Sodium and Calcium Currents in Dispersed Mammalian Septal Neurons

Sodium and Calcium Currents in Dispersed Mammalian Septal Neurons ANTONIO CASTELLANO and Jos~ L6PEZ-BARNEO From the Departamento de Fisiologfa y Bioflsica, Facultad de Medicina, 41009 Sevilla, Spain A B S T R A C T Voltage-gated Na ÷ and Ca 2÷ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na + current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 _ 0.18 ms and maximal activation of Na + conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 -+ 32 nS (~ 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 _+ 0.09 ms at 0 inV. Recovery from inactivation was complete in ~900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na ÷ tail currents had a single time constant that at -80 mV was faster than 100 p.s. Depolarization of septal neurons also elicited a Ca ~+ current that peaked in ~ 6-8 ms. Maximal peak Ca ~÷ current was obtained at 20 mV, and with 10 mM external Ca ~+ the amplitude was 0.35 - 0.22 hA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca ~+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 -+ 0.2 and 0.11 +_ 0.03 ms (25°C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons. INTRODUCTION Mammalian central neurons possess intrinsic electrical properties that make them able to generate complex firing patterns. This implies the existence of a variety of membrane ionic conductances which, with a few exceptions, are poorly characterized. Some properties of Na + channels have recently been studied in dissociated hippocampal (Sah et al., 1988) and neocortical (Huguenard et al., 1988) neurons, as well as in Purkinje cells of organotypic cerebellar cultures (G~ihwiler and Llano, 1989), but additional work is still needed to fully investigate the biophysical properties of cerebral Na ÷ channels and to ascertain whether their characteristics are homogeneous in different brain regions. Ca ~+ channels, on the other hand, have been Address reprint requests to Dr. J. L6pez-Bameo, Departamento de Fisiologia Mrdica y Bioffsica, Facuhad de Medicina, Avda. S~inchez Pizjufin, 4, 41009 Sevilla, Spain. J. GEN. PHYSIOL. © The Rockefeller University Press • 0022-1295/91/02/0303/18 $2.00 Volume 97 February 1991 303-320 303 on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 304 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 . 1991 intensively studied in neurons of the peripheral nervous system (Fedulova et al., 1985; Carbone and Lux, 1987; Fox et al., 1987; Swandulla and Armstrong, 1988), but within the brain they have been only partially characterized (Heru~indez-Cruz and Pape, 1989; Meyers and Barker, 1989). We have previously shown that intracellularly recorded septal neurons in in vitro brain slices display differential electrical responses due to the variability in the distribution of membrane ionic conductances (L6pez-Barneo et al., 1985; Alvarez de Toledo and L6pez-Bameo, 1988). This article continues our preceding work and focuses on the characterization of the ionic currents of freshly dissociated septal neurons from adult guinea pigs. Due to their size and regular geometry these cells provide an excellent preparation for studying the voltage-activated ionic channels in a well-clamped neuronal membrane using the whole-ceU variant of the patch-clamp technique (Hamill et al., 1981). This research not only has biophysical interest, it is also of importance because an accurate analysis TABLE I Compositien of Solutions NaCl KC! CaClz MgCi~ BaCI~ CoCI 2 Trizma* HEPES Ex~rnal 130 Na 130 2.7 2.5 1 -- -- -- 10 80 Na 80 2.7 2.5 1 -- -- 70 -- I0 Ca 70 2.7 10 -- -- -- 60 -- 10 Ba 70 2.7 -- -- I0 -- 60 -- Mg/Co 70 2.7 1 7.5 -- 1.5 60 -- NaCI KCI K-glutama~ KF MgCI, CsCI CsF HEPES EGTA In~rnal 130 K -- 30 80 20 2 -- -- 10 5 130 Cs .... 2 110 20 10 5 40 Na 40 -- -- -- 2 70 20 10 5 All values are given in miUimolar. pH was 7.4 in external and 7.3 to 7.35 in internal solutions *Tris (hydroxymethyl)aminomethane (pH = 7.4). of the ionic conductances of central neurons is required before undertaking the investigation of their modulation by neuromodulators and drugs. This article concentrates on the description of the properties of Na ÷ and Ca ~÷ currents; the K ÷ current is only briefly presented and therefore not studied in detail. METHODS Cells were obtained from coronal slices, 300-400 p~m thick, of the adult guinea pig septal region. The procedure followed for slicing was the same as previously described (Alvarez de Toledo and L6pez-Bameo, 1988). For cell dissociation, six slices containing only the septal nucleus were placed in a vial with 3 ml of Earle solution (Flow Laboratories, Inc., McLean, VA) containing 0.2 mg/ml of trypsin type IX and 0.05 mg/ml of DNase type II (Sigma Chemical Co., St. Louis, MO). This solution was continuously bubbled with a mixture of 95% O~ and 5% CO2 and the tissue was incubated for 20 min at 37°C. After this time period the tissue was washed on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 CASTELLANO AND I.XSPEZ-BARNEO Na + and Ca 2+ Channels of Septal Neurons 305 twice and the enzymes were removed. Cells were mechanically dispersed using fire-polished Pasteur pipettes, centrifuged, and resuspended in MEM culture medium supplemented with glutamine, antibiotics, and bovine fetal serum. Cells were plated on fragments of glass coverslips treated with poly-L-lysine and maintained in an incubator until use (between 2 and 6 h after plating). The general procedures and the set up used for recording of whole-cell currents were the same as previously described (Urefia et al., 1989a). The composition of the recording solutions is given in Table I. Juncdon potentials were corrected once the electrode was in the bath and immediately before establishing a seal. In the text and in the figure legends solutions are indicated as external//internal. Experiments were performed at room temperature (20-25°C). For recording we used a patch-clamp amplifier built in our laboratory following the design of Sigworth (1983). A fast and uniform control of the membrane potential was favored by the use of low resistance electrodes (between 0.8 and 2 Mf~), a feedback resistance of only 100 Ml'l in the current-to-voltage converter, and electronic compensation of series resistance. With this recording configuration capacity transients typically had time constant values between 40 and 50 ~s. Clamp speed was further enhanced by ballistic charging of membrane capacitance (Matteson and Armstrong, 1984). Compensation of series resistance was done empirically; in most experiments we compensated for 1.5 times electrode resistance. An IBM-PC/AT computer interfaced to the analog electronics was used for pulse generation and for acquisition, display, storage, and analysis of the data. The interface and the associated software were developed and fabricated in our laboratory (Urefia et al., 1989b) and allowed us subtraction of the linear ionic and capacity currents. Sampling interval was either 10 or 20 Ws. Tail currents were fitted with one or the sum of two exponentials using a least-squares procedure. RESULTS Cells subjected to voltage-clamp had an almost spherical shape and no appreciable processes under the light microscope. The diameter of the dispersed neurons varied between 12 and 25 Ixm. Membrane capacitance, calculated from the time integral of the transient capacity currents, was 8 - 3 pF (mean _ SD, n = 87). Input resistance was 1.5 -+ 0.6 Gf/. Whole-Cdl Currents Fig. 1 A is a representative example of the major current components recorded in septal neurons during a step depolarization to 20 mV. At the beginning of the pulse the cell generates a fast inward current that peaks in less than 0.5 ms. In the course of the next 2 ms the current changes from inward to outward and rises slowly until the end of the pulse. On return to the holding potential there is an almost instantaneous jump in the current (which in this recording lasts less than 80 V,s) and a large tail current is observed. Fig. 1 B shows the current recorded after blockade of K + channels by internal Cs +. The outward current is suppressed, and after the initial fast inward component a second, more steady component is disclosed, which, when the pulse is turned off, is followed by the tail current. External addition of tetrodotoxin (TTX, 0.5-1 ~M) abolishes the fast inward current, while the steady component and the tail remain unaltered (Fig. 1 C). It will be shown later that both the slow inward current and the tail disappear after removal of external Ca 2+. These findings indicate that the different components of the whole-cell current are due to the activity of Na ÷, Ca 2÷, and K ÷ channels. The three current components were recorded in every neuron on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 306 : / "-: / ./ u THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 • 1991 B #. 2 ms C ,.-.. FIGURE 1. Major current components in septal neurons. The three current sweeps were recorded during 6-ms voltage steps to 20 mV from a holding potential of -80 mV. Solutions: 130 Na//130 K (A), 10 Ca//130 Cs (B and C). In C, 1 IxM TIXwas added to the external solution. studied (n > 150), although the amplitude of each varied from cell to cell. In the following sections we analyze the properties of Na + and Ca *+ currents. Na + Channels Activation and conductance-voltage relations. Na + current was studied in isolation in cells dialyzed with the 130 mM Cs +'solution and bathed in an external solution with 130 mM Na +, where 90% of the Ca ~+ was replaced by Mg ~+ and Co s+ (see Table I). In these solutions depolarization elicited large Na + currents that at 0 mV had a peak amplitude of 4.6 + 1.6 nA (n = 21). Assuming no membrane infolding, current density was 0.6 mAcm -~. Most experiments were, however, performed with the 80 Na external solution (Table I) to favor an accurate control of the membrane potential. Fig. 2 illustrates a current trace obtained in low external Na +. The recording is very representative of a well-controlled Na + current with fast activation 80 Na. TTX : ' l • - -80 Na • :" 1ms • i FIGURE 2. Na + current recorded on isolation during a voltage pulse from -80 to 0 mV. The arrows indicate the onset and the termination of the pulse. Solutions were 80 Nail130 Cs. The current was completely suppressed by 1 p,M TFX. on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 CASTELLANO AND LOPEZ-BARNEO Na + and Ca 2* Channels of Septal Neurons 307 and complete inactivation in 5-6 ms. The figure also shows that addition of TTX produced a complete blockade of the current. With 80 mM external Na ÷, peak current amplitude at 0 mVwas 2.8 - 1.3 nA (n = 49). Good control of the membrane potential was also indicated by a conductance-voltage curve with a sigmoid rising phase (not shown), and because currents of different amplitude recorded at the same membrane potential (as, for instance, those in Fig. 6A) had, after scaling, almost identical time courses. Fig. 3 A is a family of Na ÷ currents generated by depolarization to the indicated membrane potentials. In this experiment we added 40 mM Na + to the internal solution to obtain a well-defined reversal potential (EN=). Current is inward at /.~ • ~ -50 to +90 :"J / "" l ." / f :), f 2 nA ~j 2 ms B I M.nA f -40 -20 20 /40 _. I I I ._[ I -0.7 -1.4 -1.4 / -0.7 6O I VM,mV FIGURE 3. Na + I-V relations. (A) Family of current sweeps elicited by depolarizations to -50, -30, -10, 10, 30, 50, 70, and 90 mV from a holding potential of -80 inV. (B) Peak current amplitude (ordinate) at various membrane potentials (abscissa). Intersection of the continuous line with the abscissa gives the value of the reversal potential (31 mV). Solutions: 130 Na//40 Na. membrane potentials more negative than EN, and outward at more positive potentials. The current turns on rapidly and inactivates completely at all voltages. Activation kinetics speed up with membrane depolarization and half-time to peak current is 0.44 + 0.18 ms at -20 mV but only 0.21 --. 0.06 ms at +20 mV (n = 10). Fig. 3 B is the I-V curve obtained by measuring the peak current at various membrane potentials. This figure shows that a measurable current can be recorded at -50 mV and that the maximal peak current is obtained between -20 and -10 mV. The reversal potential of the current occurs at +31 mV, which is similar to the Na ÷ equilibrium potential calculated by the Nernst equation. This suggests that, as in other preparations (Fenwick et al., 1982; Matteson and Armstrong, 1984; Urefia et al., 1989a), the aqueous components of the cytosol equilibrate completely with the on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 308 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 97 • 1991 pipette solution. Na + conductance (gN:), calculated by the formula gN: ---- IN:~ (I'M- ENd) (Hodgkin and Huxley, 1952), was maximal at 0 mV or more positive voltages. The average value from four septal neurons was 91 - 32 nS (~ 11 mS cm-2), and half of the total conductance was activated at -35 mV. Inactivation. During a maintained depolarization the inactivation of Na + currents could be well fitted by a single exponential function. This is illustrated by the recording of Fig. 4 A. In the range between -20 and +60 mV inactivation time constant diminishes with membrane depolarization (Fig. 4 B) and at 0 mV it has an average value of 0.44 --+ 0.09 ms (n = 5). The voltage dependence of steady-state inactivation is illustrated in Fig. 5. In A it is B 0.6- E ~0,4- .~ 0,2- A 1ms 4._ i i i i i -40 -20 0 210 40 60 V M,mv FIGURE 4. Time course of Na + current inactivation. (A) Single sweep of a Na ÷ current recorded during a depolarization to 0 inV. A single exponential extrapolated to the onset of the pulse was fitted to inactivation time course. To facilitate comparison, the exponential is drawn superimposing the current trace. (B) Dependence of inactivation time constant on the pulse membrane potential. Circles are the mean and the bars the standard deviation of values from five cells. Solutions: Mg/Co//130 Cs. shown that the amplitude of the current recorded by a test depolarization to 0 mV depends on the membrane potential during a conditioning prepulse lasting 50 ms. Fig. 5 B shows the relation between the noninactivated fraction of channels (ordinate) and the prepulse membrane potential (abscissa) with mean values obtained from six cells. At -80 mV only a small (5-10%) fraction of the channels are inactivated, but all of them are inactivated at voltages more positive than -20 inV. The average half steady-state inactivation occurs at a membrane potential of about -50 mV; this parameter varied between -45 and -70 mV in the ceils studied. Recovery from inactivation was studied by applying a 20-ms conditioning pulse to 0 on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 CASTELLANO AND L6PEZ-BARNEO Na + and Ca 2+ Channels of Septal Neurons 309 mV to inactivate the Na ÷ current and, after a variable time interval at the holding potential, applying a test pulse to open the channels already deinactivated. The traces of Fig. 6 A are Na ÷ currents elicited by depolarization to 0 mV in the absence of prepulse (control) and 10, 25, and 900 ms after the prepulse; inactivation is almost completely removed in the course of 900 ms. In Fig. 6 B are shown the time courses of recovery from inactivation when the membrane potential during the interval is held at -80 (circles), -100 (squares), or -120 mV (triangles). Recovery is about six times faster at -120 mV than at -80 inV. Interestingly, at -80 mV the recovery of the last 15-20% of the current required as much as 900 ms. This indicates the presence of a fast inactivating component that recovers extremely slowly. Closing kinetics. Closing kinetics of mammalian neuronal Na + channels have not been previously studied. We often obtained recordings where the fast Na ÷ tail A -20 / • '" 1hA 1 ms B 1,0- <E0.5- -100 -60 -20 v M , mV FIGURE 5. Steady-state inactivation of Na ÷ currents. (A) Current sweeps during depolarizations to 0 mV preceded by a 50-ms prepulse to the indicated membrane potentials. Holding potential, -80 InV. Normalized fraction of peak current recorded during the pulse (ordinate) as a function of the prepulse membrane potential. Circles and bars are the mean and standard deviation values from five cells. The inset illustrates the pulse protocol. Solutions: Mg/Co//130 Cs. currents could be resolved. The protocol consisted of the application of a brief depolarization (0.3-0.5 ms) to open Na ÷ channels that ended at the time when the current was maximal (Fig. 7 A ). At the instant of repolarization the driving force for Na ÷ suddenly increases and thus a tail current is recorded. The decay of the tail reflects the progressive deactivation of the channels open during the pulse. In our experiments Na ÷ tail currents were always well fitted by a single exponential function (Fig. 7 B), which at -80 mV had a time constant faster than 100 Izs (n = 5). on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 310 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 . 1991 A /10 ms ~~ lnA control ~ ms >. 0.5 i i t I -// I i 25 50 75 100 250 9()0 t ime. ms FIGURE 6. Recovery from inactivation of Na ÷ channels. (el) Na + currents elicited by voltage steps to 0 mV at the indicated time intervals after a prepulse to 0 mV lasting 20 ms. The control trace was obtained in the absence of prepulse. Holding potential, -80 mV. (B) Normalized pulse current amplitude (ordinate) as a function of interval duration (abscissa). Membrane potentials during the prepulse were -80 mV (circles), -100 mV (squares), and -120 mV (triangles). Lines are fitted by eye. Solutions: Mg/Co// 130 Cs. Ca 2+ Channels Identification and I-V relations. In the presence of TTX depolarization elicited in all neurons a slowly inactivating inward current that was readily identified as a Ca ~+ current. Fig. 8 A is a typical recording of the slow inward current. This trace shows that the current turns on slowly and almost reaches a maximum by the end of the 6-ms pulse. On repolarization there is a tail current due to the increase in the electrochemical gradient for Ca 2÷ ions. As in most preparations studied, the current was larger when Ba 2+ ions instead of Ca 2÷ were used as charge carrier (Fig. 8 B). Current during the pulse and at the pulse end (tail) was suppressed after removal of A • ,. / loA :: 0,5 ms gle exponential with a time constant of 60 p~s. The exponential repolarization. Solutions: Mg/Co//130 Cs. FIGURE 7. Closing kinetics of cerebral Na ÷ channels. (A) Na + current recorded during a 0.5-ms voltage pulse (indicated between the arrows) to 0 mV from a holding potential of -80 inV. When the pulse is turned off a fast tail current is recorded. (B) Fitting of the deactivation time course by a sinis extrapolated to the instant of on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 CASTELt.~O AND L6PEz-B~Eo Na + and Ca z+ Channels of Septal Neurons 311 external Ca ~+ for a mixture of Mg 2÷ and Co ~+, cations practically impermeant through Ca ~+ channels (Fig. 8 C). Fig. 9 shows a family of Ca ~÷ currents recorded on depolarization to the indicated membrane voltages. Current during the pulse, which is indicated between the arrows, increases in amplitude and activates more rapidly at more depolarized levels. After a maximum the pulse current diminishes with positive potentials due to the decrease in the electrochemical gradient for Ca 2+. Ca ~+ current measured at the end of the 6-ms steps at various membrane potentials is illustrated in Fig. 10. This/-V curve shows that the current becomes detectable at -40 to -50 mV, reaches a maximum at about +20 mV, and afterwards declines as the driving force for Ca ~+ progressively decreases. In 10 mM external Ca 2+ the amplitude of the current measured at the end of 6-ms pulses to 20 mVwas 0.35 -+ 0.22 nA (n = 12). Separation of two Ca 2÷ channel types. It is clearly seen in Fig. 9 that after small depolarizations the decay of the tails follows a simple, slow time course, but after depolarizations to potentials more positive than 0 mV a large fast component also A 10 Ca ";~"~.,,4.~_~. " ..... 1,-,;-.~-~""- FIGURE 8. Identification of Ca 2+ cur- -~'''-~ .¢ rents. (A) Current produced by depo- ' larization to 10 mV during 6 ms with Ca ~÷ as charge carrier. (B) Current B recorded in the same cell and with the 10 Ba .-, same pulse, using Ba ~+ instead of .r,,~ f Ca 2÷ in the external solution. (C) : Blockade of the current after replace- : ment of Ca 2+ by Mg ~+ and Co ~÷. : 1 nA Holding potential, -80 mV. The dif- : 2 ms ferent external solutions are indicated C in the figure; the internal solution was Mg. Co 130 Cs. ~"*"~'~"~'~"~'~'--'~.2" " appears. Both the slow and fast components of the tails grow larger as the membrane potential is made more positive. The sudden appearance of the fast component of the tails at voltages between 0 and 20 mV parallels the sharp transition seen in the I-V curve at the same voltages (Fig. 10). These observations suggest the existence in septal neurons of at least two different Ca 2÷ conductances with different activation voltages and deactivation kinetics. These conductances are probably due to the activity of two kinds of Ca ~÷ channels. Separation of the two components of the Ca ~+ current was done following the method illustrated in Fig. 11 (Cota, 1986; Matteson and Armstrong, 1986; Tabares et al., 1989). Trace A is a tail current recorded on repolarization to -80 mV after a pulse to 40 mV lasting 6 ms. An exponential is fitted to the slow component and extrapolated back to the instant of repolarization. Subtraction of the slow exponential from the total current yields a fast component on isolation which could be fitted by another exponential with a faster time constant. Thus, each exponential function represents the closing time course of slow (SD) and fast (FD) deactivating Ca 2÷ on September 22, 2014jgp.rupress.orgDownloaded from Published February 1, 1991 318 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 - 1991 transient activation of SD channels and their low activation voltage make some septal neurons capable of firing either tonically or in a burst mode, depending on the membrane potential of the cells (Alvarez de Toledo and L6pez-Barneo, 1988). It is worth mentioning that our previous data already suggested that in some septal neurons a significant proportion of FD channels might be located near the soma. The characteristic slow, high threshold Ca 2+ spikes of these cells appeared during small current injections, which is not a common finding in other CNS neurons, and it was relatively usual to obtain recordings where an initial Na + spike of small amplitude was followed by larger and broader Ca ~+ action potentials. 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