nature (Lond.), 292 (1981) 17-21. 21 Shafer, M. R. and Calabrese, R. L., Similarities and differences in the structure of segmentally homologous neurones that control the heart in the leech, hirudo medicinalis, Cell. Tissue Res., 214 (1981) 137-153.