Brief increases in intracellular Ca2+ activate K+ current and non-selective cation current in rat nucleus basalis neurons

NeuroscienceVol. 58, No. 3, pp. W-561, 1994 Elsevier Science Ltd Coovright 0 1994 IBRO

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BRIEF INCREASES IN I~~CELLULAR Ca2+ ACTIVATE K+ CURRENT AND NON-SELECTIVE CATION CURRENT IN RAT NUCLEUS BASALIS NEURONS H. TATSUMIand Y. KATAYAMA Department of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University,2-3-10Kandasurugadai Chiyoda-ku. Tokyo, 101, Japan Abstract-Neurons

were acutely dissociated from the rat nucleus ha&is, and membrane currents (whole-cell patch-clamp) and intracellular free Ca2+ concentrations (Fura-2) were measured simultaneously from large neurons (approximately 25 pm in diameter). A brief depolarizationfrom -60 to OmV for 200ms evoked an increase in intracellular free calcium and a slow outward tail cnrrent (72 + 8 pA, n = 30). The outward current reversed polarity at -75.5 k 2.7 mV (n = 14). The tail current declined and the intrac~lular calcium recovered its resting levef ex~nentia~y with tim~onst~ts of 1.Of 0.1 s and 2.5 f 0.2 s, respectively (n = 17). In neurons loaded with Cs-giuconate,a similar depolarizing pulse evoked a similar increase in intraceilular free calcium, but this was now followed by an inward tail current (118 + 8 pA, n = 44). The inward tail current reversed polarity at -27.8 i 3.8 mV (n = 7), and was suppressed by removal of external sodium ions. Neither outward nor inward tail currents were observed, when the external solution was calcium-free or when the pipette solution contained EGTA (IOmM). These results indicate that a depolarization causes a calcium entry and that this consequently increases both K+ conductor and non-selective cation conductance.

Neurons in the basal nucleus of Meynert are the major source of cholinergic afferents to wide areas of neocortex and ~p~~mpus,~4 and cortical blood flow is regulated by this nucleus.” Degeneration of neurons in the nucleus basalis is thought to be important in diseases associated with memory impairment.21 Neuronal degeneration has been linked to consequences of excessivecalcium entry which may occur in response to pathophysiolo~cal stimuli that result in prolonged membrane de~la~~~o~s.*’

More physiological stimuli that cause brief depolarizations are also associated with calcium influx and alterations in intracellular calcium concentration ([Ca2+li) resulting in the modulation of a wide variety of cellular functions. In particular, increases in [Ca2+li have been shown to activate three classes of neuronai membrane conductances: a number of distinct potassium conductances,17,‘8f7a chloride conductance” and a non-selective cation conductance ‘*J The relationship between calcium influx, [Caz+‘li and activation of these currents has been studied in most detail in invertebrate neurons and vertebrate peripheral ne~ons,i2,‘7*22but there have been few studies of these relationships in the mammalian CNS. Recently, a Caz+-dependent nonselective cation conductance has been described in neurons from the rat dorsolateral septal nucleus and this conductance has been suggested to pfay an Abbreviationr: EDTA, ethylenediaminetetra-acetic acid; EGTA, ethyleneglycol-bis(/?-aminoethylether)N,N,N’, N ‘-tetra-acetic acid; MOPS, 3-[N-morpholinolpropanesulfonic acid; TEA, tetraethylammonlum.

important role in controlling neuronal excitability.9 However, quantitative estimates of the levels of [Ca2+li required to activate this, or other currents in central neurons have not been determined. In order to determine more precisely the relationship between [Ca*+], and activation of membrane currents, it is necessary not only to measure [Caz+li and the appropriate currents but also to control the influx of Ca*+. This can be accomplished by combining voltage-clip t~hniques to initiate and terminate Ca’+ i&x with simultaneous measurements of [Ca*+],. The main purpose of the present study was to examine the relationship between [Ca2+Ii increases and the currents that such [Ca’+], increases induce in neurons of the mammalian nuckus basalis. Therefore, simult~ous recordings of membrane current and Fura- fluorescence were carried out on acutely dissociated neurons obtained from the rat nucleus basalis. EXPERIMENTAL PROCEDURIB Pre~r~~io~ and eleclro~ys~a~og~c~~ recording

Wistar rats (six to I8 days, Saitama Experimental Animals Supply Inc., Japan) were anaesthetimd with nembutal, and the whole brain was removed. A brain slice (400-5OOgm thick) containing the nucleus basalis of Meynert was cut by a Vibratome (Oxford, U.S.A.). The region of the nucleus was cut out with a razor blade under adissection microscope according to the atlas of Paxinos and Watsonz- Some slices were stained by monoclonal antibody to choline acetyhransferase to confnm the region.rqThe dissected brain tissue was treated with papain (20.3 unit/ml) in low-Ca2+ and lowMg2+ Krebs’ solution for 20min at 37”C, and triturated using pipettes. Neurons with several processes (usually less

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H. TATSUMIand Y. KATAYAMA

than 100 pm) or neurons without processes were suspended on a glass coverslip, and were allowed to adhere to the cover glass in a modified Krebs’ solution before recording. Large cells (about 25 pm in soma diameter), were chosen for recordings, since 75% of large cells are statistically cholinergic.19 We also observed that most of the typical large neurons (70-80%) isolated from the nucleus basalis were choline a~tyltr~sferase positive. Therefore, the majority of neurons chosen for recordings could be cholinergic. The ionic composition of the modified Krebs’ solution was (mM): NaCl, 117; KCI, 4.7; CaCl,, 2.5; MgCl,, 1; glucose, 11 and 3-[N-morpholinolpropanesulfonic acid IMOPS), 25; and pH 5.4 adjusted by-&aOH. The low-Ca2+ and low-Ma2+ Krebs’ solution was made bv adding EDTA (2.5 mM) to the modified Krebs’ solution.Neurons were supcrfused with modified Krebs’ solution gassed with 100% 0, at 2426°C. Membrane currents were recorded from the neurons with the whole-cell patch-clamp technique. Patch pipettes had resistances of 3-.5 Ma and were coated with enamel to reduce the pipette-bath capacitance of the recording pipettes.’ The series resistance of the recording system was compensated by the electrical circuit. Recordings of the slow outward tail current and the slow after-hype~ola~zation were made using a whole-cell pipette solution of the following composition (mM): K-gluconate (or KCI), 100; MOPS, 40; MgCl,, 1; ATP-Mg, I; phosphocreatine (Na salt), 1; creatine phosphokinase, 50 I.U./ml and Fura-2, 0.01; and pH 7.2 adjusted with KOH. In some experiments whole-cell pipettes contained nystatin (200 PM), and the membrane current was monitored without dialysis of cytoplasm. Recordings of the slow inward tail current were made using Cs-gluconate (or C&l. or ~-methyl-D~glucamine chloride in some experiments) instead of K-gluconate in the pipette solution. The extracellular solution was usually modified Krebs’ solution. Some experiments were carried out using Na+-free extracellular solution, which contained (mM); tetraethylammonium chloride (TEA-Cl, Tris-Cl, CsCl or LiCl), 140; CaCl,, 2.5; MgCl,, 1; glucose, 1t and MOPS, 5; and pH was adjusted 7.4 with KOH. Fluorescence

recordings

Fura- was excited by ultraviolet light from a Xenon lamp (300 W) passed through interference filters (340 nm, 360 nm and 380 nm, Ditric Optics, U.S.A.). A pinhole located on the excitation-light path of the inverted microscope (IMT-2, Olympus, Japan with Fluor 40 x , Nikon, Japan) focused the light on the section of about 10 pm in diameter upon which the cell body was positioned. Emission was separated by a barrier cut-off filter at 500 nm,2 and the intensity of the fluorescence was monitored by a photomultiplier tube (R1635; Hamamatsu Photonics, Japan). The fluorescence intensity and the membrane current-were filtered through a low uass electrical filter (cut-off freauencv 40-1000 Hz for Auorkscence intensity and 20,000 Hz-for membrane current) and were recorded on a thermal pen recorder (San-ei, Japan) and fed into two channels of analog-to-digital converter (Axon Instruments, U.S.A.) for further analysis. The fluorescence intensity, when excited by 380 nm, was denoted by F,80 and when excited by 340 nm by Fj4,, and so on. The [Ca’+ Ii was estimated using ratio of emissions; F3dF38~ %J&J (see belo~).~ When F3JFjW ratio was used. the lCa2+1; was estimated from [Ca*+l, = -R@, where R is the F&F,,, r&o. %{R -RAKER-When F360/F,80 ratio was used, the same equation was used though R was replaced by the Fm/Fj8,-, ratio. Since Fm did not change with changes of [Ca”+],, the [CaZ+li could be estimated from F,.,. The F&F,,,, ratio was used to estimate -_ a steady state va”i;e of the [Caz+li, and the F360/F380ratio was used to estimate a transient change in the lCa’+ 1;. The fluorescence intensity was corrected for background-‘fluorescence. The maximum ratio (R,,), the minimum ratio (R,,,) and the constants (Kd and b) were estimated in

or

_“”

separate procedures (as follows). The neurons were internally perfused with calibrated solutions of various Ca2+ concentrations through whole-ceil patch-clamp electrodes. The calibrated buffer solutions contained (mM); EGTA, 10; Fura- pentapotassium, 0.01; KCl, 100; MOPS, 10; with calculated amounts of CaCl, to give specific CaZ+ concentrations ranging from 0 to 3000 nM (PH 7.2) (details of these solutions were described elsewhere”). K, was estimated at 200 nM and b at 1.8. In cases using nystatin (see above), the neurons were stained by bathing in the modified Krebs’ solution containing 2pM fura-2/acetoxymethyl ester (AM) for 15 min at room temperature and were loaded with Fura-2. Materials

Drugs used were Fura- pentapotassium salt (Fura-Z), Fura-Z/AM (Dojin, Japan, and Molecular Probe, U.S.A.), ATP (Sigma, U.S.A.), papain ~at~in~on, U.S.A.), Nmethyl-D-glucamine (Sigma, U.S.A.). Drugs were applied by

either superfusion or intracellular dialysis through wholecell patch-clamp electrodes. Quantitative results are expressed as mean + S.E.M. for the number of observations in parentheses. RESULTS

Two types of slow tail currents

evoked

by depolariz-

ation

Simultaneous recordings of membrane current and Fura- fluorescence signal were made (Figs lA, 4A). With pipettes containing K-gluconate, 30 of 90 neurons showed slow outward tail currents (71.9 + 8.3 PA) after a depolarization from -60 mV to 0 mV for 200 ms. Other neurons (48 of 90) did not exhibit slow tail currents, or some neurons (12 of 90) had small inward tail currents. When Cs-gluconate was used, 44 of 150 neurons showed slow inward tail currents (118 & 8.2 PA); in these neurons slow outward tail currents were observed at the beginning of the whole-cell patch~lamp recording, and then slow inward tail currents became apparent after intracellular dialysis with Cs+ for more than 3 min, presumably due to the blocking of K” current increases (n = 3). Both slow outward and inward tail currents were associated with an increase in [Ca2+],, and no slow tail current was observed in Ca2+-free external solution (n = 4) or after internal dialysis with EGTA (1OmM; n = 13). Outward

tail current

A brief depolarization from - 60 to 0 mV for 10 ms increased [Ca2+], from 50 to 65 nM and was followed by a small slow outward tail current (Fig. 1Aa). Both the outward tail current and the [Ca2+ II-increase were dependent on the duration of depolarizing pulse (Fig. 1A, B). The relationship between the amplitude of the slow outward tail current and the peak [(=a’+], is shown in Fig. 1C. The amplitude of the slow outward tail current was dependent on the peak [Caz+li. However, the amplitude of the current was no longer propo~~onal to the peak [Ca’+ 1,when the [Ca’+J was large. The outward tail current decreased and the [Ca*+], returned to resting level after cessation of the

555

Ca2+ dependent tail currents

I

Duration of

d

pulse (ms)

C

ho 110 % AFIF

Im

I@l,

(nh4)

Fig. 1. (A) Slow outward tail current (I,) evoked by pulse depolarization in a voltage-clamped neuron. Pulse depolarizations from - 60 to 0 mV for 10(a), 20 (b), 120(c). and 240 ms (d) were followed by [Ca2+Ii increase (downward deflection of F,,,) and slow outward tail currents. The slow outward tail current and the [Ca2+liincrease were both augmented as the duration of the depolarization pulse increased. The zero current level is shown by arrows. (B) Relationship between the duration of the depolarization pulse (abscissa) and the amplitude of the slow outward tail current measured at 30ms after the pulse depolarization (ordinate). (C) Relationship between the peak [Ca*+],(abscissa) and the amplitude of the slow outward tail current (ordinate). All data were obtained from the same cell. depolarization.

The decay of the outward tail current

was always faster than the recovery of the [Ca2+li (Fig. 1A); the time-constant of the decay of the outward tail current was 1.0 &-0.1 s, and that of the [Ca2+]i-recovery was 2.5 * 0.2 s (n = 17). Similar re-

A

Im

B

J

Vm (mv)

n

/’

-150

IS 0

Vm

from two neurons when the nystatin method was used for whole-cell patch-clamp recordings (see Experimental Procedures). The voltage-dependence of the slow outward tail current was examined as shown in Fig. 2. The slow suits were obtained

-50

-70 t

-100

l

-50

1 -130

Fig. 2. Voltage dependence of slow outward tail current. (A) Current recordings (I,) were made using a voltage-clamp protocol shown in V,. A pulse depolarization from -70 to 0 mV for I50 ms induced a slow outward tail current, when the membrane potential was fixed at - 50 or -70 mV after the pulse depolarization. When the membrane potential was set to more hypespolarized levels ranging from -90 to - 130mV, the polarity of the slow tail current was reversed. The zero current level is shown by an arrow. (B) Relationship between the membrane potential (abscissa) and the amplitude of the slow outward tail current measured at 50 ms after the end of the depolarizing command (ordinate). The reversal potential of the slow outward tail current was - 77 mV.

H. TATSUMI and Y. KATAYAMA

5.56

outward tail current decreased in amplitude by membrane hy~rpolarization (Fig. 2A) and reversed polarity at -75.5 f 2.7 mV (ranging from -65 to - 90 mV, n = 14; see Fig. 2B). This value was slightly positive to the calculated potassium equilib~um potential under the present experimental conditions (-88 mV), perhaps because the slow inward tail current was activated simultaneously by the same depolarizing pulse (see below and Discussion). Slow afterhyperpolarization following action potentials Simultaneous recordings of membrane potential and associated fluorescence signal (F,80) were made from 33 neurons with pipettes containing Kgluconate (Fig. 3). Depolarizing current injection (50-200 PA) evoked action potentials and slow afterhyperpolarizations associated with a [Ca2+ Ii-increase in 45% of neurons (Fig. 3B). The amplitude and the half decay time of the slow afterhyperpolarization following a single action potential were 9.8 i: 1.5 mV and 0.30 & 0.04 s (n = IO), respectively, at - 50 mV (Fig. 3Aa). The [Ca2+ji increased by 7.2 It 0.8 nM (n = 10) with a single action potential, and then the [Ca”], returned to resting level exponentially with time-constant of 0.53 k 0.09 s (n = IO). The ampli-

A

a

tude of the slow afterhyperpolarization was decreased by membrane hy~rpolarization, while the [Ca2+ ],increase by every single action potential was not substantially changed (Fig. 3Ab, AC, Ad). Slow outward tail currents were observed when voltage-clamp recording was made after currentclamp recording in the same neurons which exhibited slow afterhyperpolarizations. Inward tail current A slow inward tail current was induced by depolarization to levels less negative than -40 mV, and was also associated with a [Ca2+],-increase (Fig. 4A). The maximum current was evoked by depolarization to 0 mV and then the inward tail current declined when further depolarizations (+ 10 to + 100 mV) were applied (Fig, 4B). Such a current-voltage relationship is expected in response to a Ca’+-dependent process, because the large depolarizations reached the Ca2+ equilibrium potential (i 130 mV) and CaZ+ influx became smaller. The [Ca2 +Ii-increase showed almost the same voltage-dependence as the slow inward tail current (Fig. 4B). The slow inward tail current was also observed when the pipette solution contained ~-methyl-D-glucamine (100 mF/I, n = 4).

c

B

Fig. 3. Simultaneous recordings of membrane potential (V,) and Fura- fluorescence signal (F,,,). (A) Slow aft~rh~~la~~ng potentials associated with a ]Ca2+li increase following spontaneous action the neuron was potentials (resting membrane potential of this neuron was about - 50 mV, a). #en hyperpolarized to - 60 mV (b), - 70 mV (c) and - 80 mV (d) by current injection, spontaneous firings were abolished. Depohxrizing current pulses were apphed every I s to evoke single action potentiais. Each pulse was followed by a slow afterhyperpolarization associated with an increase in [Ca’* 1,. The amplitude of the slow afterhyperpolarization decreased with membrane hyperpolarization (compare, b, c, d). Note the amplitude of action potentials increased by the hyperpolarization. (B) Slow afterhyperpolarization and [Ca2+li increase following the respective depolarizing current puise for 100 ms (50, 100 and 200 pA for a, b and c, respectively), during which action potentials were produced. Full amplitude of action potentials was not shown because of using a pen-recorder.

557

Ca’+ dependent tail currents

A

Fig. 4. (A) Slow inward tail current and [Ca2+li increase in response to various depolarizing commands in a voIta~-clam~d neuron at - 80 mV. A deplaning voltage command to - 60 to + 100mV for 100ms (v,,,) evoked a slow inward tail cnrrent (I,) and a [Ca2+) increase (F&. The zero current level is indicated by an arrow. (B) Relationship between the value of pulse depolarization (abscissa) and the value of peak [Caz+1,(upper) and the amplitude of the slow inward tail current (lower)in ordinate.

The relationship between the amplitude of the slow inward tail current and the membrane potential was linear between - I50 mV and -20 mV (Fig. 5A, B), and the reversal potential was -27 f 3.8 mV (n = 7). When Cs-gluconate in the pipette solution was replaced by C&l, the reversal potential of the slow inward tail current was not significantly changed (n = 3). After cessation of the depolarization, the inward tail current declined and the [Ca2+], returned to resting level. The decay of the slow inward tail current consisted of fast and slow components; the time~onstants of these components were 56.6 k 6.7 ms and 1.1i 0.1 s (B = 36), respectivefy (Fig. 5C), while the decay of the [Ca2+], followed a single exponential with the time constant of 2.9 f 0.2 s (n = 36). The membrane conductance measured by applying constant voltage pulse command increased from 0.5 to 6.4nS during the slow inward tail current in a typical experiment (Fig. 5D). The slow inward tail current and the [Ca*+], increase were augmented when the depolarization pulses were prolonged (Fig. 6A). The amplitude of the slow inward tail current was dependent on the [Ca*+J (40-700 nM), and was no longer proportional to the peak value of [Ca” 1, when the peak value exceeded 250 nM (Fig. 6D). When external Ca*+ was totally substituted by Ba2+, no slow inward tail current was observed (n = 3). Figure 7 shows that the slow inward tail current was blocked or a small outward tail current sometimes was left (see below) when the external Nat was replaced by TEA (or Tris), while the amplitude of the transient [CaZtIi-increase was approximately the same as control (n = 20, Fig. 7A). Small outward tail currents associated with a fCa2+],-increase were sometimes induced by pulse depolarizations to levels less negative than -40 mV (Fig. 7B). The slow outward tail current was maximum in amplitude

(20.4 f 9.2 pA, n = 8) with the pulse depolarization to 0 mV, and became smaller by a further increase in the pulse depolarization (Fig. 7B), since the Ca2+ influx became smaller. Slow inward tail currents were also observed when the external Nat was substituted with Cs+ (or Li+). The extrapolated null potential of the slow inward current was 11.6 + 6.1 mV (n = 3), when Na+ was substituted with Cs+. DISCUSSION

The present expe~ments demonstrate that slow outward and inward tail currents were associated with a (Ca2+li increase and were blocked by treatments which prevent the [Ca2+li increase. These results indicate that a change in [Ca2+li is involved in the activation of both slow tail currents. The present study suggests that about one third of the nucleus basalis neurons isolated from neonatal rats (presumably cholinergic neurons) are endowed with both Ca2+-dependent K+ channels and ~2+-de~ndent non-selective cation channels (see below), like neuroblastoma ceils3’ and Helix neurons.1’,26 Ionic mechanisms of the slow outward and inward tail currents

The present results show that the slow outward tail current was due to an increase in a Ca2+~e~ndent K’ conductance, as seen in many other preparations.“,” The reversal potential of the slow outward tail current ranged from -65 to -9OmV, suggesting the simultaneous activation of the K+ conductance and a non-selective cation conductance by the [Ca’“], increase. The typical slow inward tail current was not observed with K-gluconate pipettes, but was observed only after neurons were loaded with CS+. The most likely explanation for these results is as follows; though both the K+ conductance and the

H. TATSIJMIand Y. KATAYAMA

558

non-selective cation conductance were activated by ratio changed. Cl- conductances do not appear to be the [Ca*+], increase, the larger K+ currents masked involved in producing the slow inward tail current, the inward tail currents when pipettes contained since replacing gluconate ions with Cl- in the wholeK-gluconate; but after K+ conductances were cell pipette solution did not affect the reversal potenblocked by Cs+, the inward tail current was revealed. tial of the slow inward tail current. Furthermore, It may prove useful in future studies to examine the when Cs+ was present on both sides of the membrane effects of putative pharmacological blockerP on with Cl- in the outside and gluconate ions in the non-selective cation conductances in order to isolate inside, the reversal potential was about + 11.6 mV. more effectively the K+ conductance. Since a perfectly cation-selective channel current Reversal potentials of the slow inward tail current should reverse at slightly above 0 mV and a perfectly in various external and internal ionic conditions can anion-selective channel current at very negative pobe explained by a non-selective increase in membrane tential, the present results again indicate the existence permeability to monovalent cations including Na+, of cation selective channels. Cs* and Li+.“JO It is unlikely that the slow inward The slow inward tail current was evoked by a pulse tail current is carried only by Ca2 +, because no slow depolarization when extracellular solution contained inward tail current was observed in the Na+-free CsCl (or LiCl) instead of NaCl. Since a Na+/Ca’+ Ca2 -t- exchange system could be sup&-essed in Na +-free extracellular solution which contained (2.5 mM) and the reversal potential of the slow solution,’ it can be ruled out that the Ca*+-dependent inward tail current was not changed during the tail currents were brought about by Na+/Ca’+ ex[CaI+], recovery during which the [Caz+],/fCa2+], change system as suggested in cardiac myocytes.4~s~”

i 10 % AFIF

Im

Fig. 5. Voltage-dep~den~ of the slow inward tail current. (A) According to the voltage-clamp protocol shown in V,,, , a pulse depolarization from - 50 to -t 10 mV for 150 ms induced a slow inward tail current (I,), when the membrane potential was set to levels ranging from - 60 to --- 150 mV after the pulse depolarization. Small outward tail current was observed when the membrane potential was fixed at - 30 mV. [Ca2+Ii increased during the pulse depoiar~~tion and afterward recovered to resting level; five traces of fluorescence intensity (F,,,) are superimposed. (B) Relationship between the membrane potential (abscissa) and the amplitude of the slow inward tail current (measured at 20 ms after the termination of the conditional pulse depolarization, ordinate). The reversal potential of the slow inward tail current was -31 mV. {Cc)Slow and fast components of the tail current at -60mV. Upper and lower panets are presented in slow and fast time scale, respectively. Both components were exponential; time-constant of slow component was 840 ms (open arrowhead in upper panel) and that of the fast component was 76 ms (arrowhead in lower panel). (D) Slow inward tail current associated with an increase in membrane conductance. A pulse depola~zat~on from -45 to 0 mV for IO0 ms increased [Ca2’], (FIsO)and induced a slow inward tail current (&,,). The membrane conductance was monitored by measuring the current in response to repetitive pulse depolarizations from -45 to -25 mV for 1OOms at 1 Hz. For A, C and D, the zero current levels are shown by arrows.

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Ca*+ dependent tail currents

5

zaa

r--2

0 0

Duralian of PUISS (ms)

~:0

0

iooa Duration ofpulseIms) 500

I:’ 1

500

i

a00

Fig. 6. (A) Slow inward tail currents evoked by pulse depolarizations from -60 to 0 mV for 3, 10, 40, 80 ms, 1s, 2 s from left to right. The slow inward tail current (I,,,) and the [Ca2”], increase (F,,) were augmented as the duration of the depolarization pulse increased. It should be noted that calibration for IQ,,,is changed for the right two records. The zero current level is shown by an arrow. (B) Relationship between the duration of depolarization pulse (abscissa)and the amplitude of the slow inward tail current (ordinate) measured at 20 ms (filted circle) and at 300 ms (filled square) after the end of the depolarization. (C) Dependence of the peak [CaZ”]ion the duration of the pulse depofarization. (ID)Re~a~onship between peak vahm of [Ca”~ and the amplitude of the inward tail current measured at 2Oms Qilled circle) and at 300 ms (tilled square) after the end of the depolarizationas in 8.

Thus our results indicate that the slow inward tail current was due to an increase in Ca*+-dependent non-selective cation conductance. Ca2+ sensitivity of slow outward and inward tail currents

The [Ca2+li requisite for activating the outward tail current was about 100 nM in the present study. This value is similar to the [Ca2*li wbieh induces slow a~erh~~ola~~tions in guinea-pig myenteric neur011s.‘~The fCa2+J requisite: for activating non-seIective cation channels has been reported to be different from preparation to preparation.22 According to a majority of studies, [Ca2+li at I PM or lower can significantly increase single channel opening;2s,Mone study has shown, using cell-attached patches, that the Ca2+-dependent non-selective cation channels of mouse pancreatic acinar cells can open at 50 nM of [Ca2+]i.‘6In the present experiments the slow inward tail current was activated at aboat 100nM of [Ca2+li, and the amphtude of the current was saturated at 700 nM of[Ca2+ Ii. These results support the idea that both Ca~*~e~dent K+ and Ca2+-dependent nonselective cation channels open in the ~h~olo~~ range of fCa2+f The time course of the decay of the slow outward tail current was approximately the same as that of the slow inward tail current; however, their time courses were faster than those of the [Ca2*]1recovery associated with either current. This suggests two possibili-

ties; (i) [Ca2+li declines more rapidly near the channels than in the bulk cytoplasm, and/or (ii) the channels are activated by Ca*+ in a co-operative process (e.g. Ca2+-dependent K+ channels’). It seems likely that the fast component of the slow inward tail current reflects the rate of [Ca2+Ii-decline which may occur just beneath the cell membrane at the site of the cation channeL This suggestion is based on results obtained from previous studies using mathemat~~ai model*’ and confocal mi~ro~o~y’~ to estimate [Caz* 1 changes in the region imm~ately underneath the plasma membrane. In both studies ‘W the rate ofchange of[Ca’+k was sirnibr to the fast component of the slow inward tail current measured in our experiments. Evidence has accumulated to suggest that Ca2+ entering during Ca2+ channel opening can bind rapidly to Ca’+-binding proteins.** Thus, local [Ca*+],may well be very high beneath the membrane immediately after Ca2+ influx, concentrations which would not be accurately measured with the Fura- method. This may explain the saturation of the fast component of the inward tail current,in response to apparently relatively small Ca’* influx (e.g, Fig. 6Dj_ In contrast, the slow component of decline of the slow inward tail current is assumed to reflect the relatively slow recovery of whole-cell [C@]i and this more homogeneous Ca** gradient is well within the limits of measurement. using the fluorescence methods employed in the recent experiments.

560

H. TATSIJMIand Y. KATAYAMA

A

t3 Control

TEA “Lc”

F380 If+++

Im

120%

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+ 1

4-

100

pA

I--Vm .dk--

Fig. 7. Inhibition of slow inward tail current by total substitution of the external Na+ with TEA. (A) A control pulse depolarization from -60 to 0 mV for 500 ms (V,,,) induced a slow inward tail current (I,,,) and a [Ca’+], increase (E;80). After substitution of the external Na+ with TEA: no slow inward tail current was evoked by the same depolarization pulse, while an inward Ca2+ current and an increase in [Ca*+], were observed during the pulse. (B) Pulse depolarizations to -40, -20, and 0 mV for 300 ms (protocol is shown in V,,,) induced slow outward tail currents (I,) and [Ca*+], increase (F,,,) in Na+-free (TEA) external solution. Both were augmented as the peak value of the pulse depolarization increased up to 0 mV. However, when the peak of depolarizations was further increased beyond +60 mV, the slow outward tail current and the [Ca2+], increase were inhibited. The zero current levels were shown by arrows. Cells in A and B were different. In the present study, the time course of activation of either the Ca*+-dependent K+ or non-selective cationic current was similar (e.g. Figs 1, 4). These results differ from the situation present in Helix neurons where, although both Ca*+-dependent K+ and non-selective cationic currents are also present, the time course of activation of the two currents differ. In Helix neurons the Ca*+ influx during an action potential activates the non-selective cationic current which triggers a burst of action potentials which further elevates Ca*+ influx and [Ca*+ Ii; this additional and delayed increase in [Ca2+li activates the K+ conductance resulting in termination of the bursting pattern of firing. Physiological sign$cance

of these slow tail currents

One of the important electrophysiological characteristics of cholinergic neurons in the basal nucleus is that the neurons do not generate spontaneous tonic firings and produce burst firing only for brief periods; they adapt quickly in response to continuous depolar-

izing currents.19 Such characteristics could be easily explained from the present results; a burst of action potentials would cause a rapid [Ca*+ 1, increase which subsequently and simultaneously activates K+ and non-selective cation conductances. Because the resulting K+ current is larger than the non-selective cation current (see above), the K+ current activation terminates firing and generates a relatively small slow afterhyperpolarization. On the other hand, if the K+ channel current activated is relatively smaller, a membrane depolarization may be induced by activation of the non-selective cation current and this will cause further Ca*+ entry. This process may result in Ca2+ overload which would likely play a role in neuronal degeneration.

Acknowledgements-We thank Dr R. A. North and Dr A. Surprenant for reading this manuscript and for critical comments. This research was supported in part by the Grant-in-Aid for Scientific Research on Priority Areas, Ministry of Education, Science and Culture, Japan.

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