Effect of ethanol on subthreshold currents of Aplysia pacemaker neurons

Brain Research, 332 (1985) 337-353

337

Elsevier BRE 10684

Effect of Ethanol on Subthreshold Currents of Aplysia Pacemaker Neurons MARK H SCHWARTZ Neurosctence Program, Brain Research Instttute, Untverstty o f Cahforma at Los Angeles, Los Angeles, CA 90024 ( U S A )

(Accepted July 31st, 1984) Key words alcohol - - ethanol - - Aplysla neuron - - burstmg pacemaker - - pacemaker - - voltage clamp

The subthreshold currents in bursting pacemaker neurons of the Aplysta abdominal ganghon were indlvtdually studied with the voltage clamp technique for sensitivity to 4% ethanol The most prevalent effect of ethanol on unclamped bursting neurons was a hyperpolarization This was shown to be due to a decrease of a voltage independent inward leakage current Direct measurement of the Nadependent slow reward current showed that this current was ehminated by 4% ethanol Direct measurement of the Ca-dependent slow mward current showed that this current was substantmlly reduced by 4% ethanol Inlection of EGTA into cell bodies did not ehmmate the ethanol-reduced block of the slow inward calcmm current Thus, ethanol cannot be reducing the Ca-dependent slow reward current solely by an increase of internal calcium concentration The effect of ethanol on voltage dependent outward current was measured by blockage of all reward current The peak outward current was increased by ethanol The rate of inactivation of this outward current was also mcreased Calcium activated potassium c u r r e n t (IK(fal) IS particularly comphcated m its response to ethanol because it ~s dependent on both Ca and voltage for its activation The level of IK(ca) ehcIted m response to constant Ca mject~on was Increased by ethanol treatment The level of this current as activated by voltage clamp pulses was either increased or decreased depending on the neuron type Ca `-+ activated potassmm conductance increased e-fold for a 26 mV depolarization m membrane holding potential Ethanol decreased this voltage dependence to e-fold for a 55 mV change in potentml This result was interpreted to mean that ethanol shifted an effecttve Ca 2+ binding site of these channels from about halfway through the membrane field to one quarter of the way across The same theoretical approach allowed the further conclusion that ethanol caused an increased internal free calcium concentration probably by decreasing calcmm binding by mtracellular buffers

INTRODUCTION

rons of A p l y s t a as t h e s e cells c o n t a i n an a b u n d a n c e of subthreshold currents

T h e e a r h e s t v o l t a g e c l a m p studies on the effects of

M y p r e h m l n a r y studies s h o w e d that e t h a n o l d o e s

e t h a n o l on smgle n e u r o n a l f u n c t i o n d e m o n s t r a t e d a

alter firing p a t t e r n s of b u r s t i n g n e u r o n s via an effect

c o n d u c t i o n b l o c k of the n e r v e i m p u l s e . E x p e r i m e n t s

on their s u b t h r e s h o l d currents: 4 % e t h a n o l causes a

p e r f o r m e d on s q m d a x o n s h o w e d a specific d e c r e a s e

d e c r e a s e in the Inward c u r r e n t and an Increase in the

of the Na- and K - c o n d u c t a n c e s u n d e r l y i n g the spike

o u t w a r d c u r r e n t p r e s e n t at s u b t h r e s h o l d

currents3 49 T h e calcium c u r r e n t c o n t r i b u t i n g to the

levels 55 T h e s e d e m o n s t r a t i o n s , h o w e v e r , e m p l o y e d

spike in the s o m a of s o m e n e u r o n s of A p l y s l a califor-

t i m e - v a r y i n g v o l t a g e c o m m a n d s which did not allow

inca is also d e c r e a s e d by ethanol6

B u t the b l o c k a g e

the s e p a r a t i o n m t i m e of the p a c e m a k e r currents. In

of spike currents and a x o n a l c o n d u c t i o n at large etha-

a d d m o n , t h e small m a g n i t u d e of the c u r r e n t s , the

nol

profound

fact that t h e y o c c u r In an o v e r l a p p i n g r a n g e of p o t e n -

changes in n e u r o n a l firing p a t t e r n s s h o w n in n u m e r -

tial and the fact that t h e y are all active at typical hold-

ous stu&es 7 I8 T h e study of s p o n t a n e o u s l y active

ing p o t e n t i a l s , m a k e s it i m p o s s i b l e to d e t e r m i n e f r o m

cells has s h o w n that c h a n g e s in firing p a t t e r n m u s t be

the steady-state c u r r e n t - v o l t a g e r e l a t i o n ( I - V ) in n o r m a l solution which c u r r e n t s are a f f e c t e d by etha-

concentrations

cannot

explain

the

e x p l a i n e d via effects on the s u b t h r e s h o l d currents37 42,61. It was t h e r e f o r e d e c i d e d to re-investigate the effect of e t h a n o l on b u r s t i n g p a c e m a k e r n e u -

nol

potential

T h e studies d e s c r i b e d b e l o w e m p l o y e d special

ion b l o c k i n g p r o c e d u r e s and c u r r e n t s e p a r a t i o n tech-

Correspondence M H Schwartz, Neuroscience Program, Brain Research Instttute, Umverslty of Cahfornla at Los Angeles, Los Angeles, CA 90024, U S A

0006-8993/85/$03 30 © 1985 Elsevier Science Publishers B V (Blome&cal Division)

33,~ ntques along with both ramp voltage and pulse xoltage clamp (VC) techmque~ to facd~tate analysis of

tim sensing input so that large ionic currcqtb dt~ 1;o~, cause polarization oI the sensing electrode Ser~c~ rc s~stance compensation was not employed a~ the rc,>tance in series with the m e m b r a n e ha~ been :,hm~p. ;~

all subthreshold currents known to bc present m bursting pacemaker neurons

be about 1-3 k.Q 3a Fhe ser~e~ res~stan,.~ c~,)i , n r c MATERIALS AND METHODS

corded m e m b r a n e potenmtt would ha~t been lc,s than 15 mV during the largest measarcment~ oI m e m b r a n e current and less than 0 1 m'v m most ex-

Experiments were performed on the ldenttfied bursting pacemaker neurons, L 2 - L 4 . L6 and R15. m

periments The measured current was hhered with a passive filter with a cutoff trequenc~ ¢~! ~'~ Hz '\11

the abdominal ganglion of Aplysta cahforntca 2o The ganghon was removed from the antmal and the con-

measurements were monitored on an ,,sClllOscopc and p e r m a n e n t records were made on a stop chart re-

nectwe t~ssue was softened by soaking m a solution ot 10 mg/ml pronase (B-grade, Calblochem-Behrmg) In

corder (Gould 2200S or Hewlett-Packard 7402A)

normal amficlal sea water (NASW) for 15 n u n Mild pronase treatment does not affect the bursting propertms of A p l y s m neurons3J The ganglion was then

For mjechon of calcmm ~ons, electlodes were filled with 0 5 M CaCI e solunon and tot mjectum of ethylene glycol bls (B-ammoethyl ether I-N.N'-tetracetlc acid ( E G T A , Sigma). the electrodes were filled with a 0.5 M K E G T A solution Iontophoresls was

pruned to the bottom of a stlasttc-lined recording chamber Neurons were exposed by carefully dlssectmg away the sheath above the cells using very fine forceps and tridectomy scissors

performed under VC c o n d m o n s so that the mlect~on current would not disturb the m e m b r a n e potential The mntophoresls unit was a modified Howland current pump design which could pass a constant current up to 150 n A through a resistance of 100 NI£2~ The composttton of the ~olutlons used 1~ ~hown in Table I. All solutions contained at least 10 mM T n s and were adjusted to pH 7 (>-7 8 with HC1 An lSOosmotic concentrahon of T n s replaced Na m low Na so-

Mlcroelectrodes were made from thin-walled glass blanks (TW150-F, WP Instruments) W h e n filled with 3 M KC1, electrodes had a resistance of 2 - 4 Mr2 Two electrodes were connected to a conventional VC circuit modified for m e a s u r e m e n t of currents to a tenth of a n a n o a m p e r e . By addition of an adjustable lead network to the gain stage of the VC, the cutoff frequency can be conveniently adjusted to reduce noise to an appropriate level 60. The potential of the

lut,ons It was noticed that solutions containing low Na and some Mn change color from hght r , s e to amber m about 20 mm. This color change probably slg-

bath was clamped to clrcmt ground by a negative feedback circuit. This arrangement has the advantage of not allowing current flow through the poten-

mfies chelation of Mn by TnsJ~ These solutums were used before this color change became ver} apparent.

TABLE I Composmon oJ ~oluuons (mM)

NASW 0 Na 0 2 Na TFX Test* ** Cs Test** Z Mg Test High Mg Test Y*** **

NaCI

KC1

450

i0 10 10 10

90 350 10 90 10 10 10

* 30uM TTX ** 100 mM TEA 4 mM-4 AP *** 100,uM verapamfl 4 mM NICI. g

.

C~CI

CaCl ,

10

11 11 11 2 2 2 2 2 2

10 10 10 10 .

.

.

.

.

BaCL

MgCL

20

49 49 49 49 49 49 58 194 49

.

.

MnCI.

4(1 40

Trts

1{I 494 397 I[) 376 397 483 283 483

339 The l o n o p h o r e nystatin (Sigma) was used to replace internal potassium with cesium following the procedure described by TIllotson 63. The voltage recording and current passing microelectrodes were filled with 3 M CsC1 in this series of experiments in o r d e r to avoid leakage of K into the cells. Strophanthldln (Sigma) and o u a b a m (Calbxochem-Behring) were added to test solutxons at a concentration of 0 5 raM. Cobalt was a d d e d directly to test solutions. T e t r o d o toxin (TTX, Calblochem-Behrlng) was applied from a stock solution of 10 -3 g/ml diluted 100 x to give a final concentration of 30 BM. Solutions containing 100 u M verapamll hydrochlorlde were m a d e by adding one ampule of Calan (Searle Pharmaceuticals) containlng 5 mg verapamll to 98 ml test solution. Ethanol concentration was m e a s u r e d as the percentage of final solution by volume (v/v) All solution changes were made by flowing at least 30 ml solution through the 1 5 ml c h a m b e r over a p e r i o d of a p p r o x i m a t e l y 10 mnn Solutions were flowed until m e a s u r e m e n t of a steady level of holding current. All experiments were carried out at room t e m p e r a t u r e (20-23 °C) RESULTS

Effect on n-shape m Na-free solutton All bursting neurons have an n-shaped steadystate I - V relatlonZ3,~, 64 The inward portion of the I - V curve represents the depolarizing drive for this oscillatory p h e n o m e n o n The ionic basis for this slow inward current (I~l) has been attributed to Na 11 59, Na and Ca~% and Ca 1~ Currents m e a s u r e d in R15 at the end of a 5 s VC c o m m a n d pulse are shown in Fig 1 At this time, the current had reached a quasi-steadystate The I - V relation exhibited the classic n-shape The inward current reached a m a x i m u m of 3 5 n A and was centered at a c o m m a n d voltage o f - 4 2 mV, As shown by several researchers, m 0 Na solution the n-shape is almost abolished leaving a plateau in this region of the steady-state I - V : : ~ The a d d m o n of 4% ethanol eliminated the plateau replacing It with an outward current (lout) It is clear that the increased Iou t c a n n o t be explained by a decrease in steady-state Na current as this current should be negligible in 0 Na solution The I - V curves below - 6 0 m V are seen to be unaffected in slope by either 0 N a or ethanol treatment Carnevale and Wachtel d e m o n s t r a t e d the potassium d e p e n d e n c e of this region of the I - V t: The

/

25

20

]5

/-'°/

o/° / .//

•

e J

lo

5

V~ (mV) -80I

360~/0~o I

I//# lot

• ,

Ic

°

(hA}

-4,0 0

i

L

©

-5

-10

-15 aNASW eONa + ~ONo + +

-20 4%

EtOH

a

-25

Fig, 1 Effect of 4 % E t O H on the s t e a d y - s t a t e I - V c u r v e of cell R15 in 0 N a solutton The c l a m p c u r r e n t I c was m e a s u r e d at the end of a 5 s V C p u l s e to m e m b r a n e p o t e n t i a l V m T h e h o l d i n g p o t e n t i a l , V H was - 5 5 m V

effect of ethanol in 0 Na solution was tested on 12 other cells with similar results

Effect on n-shape m cobalt-containing soluuon The VC was unable in N A S W to control large currents and eliminate axonal spiking A solution with a Na concentration of 0.2 x normal (0 2 Na) was found to be convenient to use for two reasons It allowed voltage clamping of the cells at lower gains without escape of potential or spiking, and with improved spatial control It was also found to provide better recoveries after long experiments than did very low Na solutions This is p r o b a b l y because of a blockage of a N a - C a exchange p u m p and a build-up of internal calcmm as shown in 0 Na solutlons2,s4 In Fig 2 A the results of r a m p voltage c o m m a n d s with several different solution changes are shown Fig 2 A a shows a substantial n-shape in (l.2 Na Johnston has shown that cobalt will reduce Ca-de-

14{) /2

p e n d e n t tall currents to zero implying , b l o c k a g e ~r, 2

b

J

calcium entry ~' In Fig 2 A b ~t ts ~ecn th,~i the ,tdd~t,on

l]na

o t 2 5 m M Co blocks all n-shape o f l h c Lu n e n t r~'

sponse

24~

Fig 2 A c shows that a d d m o n o: 4 ~, e t h a n o l

to th~s solution caused an a p p a r e n t m¢rcasc m 1.,,~ -25mg

with a h n e a r l z a t l o n of the I - V

75m~,

! 1(}

•

T h e effec,~, of ( ' o and

e t h a n o l w e r e fully r e v e r s i b l e [Fig 2Ad}

T h e s e re~

sults w e r e seen in 4 s e p a r a t e e x p e r i m e n t s

Fhe c o m -

plete r e p l a c e m e n t of Ca with ( ' o . howe~ el or the use of 2 m M Cd m s t e a d of 25 m M Co gave s u m l a r results

5 I,

m 5 e x p e r i m e n t s but with p o o r e r r e c o ~ c l y

'/m ( m Y )

Fig 2B

shows the steady-state I - V curves which x~ere meas•

•

•

%

•

~o

o

~

8'

•

8

•

•

I

Ic

o

o

• ~o 2 N o + ~b •

~-I0

n o u n c e d effect on the inward current b e t w e e n -4H

I

and - 2 0 m V

2No+ + 2 5 r a M Co++

~,c 2 N o + + 2 5 r a M Co ++ + 4 % EtOH @ 1 5 I od 2No +

o

I.

end of a 5 s c o m m a n d pulse C o b a l t has the m o s t p l o -

i

o

'\~ m Fig

each p o i n t r e p r e s e n t s the current m a g n i t u d e at the

l-~,

A o

ured using s q u a r e pulse \ ' C c o m m a n d s

Fig 2 Effect of 4% EtOH upon the I - V cur,,es ot cell R15 during block of Ica with 25 mM Co A effect on nine varying I - V curves Inset shows typical recording of membrane potentml The cell was held at -75 mV and clamped to -25 mV by a triangular voltage ramp (dV/dt = 1 mV/s). (a) 0 2 Na solution, (b) 0 2 Na + 25 mM Co solution, (c) 0 2 Na + 25 mM Co + 4% EtOH solution, (d) after recovery in 0 2 Na soluUon B plot of steady-state I - V curves made subsequent to corresponding measurements shown above m A

It changes this region f r o m an n-shape

to a fiat p l a t e a u

T h e residual n-shape m 0 2 N a Is

therefore Ca-dependent

A d d i t i o n ol

4%

e t h a n o l ef-

fectively i n c r e a s e d Iout b e y o n d that which occurs with a block of calcium current

T h e steadv-~tate 1 - V ~s

c h a n g e d f r o m a p l a t e a u n e a r 0 n A b e t w e e n --4(I and - 2 0 m V to a relatively large l,ut m c r e a ~ m g in m a g m t u d e h n e a r l y with d e p o l a r i z a t i o n

T h e m c l e a s e d I,,~

c a n n o t be solely e x p l a i n e d b~ a d e c r e a s e in stead,,state Ca current as this current should be blocked by 25 m M C o T h e r e is also a very o b v i o u s shift m the I - V in the h y p e r p o l a r l z m g d i r e c t i o n smaller but also clear m Fig

ples of the actual m e a s u r e m e n t s of steady-state era-

V T (mY)

VH = - S b m V ~

£hls shdt is

I Fig 2C shows e x a m -

5~A~_~

l~

rent from which the curves in Fig 2B w e r e p r e p a r e d

2 sec

It is seen that 0 2 Na e f f e c t i v e l y e h m i n a t e d axonal esNASW

2 No +

2 No + +25raM

Co ++

2 No +

2 NO +

+ 2 5 r a M Co ++ + 4 % E*OH

cape and s p N m g seen in N A S W w i t h o u t having cornpletely e h m m a t e d the inward c u r r e n t

Effect on Na-dependent I,, VT : - 6 9

- 68

- 69

," ~

69

T h e Na spike c u r r e n t s m

Aplysta

h a v e b e e n shown

to be inhibited by T T X 42 0[ N a - d e p e n d e n t 1,, channels s e e m to be q m t e distinct f r o m Na spike channels because cells c o n t i n u e to display p e r i o d i c w a v e s of potential e v e n w h e n spikes h a v e b e e n b l o c k e d with -35

-36

-34

35

Fig 2C Sample measurements of currents in response to step VC pulses The cell was stepped to test voltage V r from V H = -55 mV Examples are shown of currents measured m response to depolarized (lower traces) and hyperpolartzed voltages (upper traces) Current measurements were made at the end of the 5 s pulses Note that 0 2 Na ehmlnates the uncontrolled spiking seen in NASW

T T X 3v T h e results of Fig

1 d e m o n s t r a t e d that N a

was not r e q m r e d in the bath for a g e n e r a l e t h a n o l etfect to be seen It was n e c e s s a r y to s e p a r a t e l y test the e t h a n o l sensitivity of N a - d e p e n d e n t I~, P r e v i o u s w o r k on effects o f e t h a n o l on N a spike c u r r e n t s gave indication that s o m e N a c h a n n e l s are particularly sensitive to e t h a n o p 4 ~ In o r d e r to m v e s n g a t e the

341 sensitivity of the N a - d e p e n d e n t Is, to ethanol it was

+30

A

• TTX Test

necessary to block the inward calcium current (Ica). G o r m a n and H e r m a n n found leakage conductance to increase greatly In 0 Ca solutions25. Large changes in

zx TTX Test + 4 % EtOH o TTX Test

,,/

~ zs

+20

leakage conductance are unsatisfactory in the investigation of small pacemaker currents as changes in leakage tend to be progressive with time and can eas-

7 +10 ]Na (nA)

ily swamp out m e a s u r e m e n t of true currents. As suggested from low Na experiments, the cells responded

X2:,:

to a solution with reduced Ca and Ca blockers with a much greater recovery and no change in leakage current The experimental m a n i p u l a t i o n therefore used for ehmlnatlon of calcium current was reduction of Ca from 11 m M to 2 mM and addition of 40 m M Mn. The T T X Test solution also contained 100 m M tet r a e t h y l a m m o n m m ( T E A ) and 4 m M 4-amlnopyrldine (4-AP) to block I~lv), the voltage d e p e n d e n t potassium current, and I a, the fast outward potassium currenP 2. Residual potassium current would be expected to be small, especially over the range of potentials used to Investigate Is,. H e r m a n n and G o r m a n have shown that 100 m M T E A blocks 90% of I~(v) 35, which at any rate does not activate significantly be-

L

i

I__

-80

L

I

-60

~

-40 Vm

I

I

0

I J-lO

I

-20 ©

I

-20

(rnV)

B Vq- : - 2 0 mV VH = - 5 0 mV _._~

jl~ 5s

low 0 mV The blockage of inward calcium current would by itself prevent activation of the IK(ca) In addition, H e r m a n n and G o r m a n have shown that I~(c~) is even more sensitive to external T E A than is IKIV)32. The fast inward Na currents are blocked with 30 u M T T X This is necessary to allow spatial control of potential without axonal spiking as Na concentration is kept high to maximize the N a - d e p e n d e n t Isl. The work of Futamachl and Smith showed that the Na-dep e n d e n t I~ channel was sensmve to very high concentrations of TTX2~ However. N a - d e p e n d e n t Is, was not markedly affected by 30/~M T T X m the present study' whtle T T X completely blocked spiking, the steady-state I - V relation in T T X (Fig. 3A) still retains ItS n-shape. Therefore if N a - d e p e n d e n t I~, is sensitive to T T X It is much less so than is Na spike current. Fig. 3A demonstrates that 4% ethanol reversibly blocks the N a - d e p e n d e n t Is, The decrease in T T X Test solution of i~, with voltage c o m m a n d s to potentials more positive than - 3 0 mV is not apparent in the recovery curve This decrease is most probably due to incomplete blockage of the C a - d e p e n d e n t I~, This is also suggested by the outward tail current in Fig. 3Ba which is usually indicative of IK(Ca) (ref 48) This tall current was not observed in the subsequent

a

b

c

Fig 3 A effect of 4% EtOH upon Na-dependent I,, of cell R15 TI'X Test solution was designed to block all voltage-dependent and calcium activated outward current and inward Ic~ (see text) Since the solution contained high Na concentration, TI'X was added to allow voltage clamping w~thout axonal escape The level of sodium current, IN~, was measured in TI'X Test solution (filled circles) after addmon of 4% EtOH (triangles) and after removal of the ethanol (open circles) B sample records of measurements from which curves m A were made Points in A were the current level at 5 s Note in (b) the increased outward holding current m 4% EtOH The inward current decays m (a) with time, which it does not after recovery in (e) This was ascribed to continued presence of IK~Cd~as evidenced by the outward taft current after the end of the command pulse m (a)

solutions. In Fig. 3Bc, the sample curve for recovery did not possess a tall current and the Is, fully recovered The N a - d e p e n d e n t Is, did not noticeably inactivate over the 5 s c o m m a n d pulse. This indicated that the Icd was fully blocked and that the IK~caI was not activated The steady-state lout measured in T T X Test + 4% ethanol, as seen in Fig. 3Bb, was therefore mainly leakage current. In a similar experiment

4_ on an L4 cell, 4% ethanol slgnlficantl~ diminished -Stl

the N a - d e p e n d e n t I,.

Effect on Ca-dependent 1,,

GO

4qq

• o"

d'

I,

\ \X

I J ~t,

The C a - d e p e n d e n t c o m p o n e n t ot I,, was tested independently for sensitivity to ethanol

IOn~,[

In order to

prevent the posstbihty of a change in lore masking the

];

true effect on C a - d e p e n d e n t Is,, extensive precautions were taken to minimize all sources ot l,,ut As

10nA[ ~ / / ~

described above, 100 m M T E A and 4 mM 4-AP were again employed to block IK(Vl, I~, and IK(C~, Further

40 nA[ ~ / ~ - 0

precaution against IKicaI was taken by replacing calcium with barium. Barium has been shown to substitute for calcium in carrying reward current 27 but not to substitute in activation of IKcc,, ~ G o r m a n , Hermann and Thomas previously studied l~i wtth inJection of E G T A and external T E A and 4-AP :(~. They found this procedure causes a 10-fold Increase in the magmtude of the I~, suggesting that the I,, ~s normally masked by I,,ut. Calcium channels at rest could be at least partially inactivated, as Tillotson has shown that calcium channels undergo an inactivation d e p e n d e n t on internal Ca concentration6a The posslbthty existed that the experiments which showed the anomalously large I,, were erroneous m that the mlected E G T A might act to reduce internal calcium concentration and remove e n d o g e n o u s inactivatton of the calcium channels In order to avoid this posslblhty, outward potassium current was blocked by removal of internal K and replacement with cesium Cesium IS not permeable through potassium channels ~ Nystatln. a bacterial lonophore, was used to allow cestum to enter the cell as the potassium was washed out The procedure used was identical to that described by Tillotson 63 The possibility that this procedure did not completely elimmate outward potassium currents can be seen in the msets to Fig 4A. The fast mltlal decay of this current could indicate turn on of outward currents due to incomplete blockage or more probably inactwatlon of calcium channels Repetition of pulses at an interval of less than 1 rain did show a significant mactivatlon of this current The outward tad currents were probably due to residual lj¢~c,,) The apparent decay of inward current was therefore probably at least partially due to activation of IK(ca) Fig 4B shows the difference between the I~, measured m Cs Test solution and that m Cs Test + 4% ethanol. The difference current or the blocked

1

• r s Test

\5 '

ACs Test + 4% EtGH

'-'~L

~0

if4- 2(10

C5 Test

5~ m,,' ~ J ~ L 4

B t

V~ -80 • ,it/•

60

,mT~ -4u

-2CJ

•\ \

I 4,,

\

\

,~AJ 4 -6CJ

6'0 \

I

Fig 4. Effect of 4% EtOH upon Ca-dependent i,, of cell L3 A cesium loading and external TEA and 4-AP were used for blockage of outward current and external soluuon contained 10 mM Na to effectively ehmlnate reward sodium current The cell was g~ven 1 s command pulses to potential Vmat 1 mm intervals The points plot the maximum reward current measured. This procedure revealed a large Ca-dependent I~, m Cs Test solutmn (closed circles), Cs Test + 4% EtOH (mangles). and after removal of the alcohol (open circles) Insets shot the results of a voltage step to -24 5 mV m the 3 solunons The decay of the Is, and the outward taft current are explained m the text B. the I~,ehmlnated by 4% EtOH The points plot the difference between current measured m the original Cs Test solution and after addmon of 4% EtOH The current ehmmated appeared to have the voltage dependence of Ca-dependent I,, V~ = -55 mV

current has a threshold of about - 4 0 mV and mcreases monotonically to 0 mV. It ts clear that ethanol acts in this solution to block the C a - d e p e n d e n t 1~, The It, is reduced to almost half normal size in 4% ethanol The decrease is seen in Fig. 4A to be fully reversible. This same procedure was used in two other experiments with similar results. In 3 other experiments Cs was loaded into the cells by depolarizing them in a saline m which Cs was substituted for Na and K. Although this procedure did not ehcit as complete a block of the outward currents, it also showed that ethanol decreases C a - d e p e n d e n t It,

343

Effect on ll~k M o o r e et al. did not find an effect of ethanol on resting m e m b r a n e potential in squid axons 49 A r m strong and BInstock found a depolarization of resting potential which they ascribed to a decrease in resting K conductance 3 B e r g m a n n et al m e a s u r e d changes in resting m e m b r a n e potentialS. In the u p p e r left q u a d r a n t bursters, L 2 - L 6 , they found 7 out of 12 cells were depolarized by ethanol while the remainder were unaffected. This was in contrast to their finding that 4% ethanol caused a hyperpolarlzatxon of 5 - 1 0 mV in R15 In almost all the cells tested, when 4% ethanol was a d d e d to the bath there was a noticeable hyperpolarlzatlon or under VC an increased Iota. F o r e x a m p l e , in Figs 1, 2 and 3 there is an a p p a r e n t hyperpolarizxng shift in the I - V relation with ethanol application. The fact that this 'shift' was seen in 0 Na, Ca 2+ blocking, and T E A containing solutmns implied that It was not exclusively d e p e n d e n t on a decrease in Na or Cad e p e n d e n t I~, or on an increase in IK(Ca) or IKIVl. Five possiblhties were tested in turn as explanations for this a p p a r e n t shift in the I - V : 1 0 s m o t w effect. A 4% ethanol solution is 688 m M m o r e concentrated than N A S W If the a d d e d ethanol acted as an ordinary i m p e r m e a n t solute it would draw water out of the neurons This would cause a sharp rise in internal calcium concentration The inward driving force on calcium would be decreased and calcium current would be expected to decrease The internal potassium concentration would be mcreased This would increase the driving force on outward potassium current, increasing the magnitude of all potassium currents The cell therefore would be h y p e r p o l a n z e d at rest F r o m the results p r e s e n t e d above ~t would a p p e a r that the osmotic effect is an attractive hypothesis for ethanol action This was ruled out for two main reasons' (1) N A S W was m a d e hyp e r o s m o l a r with 680 m M sucrose. This solution caused the cells to be severely injured as they contracted and tore their m e m b r a n e s against the microelectrodes This h~gh concentratmn was a t t e m p t e d on 3 cells, none of which could be r e c o r d e d from for more than a few m m This p r o b l e m was never encountered with ethanol which was tolerated by cells for up to 6 h (2) The partition coefficient of ethanol in lipid is about 0 04 (ref 51) The fact that ethanol is so soluble in hpld means that it should equilibrate

across the plasma m e m b r a n e with no dlfflcult~ LI also concluded that ethanol does not act via an osmotto effect as it freely diffuses across cell membranes 40

2 Frankenhaeuser-Hodgkm shtft F r a n k e n h a e u s er and H o d g k i n showed that for a decreased external Ca 2+ concentration the I - V curve is shifted in the hyperpolarizlng direction > It has been p r o p o s e d that external Ca > decreases the effecUve transmembrahe potential by bandangl9 2_" or screening ~4 The depolarization n e e d e d to activate the same amount of current is therefore reduced Ross has reviewed the evidence that ethanol affects the surface charge density of Nologmal m e m b r a n e s > The diffuse double layer theory predicts that an increase m negative surface charge density would increase the surface potential 4a The hypothesis that ethanol decreases binding of Ca 2+ to the external m e m b r a n e of Aplysm neurons was tested by examining the change m resting m e m b r a n e potential between the solution Mg Test + 4% ethanol and the solution High Mg Test + 4% ethanol High Mg Test solution should c o m p e n s a t e for increased surface charge due to ethanol by increasing the divalent concentration, greatly increasing the screening of surface charge (without adding inward Ic~). Although the total &valent concentration m the High Mg Test solution was nearly 200 raM, there was no change in cell resting m e m b r a n e p o t e n u a l between these two solutions H o w e v e r , the~ both caused a hyperpolarizatlon when c o m p a r e d to these solutions without ethanol It was concluded that the ethanol induced h y p e r p o l a n z a n o n was not due to a F r a n k e n h a e u s e r - H o d g k l n shift 3. Effect on electrogentc pump The active N a - K exchange mechanism contributes significantly to the resting m e m b r a n e potential in Aplysm neurons12 The hyperpolarIzatlon caused by alcohol was still present in a solution containing both 0 5 mM o u a b a i n and 0.5 m M strophanthldln. It was concluded that the increased Iout w a s not attributable to ethanol stimulation of the electrogemc p u m p This does not preclude the possibility that some unknown electrogemc p u m p might be affected by ethanol 4. Effect on leakage current A n o t h e r possible explanation for the hyperpolarIzatlon would be a change in leakage current. In order to test this hypothesis it was necessary to block all other currents The test solution had 10 m M Na to e h m m a t e IN~ and 2

344 A

the h y p e r p o l a n z m g shift in the l-'V cur'-.'s is an Intct

'1-~

2ic

nal release o I calcium lonb

i

__~.--

~.~/

~ o

7-

_>.~d/oA:2---io

.f.fo._-o__o.~~

tive surface charge on the inside surface ol the plas-

~

'-°

.~le~ >"

I~ {nA 1

4- 5

ma m e m b r a n e - - d e c r e a s i n g the t r a n s m e m b r a n e potential. Less d e p o l a r t z a t l o n might then be n e e d e d to

d"o¢

/

(dlClllIll

n o n site It could also act directly by s c r e e n i n g nega-

-v . . ~ "

,

increase in

IK(Ca ~activated at rest by direct b m d m g to the activa-

v. ImV,

-x_

tktl

c o n c e n t r a t i o n w o u l d c h a n g e the stead}-'date le~ el ol

acttvate lK(Ca I channels. T h a t IK{C,~ c o n t r i b u t e s to the resting p o t e n t i a l had b e e n s h o w n with E G T A inlec-

°&¢ o/

o

!

a y + 4% EtOH

I

A •

2No

A 2 No + 4.% EtOH

B ~

- ~

-61O

Vm (mk,) -40

o 2Na

.. • • "" . . . .o o - .o- - o - -o~

--o• •

lr

-20 '--'--I -

/

o--°--°--~-'8--~

~-5 I0

•

i

0

1

tnA)

y(o) - (Y+ 4% EtOH)o

1-10

Y(ol - (Y+ 4% EIOH)o

-15

Fig 5 Effect of 4% EtOH upon leakage current of cell L4 Solution Y contained low Na to eliminate active Na current and low Ca, 4 mM Nl, and 100#M verapamd to block Ic. Outward current was blocked with 100 mM TEA and 4 mM 4-AP A. VC pulses of 5 s were used to determine the steady-state I - V of the leakage current in solution Y (open circles), Y + 4% EtOH (triangles), and after removal of the alcohol (closed circles) B the plots of the leakage current in solution Y minus the leakage current in Y + 4% EtOH are shown for the two cases above The broken line intersects the voltage axis at +50 mV V H = -55 mV

/ / o

,d p

~•

%.

\

/-g

o

~- IU

\

/,'// //

\

,

.<

\./

o

- a("

-80

-60

-4(~

-20

Vm (rnV ;, m M Ca and 4 m M Ni to b l o c k Ic~. V e r a p a m i l was a d d e d to f u r t h e r aid in b l o c k i n g Ica 1. S o l u t i o n Y furt h e r c o n t a i n e d 100 m M T E A

and 4 m M 4 - A P to

B

Vr : - 3 2 5 mV I--L__ '~/H : --50 mV - - ~ 5s

block IK(V) and I A. Fig 5 shows the results o f an exp e r i m e n t on an L4 cell. In test solution Y the cell was d e p o l a r i z e d to a l m o s t - 1 8 m V . T h e resting p o t e n t i a l b e c a m e - 5 3 m V in Y + 4 % e t h a n o l with a c h a n g e in the I - V o v e r the full r a n g e of p o t e n t i a l

This effect

on l e a k a g e c u r r e n t is s e e n to be quite r e v e r s i b l e Fig. 5B plots the c o n t r o l l e a k a g e c u r r e n t m i n u s the l e a k a g e c u r r e n t m 4 % e t h a n o l . This d i f f e r e n c e s h o w s that e t h a n o l did r e d u c e an i n w a r d l e a k a g e c u r r e n t . All 11 cells t e s t e d s h o w e d a d e c r e a s e d l e a k a g e cond u c t a n c e with 4 % e t h a n o l application.

5. Effect on znternal Ca 2+. A n o t h e r possibility for

2 No o

2 No + 4 % EtUH b

2 No c

Fig 6 Effect of 4% EtOH upon [ C a 2 + ] , of cell R15 lontophoresls was used to inject 0 5 M EGTA into the R15 cell InJection current was approximately 700 nA over 7 rain During iontophoresls, the holding current gradually became less outward A. plot of the maximum inward current elicited dunng a 5 s VC pulse B. sample records of traces from which the curves in (A) were made Note the increased outward holding current with ethanol application V H = -50 mV

345 tlons causing a d e c r e a s e in i n t e r n a l C a -'+ c o n c e n t r a -

A

600

tion with an a t t e n d a n t inward shift in h o l d i n g cur-

oZ

rent 5 F o u r bursting p a c e m a k e r s w e r e i n j e c t e d with

oz

E G T A and gave s i m d a r r e s p o n s e s to 4 % e t h a n o l In Fig. 6 an R15 cell was held at - 5 0 m V and 0 5 M EGTA

was

injected.

Iontophoresis

current

n

/

/y

,5 Z + 4 % EtOH

/

400

was I C

a b o u t 700 n A for a b o u t 6 mln T h r o u g h o u t the Injec-

(hA)

tion p e r i o d , the h o l d i n g current steadily d e c r e a s e d This i n d i c a t e d that the E G T A

had e n t e r e d the cell

200

and d e c r e a s e d the e n d o g e n o u s IN(Ca) T h e results s h o w that t h e r e was a large shift in the I - V In 4 % ethanol. In b o t h Fig. 6 A and 6B It is s e e n that e t h a n o l also caused a large d e c r e a s e m C a - d e p e n d e n t I,, If the E G T A

I

is sufficiently high in c o n c e n t r a t i o n it

A,~-~CDAa--a"~'-40

should act to c o m p l e x Ca -`+ b e f o r e it can activate iKic ~a 4; T h e fact that in Fig. 6B t h e r e are no large o u t w a r d tail currents s u p p o r t s this c o n c l u s i o n

As

E G T A should act to p r e v e n t an increase m internal

Ca-'*, the fact that the h y p e r p o l a n z m g shift still oc-

I

__ L

-20 Vm

t 0

(mY)

B VT : -14 rnVl VH : - 5 0 m V ~

c u r r e d l m p h e s that the shift is not c a u s e d by an m-

l

05s

?~-

c r e a s e d internal Ca 2+ c o n c e n t r a t i o n activating IKICa) or d e c r e a s i n g I Q

T h e r e d u c t i o n in Ic~ as s e e n in

Fig. 4, t h e r e f o r e , also c a n n o t be e x p l a i n e d solel) b) i n c r e a s e d internal C a 2+ c o n c e n t r a t i o n causing dec r e a s e d Ca -'+ driving force or i n c r e a s e d C a - d e p e n d ent i n a c t i v a t i o n of c a l c m m c h a n n e l s (,~ T h e g e n e r a l i z e d effect of e t h a n o l to cause a s e e m mg shift in the I - V is not due to an effect on the osmotic strength of the e x t e r n a l solution, the e l e c t r o g e ntc p u m p , the internal Ca -'+ c o n c e n t r a t i o n , o r the external surface c h a r g e

The most reasonable mecha-

nism for the h y p e r p o l a r l z a t i o n of u n c l a m p e d n e u r o n s is the d e c r e a s e of a non-specific inward l e a k a g e conductance

Effect on voltage-dependent I.., T h e large I~, in Fig 4 is usually almost c o m p l e t e l )

a

b

c

Fig 7 Effect of 4% EtOH upon ,~oltage-dependent potassium current of cell R15 Test solution Z contained low Na to decrease INa and low Ca and some Mn to block Icd The outward current then consisted of contributions ot I~,, Ik(v~ and leakage current A points plotted show the peak outward current during the 0 5 s test command pulse in solution Z (closed circles), Z + 4% EtOH (triangles) and after removal of the alcohol (open circles) B sample records of measurements from which curves m A were made Ba outward current m test solution Z The lack of tall current showed full block of Ik( C~} The outward current decayed sigmficantly urmg the 0 5 s pulse Bb outward current after addmon of 4,c¢cEtOH The current peaks were mcreased and decayed at a more raDd rate Bc recovery of outward current levels to control values after remo,~al of EtOH V H = -511 mV

o p p o s e d by a large o u t w a r d c u r r e n t -'6 This raised the posslblhty that the o b s e r v e d d e c r e a s e in Is~ c o u l d explain the i n c r e a s e d Iout as s h o w n for e x a m p l e in

without axonal escape R e s i d u a l N a c u r r e n t w o u l d be

Fig 1 T h e effect of e t h a n o l on v o l t a g e d e p e n d e n t

t o o low to h a v e an effect on the results shown. T h e

Iou~ was t h e r e f o r e i n v e s t i g a t e d in 4 cells in which alm o s t all inward c u r r e n t was e l i m i n a t e d by use of a so-

p e a k Iou t is p l o t t e d in Fig 7 A lou t was greatly increased in the cell with application of 4 % e t h a n o l

lution containing 90 m M Na, 2 m M Ca and 40 m M M n

T h e cell also s h o w e d a full r e c o v e r y Fig. 7B shows

Fig 7 shows the effect of e t h a n o l on Ioot of an R15

e x a m p l e s of c u r r e n t m e a s u r e m e n t s for this cell T h e

cell T h e control s o l u t i o n b l o c k e d i n w a r d Ica so that

c o m m a n d pulses w e r e 0.5 s l o n g and lout d e c a y e d to

Iota is a s u m m a t i o n of Ileak, IK(Vl, and I A T h e N a conc e n t r a t i o n was sufficiently low to allow V C control

less than half of its p e a k v a l u e in this t i m e T h e t i m e for lout to fall to l/: its p e a k v a l u e (t,_3 was 0 28 s in

34h 7Ba. 4% ethanol increased the lnactlvatum rate m 7Bb decreasing t,, to 0 18 s W,th remowd ~d ethanol t recovered to 0.34 s. All 4 R15 cells tested showed an Increased p e a k current and an Increased rate of mactivation Bergmann et al , working on unldentlhed Aplysm neurons, also found an increased macnvatlon rate of iout in ethanol but described ~ts peak magmtude as relatively unchanged ~

E f f eonc tIKf ¢aj IKIC~~has been shown to be activated by accumulation of calcium ions in the cytoplasm directly Inside the cell m e m b r a n e 2~. It was necessary to use VC techmques to d e t e r m i n e the effect of ethanol on this current in Aplysta neurons. This current can be studmd under VC by ellciting calcium entry through the celt m e m b r a n e with depolarization 48 or by intracellular injection of calcium ions 4s,a~ While these techniques activate Identical channels they do not ymld identical ln/ormatton. R e c e n t work by Barish and T h o m p s o n for example showed that carbonyl cyanide m-chlorophenyl hydrazone ( C C C P ) , whmh uncouples mltochondrlal oxidative p h o s p h o r y l a t l o n , did not alter the time-course of tall currents following step depolarizations, while it did alter the ninecourse of 1KIC~} activated by Ca 2+ lontophorests 5 The question arose as to how ethanol would effect IK(Cat and whether It, like CCCP, would affect IK(C,,~differently d e p e n d i n g on the m e t h o d of ehcltation IKIC,~as activated with a voltage c o m m a n d is p r o b a b l y terminated by buffering systems akin to those which buffer a calcium load provided by a burst of action potentials This ehcltatlon of IKICa~ ordinarily provides a more realistic calcium load to the m e m b r a n e than a Ca injection Results with 1K(CaI activated b y a voltage pulse, however, are distorted by the specific m a r k e d decrease in [Ca during the study of ethanol effects Both methods were used to trx to cancel the drawbacks inherent in each 1 IK~C~J activated by Ca mjecnon A third electrode containing 0.5 M CaCI 2 was inserted into a cell which was then voltage clamped Ca was injected iontophoretlcally. InJection current pulses ranged from 100 to 500 n A and were 5 s in duration Several backing pulses of this same magnitude were applied before each injection pulse. This p r o c e d u r e aided in clearing the tips of the lontophoresls electrode and p r o d u c e d cleaner current records

Two examples of this type ot cxpcrmlent _tie shown In Fig 8A1 and 8BI Both celN x~ere held :it -40 mV and given a Ca2* intecnon tor "~ , Fhe ttmtt> phoretlc current was 200 nA. In both cell- the nlagmtude of Ik(fa ) was greatly increased b~, apphcatum of 4% ethanol This result was tully rever~tbtc The mcrease in lK(ca ) elicited by lontophoresls ~as {ound no matter what the holding potentml I~,) cells were held at - 5 0 mV, 3 cells at -40 lnV, t ~ o ceils at 45 mV, and one cell at-2(} mV l'he average increase by ethanol in the 9 cells was 3 3 2 l 7 (S D ~t an observation) times the control level. The time lnter~al from the cessation ol lontophoretlc current until the peak IK<,, }was increased in all 9 cells. This effect was not as reversible as was the effect on magmtudc The time to peak is an indication of the rate ot activation of 1K¢C~~, the buffering properties of the cytoplasm. and numerous other factors such as poslt~on ol the lontophoresis electrode :4 Also lndlcah~e of the butferlng properties of the cytoplasm is the rate ot decay of IK(ca ) Potassium accumulation shouM not affect the rate ot decay as E a t o n showed that accumulation does not occur for outward currents les~ than 200 n A 16 Most mathematical models ot the decay ot IK(Ca} describe ~t as falling with twe tmle constants 2",5s The time for the current to tall to ~,2 its peak value (t~2) was considered here to b c a general measure of the Ca e+ removal processes In Fig 8A1 the t~: of R15 was increased b~ a factor o t t 4 by ethanol. In 8B1 the t,, of L6 was increased b~ the same factor. In the 9 cells tested, t,~ was Increased 3.i _+_ 2 6 (S D of an observatlonl times b~ 4% ethanol Therefore ethanol increased the magnitude of l~{c~ and decreased its rate of decay when activated by a constant Ca 2+ injection 2. IK(c,. acm,ated t)3' VC c o m m a n d While it was clear that a constant Ca > injection ehcJts a much larger IK(C~I In an e t h a n o l - t r e a t e d cell, ~t would not be possible to predict how ethanol would affect this current when activated by a VC c o m m a n d A~ shown in Fig 4 the Ic~ activated by a VC c o m m a n d would be greatly decreased by ethanol As shown above, howe v e r , IK(ca ) lS more sensmve to a given dose of Ca 2+ in ethanol-containing solution The net effect ot ethanol on IKic~ I elicited by a VC c o m m a n d would be a summation of those opposing factors Fag 8 shows that L6 and R I 5 exemplify the two opposite posslbfl> ties As with Ca 2+ mlectlon, m L6 ethanol increased

347

R]5 2,

B]

L6

L OOn,~

__]

[200 nA

/X

c5 S

B2

A2 -30 mV -40 mV-"-~,5s

-33 mV - 40 mV"--'I 5s ,'-'-"

As

Bs - 3 0 mV

-4o v

- 2 0 mV

-4o v

5s

5s

lOnA

NASW

N A S W + 4 % EtOH

NASW

N A S W + 4 % EtOH

Fig 8 Effect of 4% EtOH upon IK(Ca) of ceils R15 and L6 A1, B I the effect on IK~c~I ehclted by lontophorests Calcmm ~ as injected for 5 s into the cells with a 200 nA pulse Measurements were made of the peak reached after cessation of the rejection pulse A2 Iklcd j was ehoted by a VC pulse m the same cell seen m (At) and in another cell R15 (A3) IK~c~)tollowmg the pulse ~as non-measurable m both cells B2 Ir, lc~) was ehclted by a VC pulse m the same cell seen m (BI) and in another cell L(~ (B3) Amphtude and rate ol decay of I~,<4 increased m both cells Measurements of t~: were made starting from the time ot the peak The full records are no~ shown here for space reasons The mserts show the value of the VC command pulse and the t~me scale V H = -49 mV

t h e p e a k lKica ~ 6.3 t i m e s m Fig. 8 B 2 a n d 1 6 t i m e s in

r e d u c e d to a level t o o s m a l l to m e a s u r e t h e t l m e c o n -

F~g 8B3 E t h a n o l also i n c r e a s e d t h e r a t e of IK~c4 de-

stant

cay

In Fig 8B2 tl,2 d e c r e a s e d b y a f a c t o r of 0 28,

3. Effect on Ix(c~) as a funcUon of holding potential

while m 8B3 t~,~d e c r e a s e d b y a f a c t o r of 0 42 T h e re-

IKICd~ a c t w a t l o n is d i r e c t l y d e p e n d e n t o n v o l t a g e in

sults s e e n m R15 w e r e

addltxon to [Ca2+], (refs 30, 41)

much

different

In b o t h

A full u n d e r s t a n d -

current

ing of e t h a n o l ' s a c t i o n o n IK(c~ ~t h e r e f o r e r e q u i r e d in-

during the command pulse was greatly increased, the

v e s t i g a t i o n of a l t e r a t i o n s in t h i s c u r r e n t ' s v o l t a g e de-

tail c u r r e n t m a g n i t u d e at t h e e n d o f t h e V C p u l s e w a s

pendence

Fig 8 A 2 a n d 8 A 3 , a l t h o u g h t h e o u t w a r d

Gorman

and Thomas

m a d e C a -`+ m j e c -

348 uons and measured IK(c~~ and arsenazo II1 light absorbance (related to internal Ca :+ concentration) as a function of VC holding potentlaP(' There was no effect of voltage on the a m o u n t of dye absorbance, but the magmtude of IK(c~) was profoundly affected Ca 2+ activated potassmm conductance (GKIt,H) and had an exponential variation with holding potential These researchers concluded that the Ca 2~ binding site of the IK(Ca) channels is halfway through the m e m b r a n e and therefore subJect to potential effects

G o r m a n and Thomas were able to quant~tatp, el) ~clate GKIC,,I to holding potential (VI, ,Iv. frachonal distance of the Ca z+ binding site m th~ metnbranc (6), and a measure of the internal calcmm concentr,, tlon (zlA) x0 Their equation {3) In G~qc,a~ = z F d V / R T + lmJA

tl}

(where z is the charge of the Ca '+ ion and k, R and T are the usual thermodynamic quantities) is derived from the simple assumption that the reaction between Ca 2+ and the binding site is a first order reaction and that each K + channel is opened by a single

ffso

A

0 Z + 4%

Ca 2+ It is clear from equation (1) that the slope of the line In GKICa/ VS V may be used to obtain d or the

EIOH

/

~K(CcI) (nA)

dmtance of the site through the m e m b r a n e , while the zero-intercept of the line will give the measure of internal Ca 2+ concentration It was therefore possible to test the effect of 4% ethanol on 6 and [Ca2+], independently by measuring 1K((.~ as a function of holdmg potential Fig. 9A shows the results ot measuring peak IK(Ca) magnitude as a function of holding potential Current was measured not as absolute current but as the Increase above holding current Slmdarlv to Fig 8, IK(Ca) can he seen to be greatly increased w~th alcohol treatment Fig. 9B shows typical current measurements from which the curves were made.

-60

-40

-20

0

The potassium reversal potential (EK) was assumed

VH (mY)

to be equal to - 7 0 mV With the further assumptmn that the Instantaneous I - V for IK~C~~IS hnear ~u use ol

B

the e q u a h o n GKICa) = IK~c.{(V-EK) ~s valid and it was possible to convert the information m Fig 9A to that in Fig 10 Straight hnes were fit to the curves by eye As A A is independent of holding potenttal then

F ] + ] 7 5 nA Iionto--~JL-5S VH =- 40 mV

] 4 nA

-/b-VH=-20mV

#~~

Z

if GK(c. I increases e-fold for a change in holding potentml of AV ] j4nA

~ X j~ ~

A

Z + 4% EtOH

Fig 9 Effect of 4% E t O H upon voltage dependence ot IKtca ) Of cell R15 The cell was clamped to a p a m c u l a r V H and then rejected with 0 5 M CaCI 2. Iontophoretlc current was 175 n A

for 5 s After IK/C~) had returned to baseline or a steady level, Vu was mcreased to the next value Test solution Z contained Mn to prevent entry of external Ca as the cell was depolanzed The points plot the peak IK(C4measured after the end of the injectmn pulse in solution Z (closed circles) and m Z + 4% EtOH (open circles) B' sample records of traces for VH = -40 mV and-20 mV

6 = RT/zFA V

(2)

At 22 °C 6 = 12 7 mV/A V The slope of the line is decreased in 4% ethanol from an e-fold change in conductance in 19.9 m V to an e-fold change in 50 5 mV This ts ln&catwe of 6 being decreased or the Ca2+ binding site being shifted closer to the surface of the membrane. From equation (2) 6 changes from 0.64 to 0.25. The zero-intercept changes from 6.0 × 10-7 to 1.2 x 10-6 This imphes a d o u b h n g in [Ca-'+],. This experiment was performed on a total of 5 cells with

349 and added M n do effectively block IK(Ca), as was indi-

y

o~

cated by the lack of tall current in Fig 3Bb and 3Bc. Even if there was a small residual I~/c~ }as ehclted by voltage commands, the results in Fig 8 show this

, '°-6

jol

should be further reduced by ethanol in R15 cells.

f/

Second, T T X Test solution, which contains 100 mM

GK(co) o J

°

(s)

T E A , was expected to reduce outward currents over 90% 35. Fig. 7 showed that in 0.5 s. lout decayed quite sigmflcantly and while ethanol increased the magnitude of IouI it also increased its decay rate Thus. it would be of a comparable size or smaller at 5 s than Iout without ethanol. It is concluded then that Fig 3 shows that the contribution of N a - d e p e n d e n t I,, channels to neuronal activity is directly decreased by etha-

oZ oZ + 4 % Et0H I

-60

I

[

I

-40

I

-20

nol

I

10 -8

0

V H (mY) Fig 10 Effect of 4% EtOH upon voltage dependence of

OK~c~~of cell R15 The data m Fig 10 were analyzed as described m the text and replotted as conductance values Note the log scale for GK(c.) The line drawn for GK(Ca) m solution Z (closed circles) shows an e-fold change for a 19 9 mV increase m VH The hne drawn for GK~c~) in Z + 4% EtOH shows an e-fold change for a 50 5 mV increase m VH

essentially identical results. In the control solution d was 0 49 + 0 16 (S.D. of an observation) and after 4% ethanol application was 0 23 + 0 04. [Ca2+], was increased 56% in the 5 cells from 11.4 x 10-7 + 11 2 x 10-7 to 17.8 x 10-7 + 16.2 x 10-7. DISCUSSION

G o r m a n et al revealed a C a - d e p e n d e n t lsmof up to 300 n A in R15 by blockage of lout with T E A and 4-AP and further removal of IKcc~/ with injection of EGTA26 This current was more than an order of magnitude greater than an)' previously measured I,, In these cells They concluded that the N a - d e p e n d e n t Is, was u n i m p o r t a n t when compared to the Ca-dep e n d e n t component. The Cs loadmg procedure "~ combined with barium substitution for calcium gave a solution in which inward current was very similar in size to that measured by E G T A injection (up to 220 n A ) This confirmed the result that the magnitude of the Ca c o m p o n e n t of Is, is much larger than the Na component. With the outward currents so efficiently blocked. Fig. 4 showed that the slow inward calcium current is also greatly reduced by 4% ethanol Therefore. the inactivation with time and the large size of the C a - d e p e n d e n t I~, are the major differences be-

Effect on reward currents Researchers have claimed the Is, to have both Na and Ca dependencies. The effect on the I~, therefore was examined by blocking all outward current and then eliminating either Na or Ca flow. Blockage of Ca current revealed that there is a large Na-dependent Is, This current can be a constant source of depolarizing drive at subthreshold potentials as it does not appear to inactivate (Fig 3B) There was a total elimination of N a - d e p e n d e n t Is, with 4% ethanol and it was replaced with an outward current. It is unlikely that all of the disappearance of N a - d e p e n d e n t Is, can be explained as a specific increase in outward current for two reasons First, outward current is expected to be blocked in this solution Solutions with reduced Ca

tween it and the N a - d e p e n d e n t Is, Ethanol could act (see below) to cause lntracellular release of calcium. This could cause an increase in [Ca2+]~ which would increase inactivation of Ica~ These effects could not explain all of the ethanol-induced decrease in Ca-dependent Is, as this current was shown in Fig 6 to decrease even when an intracellular increase in [Ca 2+] is prevented by mlectlon of E G T A Ethanol must therefore act directly on calcium channels to decrease calcium current

Effect on leakage current As described above, the most pervasive result in all cells tested under all ionic conditions was the apparent hyperpolarlzing shift in the I - V Several pos-

750 slbihtles were ruled out as the basis for this p h e n o m e non, which was hnally ascribed to a decrease m leakage conductance. Fig 5A showed that 4% ethanol ehclted a large shift in the 1 - V lor leakage current The straight line fit to the closed circle difference points in Fig 5B gave a reversal potential for the mward leakage current (El~ak) of +5(I mV The leakage conductance for the m e m b r a n e can then be modelled with the parallel conductance model with Rleak being the resistance of the leakage path tor reward current and R K being the resistance to leakage for outward current. If Eledk IS +50 mV and it is assumed E K is - 7 0 mV an RI~k/RK ratio of I 3 would give a resting m e m b r a n e potential o f - 1 8 mV corresponding to that found in solution Y A n increase in Rl~k by a factor of 4 7 to give an Rleak/R K ratio of 6 1 would be sufficient to explain the hyperpolarlzlng shift of resting membrane potential to - 5 3 m V This non-inactivating inward leakage conductance supplies constant depolarizing drive to the p a c e m a k e r neurons Silver and Trelstman tested the effect ot ethanol on silent neurons 57 A n examination ot their l - V curves of silent cells did not show a hyperpolarization shift This is as expected as silent cells do not possess this steady depolarizing current. A similar parallel conductance model was p r o p o s e d by Noble for the background current in Purklnle fibers s° He postulated that the persistence of a d e p o l a r l z a n o n in these fibers in Na free solutions was due to a p e r m e a b l h t y of the inward leakage channel to Na substitutes Recent work on cultured cardiac cells ~~ and mouse n e u r o b l a s t o m a 65 has shown the existence of single Ca :+ activated channels which are essentially non-selective in permeability to m o n o v a l e n t cations and are n o n - v o l t a g e - d e p e n d e n t These channels could support an inward current identical to the reward leakage current in Aplysta bursting cells F u r t h e r study is necessary to d e t e r m i n e if ethanol decreases the leakage current by an effect on such channels

Effect on outward current magnitude Figs 7 and 8 show the effect of 4% ethanol on outward current. Fig 7 shows that the total current IK(v~ + 1A + Ileak IS increased, and that the rate of decay of this current IS also increased so that aster about 0.5 s both control and ethanol t r e a t e d o u t w a r d currents are of the same magnitude This IS an i m p o r t a n t result for the Interpretation of o t h e r experiments as it

demonstrates that the increased outward currcn~ ;v the steady-state I - V is not due to these cu~ rents Ethanol was shown by C,t ~'* mjecnon ~o mcrea,~e the IK~(a ~ ehclted by a constant dose ,,J calcmm While this result lmphes that 1~,1¢dJ channels are more sensitive to Ca -,+ in the presence of ethanol, the decrease ol Ic,, by ethanol is more than sufficient m some cases to actually decrease IKIc,,I elicited b~ a voltage command. This is probably the case In R15 cells Recent work by K r a m e r and Z u c k e r has provided evidence that the h y p e r p o l a n z a t l o n in the interburst interval is due to inactivation of endogenous I t,,~') These researchers have shown that the delayed outward tail current after a short depolarizing voltage clamp step is for the most part due to limCtlvation ot inward current The outward tail currents seen in Fig. 8 might be caused by such a mechanism and not by IK(Ca) as suggested above The increased Iota, as seen in Fig 8B2 and 8B3, could therefore be an indirect effect due to an increase in [Ca2+ L caused by ethanol (see below). Ica channels would then be partially inactivated and the influx of Ca n+ during the voltage c o m m a n d would ehclt an increased inactivation oI the Ic~ channels Alternatively, alcohol might act directly to Increase the affinity of the C a - d e p e n d e n t inactivation sites of the Ca 2+ channel for Ca -'~ There is an alternate means by which an ethanolelicited increase in [Ca 2+], might increase the measured outward current without affecting a potassium current. Byerly et al. have p r o p o s e d that increases in [Ca2+]~ ehclt Ca exchange for hydrogen ions at lntracellular sites causing a decrease in internal p H jo They further have shown in perfused snarl neurons that a decrease in internal p H will turn on a rapidly activating o u t w a r d hydrogen ion current

Effect on IK(Ca~decay Seeman et al r e p o r t e d that ethanol causes an mcreased binding of 45Ca to erythrocyte m e m b r a n e s 50 Ross, however, r e p o r t e d that ethanol causes a decreased 45Ca binding capacity of rat synaptlc membranes 52 H e hypothesized that the initial response to ethanol is a release of m e m b r a n e - b o u n d calcium with a subsequent increase in the [CaZ+], Ross's release hypothesis seems to afford an explanation for some of the results found in Aplysta bursting pacemakers: ethanol could decrease the binding of ('a2~ to the in-

351 tracellular buffers such as plasma membrane, soluble Ca x+ binding proteins, and calmoduhn The increased [Ca2+], near the membrane could summate with the Ca 2+ elicited by lnlectlon or by a VC pulse. It was possible to use electrophysiologlcal techniques to verify this theory as Barlsh and Thompson showed by study of I~lc~ I tall currents that inferences could be made about the type of buffering systems revolved in decay of [Cae+]l-S The binding of Ca 2+ by soluble buffers close to the plasma membrane controls the Irc/c~ response to a VC-lnduced Ca 2+ influx. The efficiency of mltochondrial buffering, however, controls the response of IKic~) to a Ca injection which provldes a Ca load deeper in the soma. A decrease in Ca 2+ buffer capacity would cause an increased magnitude and an increased rate of fall of I~(c~ ) as elicited by VC pulses 5 In upper left quadrant bursters, I found that 4% ethanol increased the magmtude and increased the decay rate when IK(c~) was activated by a step VC c o m m a n d The results for VC elicltation of Ix(ca ) in R15. however, did not appear to be consistent with the release hypothesis It is proposed that R15 cells are more susceptible to blockage of Ic~ by ethanol than are the left upper quadrant bursters The level of lc~ activated in ethanol would become so small that it would not be possible to observe the effect on kinetics of decay or buffering The mltochondrial uncoupler drug CCCP causes I~¢/c~ to increase in magnitude along with a decrease in rate of fall when elicited with CaX+ injectionS. I found 4% ethanol also increased the magnitude and decreased the decay rate of I~(c~ ) when activated by iontophores~s injection This is further evidence that ethanol has a similar action on [Ca2+], to that of CCCP. as would be predicted by the Internal release hypothesis It was shown above that neither the hyperpolanzatlon shift nor the decreased Ic~ are dependent on an increased [Ca2+], for their occurrence Ethanol. at least m part, directly causes these effects. However, the hypothesis as described by Ross 53 and as elaborated here proposes that ethanol will increase restlng [Ca2+]l by decreasing Ca :+ binding to soluble cytoplasmic buffers and inhibiting uptake of Ca2+ by mitochondrla Changing Vho~dwhen measuring IKCC.,) gave additional results supporting the hypothesis [Ca2+], was found to double in these studies In a separate preliminary experiment, an R15 cell under VC was impaled with a Ca2+-sensltIVe electrode Addl-

tion of 4% ethanol to N A S W caused an increase in [Ca2+]l which was fully reversible (M H Schwartz, S Levy and D Tillotson. unpubhshed)

Effect on voltage dependence of l ~ . ~ mechamsms oJ action G o r m a n and Thomas demonstrated the dependence of GKtc~) on potentlaP 0 They explained this dependence by reasoning that the site to which Ca 2+ must bind in order to activate the l~lc, o channels must be electrically charged in order to attract the divalent cation If the charged binding site then is physically located within the membrane field it would be subject to potential effects This physical Interpretation ol the voltage dependence led to a theoretical model as shown in the Results Given the relation between GKICaI and holding potentml, the voltage-dependence of I~lc~ / can be modelled, resulting in the concluslon that the Ca 2+ binding site is located halfway through the membrane As seen in Fig 10, ethanol decreased the slope of the plot of GKIca ~as a function of holding potential. The decreased sensmvltv ol G~/c~ ) to applied voltage implies that the channel's binding sites are subjected to less of the voltage drop across the membrane As calculated above, 4% ethanol can be thought of as shifting the Ca 2+ binding site to one quarter of the distance from the surface of the membrane However, ethanol might increase IK/c,,) by directly acting on the channel increasing its affinity for Ca 2 (ref 30) An example of ethanol acting in this alternative fashion was the evidence of Baker and Schaplra that ethanol increases the hght emission of aequorln by directly increasing the Ca binding affinity ot this protein 4 In conclusion, all bursting pacemaker currents are sensitive to ethanol .All inward pacemaker currents are decreased and all outward pacemaker currents are increased by ethanol The effects on these currents appear to be at least m part direct Theoretical analysis of the results reported here for Ik~c,,) are consistent with the hypothesis that ethanol affects this current via two means' (1) altering calcium butfers causing an increase of internal free Ca 2+ concentration, (,2) shifting the effecm'e position of the channel's Ca 2+ binding site in the membrane field thereby changing the voltage-dependent properties ot these channels

352 A n u m b e r o f r e s e a r c h e r s h a v e sht,wn that g e n e r a l

e l e c t r o p h y s l o l o g l c a l m e a s u r e m e n t s w h i c h ~ho~ etl~a-

a n e s t h e t i c s a n d e t h a n o l m pait~cular act by p e r t u r b -

nol acting d i r e c t l y on tom~ c u r r e n t s ',~,~ ,~ ph},s~c,d

ing t h e m e m b r a n e a n d a l t e r i n g t h e r e l a t i o n b e t w e e n

p e r t u r b a t i o n ol c h a n n e l s m the m e m b r a n e Ileld t-ut-

h p t d s a n d e m b e d d e d p r o t e i n s M o r e d e t a i l e d w o r k is

t h e r analysts ts d i r e c t e d tov~ards dete~ m m m g whetl~-

aimed towards dtscovermg exactly how a hpM pertur-

er e t h a n o l affects t h e o t h e r p d c e m , t k e r ~ u r r e n l s ~ ~,~

b a t i o n affects t h e m e m b r a n e - p r o t e i n

similar m e c h a m s m s

relationship.

Borochov and Shmttzsky demonstrated a change m vertical d i s p l a c e m e n t o f m e m b r a n e - b o u n d m human erythrocyte membrane

proteins

ACKNOWLEDGEMENTS

wtth c h a n g e s m

m e m b r a n e m l c r o v t s c o s t t y ' T h e y p r o p o s e d t h a t ver-

I thank T Sable and Drs

D. J u n g e a n d R. H o r n

tical m o v e m e n t o f p r o t e m s m~ght play a significant

for t h e t r g u i d a n c e t h r o u g h o u t this p r o j e c t

role m m o d u l a t i o n o f t h e i r t u n c t t o n , b u t l a c k e d the

t h a n k D r s M B J a c k s o n a n d L. O T r u s s e l l for t h e i r

e x p e r i m e n t a l e v M e n c e to s u p p o r t this h y p o t h e s i s

careful r e a d m g o f t h e m a n u s c r i p t

I also

T h e results d e s c r i b e d a b o v e for I~.,~,,t are t h e first

REFERENCES 1 Akalke, N , Lee. K S and Brown, A M . The calcmm current of Hehx neuron, J gen Physiol, 71 (1978) 509- 531 2 Apland, J P and Llvengood, D R . Effect of low-sodmm solunon conductance in the giant abdominal neuron (R2) of Aplysta, Soc Neurosci Abstr , 3 (1977) 213 3 Armstrong, C M and Binstock. L , The effects ot several alcohols on the properties of the squid giant axon. J gen Physlol, 48 (1964) 265-277 4 Baker. P F and Scbaptra. A H V . Anaesthetics increase light emission from aequorln at constant lomsed calcium Nature (Lond), 284 (1980) 168-169 5 Barlsh, M E and Thompson, S H . Calcium buffering and slow recovery kinetics of calcmm-dependent outward current in molluscan neurones. J Phwtol fLond ), 338 (1983) 201-219 6 Bergmann, M C , Klee, M R and Faber, D S . Different sensitlvmes to ethanol of three early transient voltage clamp currents of Aplysta neurons, Pflugers Arch ees Physzol, 348 (1974) 139-153 7 Berry. M S and Pentreath, V M . The neurophysJology ot alcohol In M Sandier ( E d ) , Psychopharmacologv of Altohol, Raven Press, New York. 1980. pp 43-72 8 Bezanllla, F and Armstrong. C M , Negative conductance caused by entry of sodmm and cesium ions into the potassium channel~ of ~qmd axons, J gen Physlol, 60 (1972) 588608 9 Borocho,~. H and Shimtzky, M . Vertical displacement of membrane proteins me&ated by changes in mlcrovlscos~ty. Prot nat Acad Sct U S A , 73 (1976) 4526-4530 10 Byerly, L . Meech, R W and Moody, W J Hydrogen Ion currents in snail neurones. Soc Neurosci Abstr, O (1983) ,

1189

11 Carnevale. N T and Wachtel. H . Two reciprocating current components underlying slow oscillations in Aplysta bursting neurons, Brain Res Rev , 2 (1980) 45-68 12 Carpenter. D O and Alvmg, B O , A contribution of an electrogenlc Na ÷ pump to membrane potentml m Aplysm neurons. J gen Phystol, 52 (1968) 1-21 13 Colquhoun, D , Neher, E , Reuter, H and Stevens. C F . Inward current channels activated by mtracellular Ca in cultured cardiac cells. Nature (Lond), 294 (10811 752-754

14 Connor, J A , Calcium current in molluscan neurones measurement under conditions which maximize ItS VlSlbihty, J Phystol (Lond), 286 (1979) 41-60 15 Cotton. F A and Wilkinson, G , Advanced Inorganic Chemistry, 2nd e d n . John Wiley and Sons. New York. 1966. p 836 t6 Eaton, D C , Potassium ion accumulaUon near a pacemaking cell of Aplysta, J Phystol (Lond l, 224 (1972) 421440 17 Eckert. R and Lux, H D , A voltage-sensm~e persistent calcmm conductance in neuronal somata of Heh.t, J Phystol (Lond), 254 (1976) 129-151 18 Faber. D S and Klee. M R , Actions of ethanol on neuronal membrane propemes and synapuc transmission In K Blum ( E d ) , Alcohol and Opmtes Neurochemical and Behavioral Mechanisms, Academic Press. New York, lC)77 pp 41-63 19 Frankenhaeuser, B and Hodgkm, A L . The action ot c,tlcium on the electrical properties ol squid axons J Phystol (Lond), 137 (1957) 218-244 20 Frazier, W I , Kandel, E R . Kupfermann, 1 Wazm. R and Coggeshall, R E., Morphological and functional propernes of ~dentlfled neurons in the abdominal ganglion of Aplysta caltforntca, J Neurophy.stol , 30 (1967) 1288-1351 21 Futamacht, K and Smith, T G . Jr , Action oftetrodotoxln on pacemaker conductances m Aplvsta neurons. Brain Research, 223 (1982) 424-430 22 Gilbert. D L and Ehrensteln, G . Effect ot &valent canons on potassium conductance of sqmd axons dctermmauon ol surface charge, Btophys J , 9 (1969) 447-463 23 Gola, M . Neurones a ondes-salves des mollusques, ~anatlons cycllques lentes des conductances lomqucs. Pfluger~ Arch ges Phystol , 352 (1974) 17-36 24 Gormam A L F and Hermann. A . Internal etlects ot divalent cations on potassium permeability in molluscan neurons, J Phystol (Lond), 296 (1979) 393-410 25 Gorman. A k F and Hermann. A . Quanmatlve dillerences in the currents ot bursting and beating molluscan pace-maker neurones. J Physlol (Lond), 333 (1982) 681 - 699 26 Gorman, A L F , H e r m a n n , A and Fhomas. M "v . l n tracellular calcium and the control of neuronal pacemaker activity. Fed Proc. 40 (198t) 2233-2239

353 27 Gorman, A L F , Hermann. A and Thomas, M V , Ionic requirements for membrane oscillations and their dependence on the calcium concentration in a molluscan pacemaker neurone, J Phvslol (Lond), 327 (1982) 185-217 28 Gorman, A L F and Thomas, M V , Changes in the lntracellular concentration of tree calcium ions in a pacemaker neurone, measured with the metallochromic indicator dye arsenazo III, J Phvsiol (Lond), 275 (1978) 356-376 29 Gorman, A L F and Thomas, M V , Intracellular calcium acctimulation during depolarization m a molluscan neurone, J Phystol (Lond ), 308(1980)259-285 30 Gorman, A L F and Thomas, M V , Potassium conductance and internal calcium accumulation in a molluscan neurone J Physiol (Lond), 308 (1980) 287-313 31 Hafemann, D R and Miller, S L , Enzymatic softening of connective tissue sheaths to a~d mlcroelectrode penetration, Comp Bzochem Physlol, 22 (1967) 303-307 32 Hermann. A and Gorman, A L F , External and internal elfems of tetraethylammonlum on voltage-dependent and Ca-dependent K- currents components in molluscan pacemaker neurons, Neurosct Lett, 12 (1979) 87-92 33 Hermann, A and Gorman, A L F , Blockade ot voltagedependent and CaZ+-dependent K + current components of internal Ba 2+ m molluscan pacemaker neurons, Experlentin, 35 (1979) 229-231 34 Hermann, A and Gorman, A L F , Effects of 4-aminopyrldlne on potassium currents in a molluscan neuron, J gen, Phvstol, 78 (1981) 63-86 35 Hcrmann, A and Gorman, A L F , Effects of tetraethvlammonlum on potassmm currents in a molluscan neuron, J gen Phys*ol , 78 (1981) 87-110 36 Johnston, D , Voltage clamp reveals basis for calcmm regulation ot bursting pacemaker potentials in Aplysta neurons. Brain Research, 1/)7 (197~) 418-423 37 Junge, D and Stephens, C L , Cyclic variation of potassium ~.onductance in a burst-generating neurone in Aplysta, J Phystol (Lond ~, 235 (1973) 155-181 38 Katz, G M and Steinberg, S , Mlcroelectrophoresls and constant current sources In J Reuben, D Purpura, M Bennett and E Kandel ( E d s ) , Electroblology oj Nerve, Synapse and Muscle, Raven Press, New York, 1976, pp 367-377 39 Kramer, R H and Zucker, R S , Inactivation of persistant mward current mediates post-burst hyperpolarizatlon in Apl.~ ;la bursting pacemaker neurons, Soc Neuro,sct Abstr, 9 (1983) 510 40 L1, T K , The absorption, distribution and metabolism of ethanol and its effects on nutrition and hepatic function In B "fabakoff, P B Stuker and C L Randall ( E d s ) , Me&cal and Social Aspects of Alcohol Abuse, Plenum Press. N e w Y o r k , 1982, pp 101-131 41 Lux, H D , Voltage dependence of Ca++-actlvated K+ conductance In J Koester and J Byrne ( E d s ) , Molluscan N e n e Cella From Bzophvstcs to Behavior, Cold Spring Harbor Press, New York, 1980, pp 105-114 42 Math~eu, P A and Roberge, F A , Characteristics of pacemaker oscillations in Aplysla neurons, Canad J Physlol Pharmacol , 49 (1971) 787-795 43 McLaughhn, S G A , Electrostatic potentials at membrane-soluuon interfaces, Current Topics In Membranes and Transport, 9 (1977) 71-144 44 McLaughhn, S G A , Szabo, G and Elsenman, G . DJvalent ions and the surface potential of charged phosphohpld membranes, J ,gen Phvstol, 58 (1971) 667-687

45 Meech, R W , Intracellular calcium injection causes increased potassium conductance in Aplysla nerve cells, Comp Bzochem Phystol , 42A (1972) 493-499 46 Meech, R W , The sensitivity of Helix aspersa neurones to injected calcium ions, J Phvslol (Lond), 237 (1974) 259-277 47 Meech, R W , Calcium-dependent potassium activation m nervous tissues, Ann Rev Btophys Btoeng, 7 (1978) 1-18 48 Meech, R W and Standen, N B , Potassmm activation in Helix a~persa neurones under voltage clamp, a component mediated by calcium influx, J Physiol (Lond ~, 249 (1975) 211-239 49 Moore, J W , Ulbrlcht, W and Takata, M , Eltect ot ethanol on the sodium and potassium conductances of the squid axon membrane, J gen Phvstol, 48 (1964) 279-295 50 Noble, D , The lnmatton of the Heartbeat, Oxford Press, New York, 1979, pp 120-123 51 Overton, E , Studten uber die Narkose zugleich eln Beltrag zur allgemeInen Pharmakologle, Gustav Fischer, Jena, Germany, 1901,p 101 52 Ross, D H , Adaptive changes m Ca++-membrane interactions following chronic ethanol exposure, Adv cap Med Btol, 83 (1977) 459-471 53 Ross, D H . Molecular aspects of calcium-membrane interactions a model for cellular adaptation to ethanol In H Rlgter and J Crabbe ( E d s ) , Alcohol Tolerance and Dependence, Else,eler, Amsterdam, 1980, pp 227-239 54 Satin, L S . Sodium dependent calcium efflux from single Aplysla neurons, Bram Research, 300 (1984) 392-395 55 Schwartz, M H , Voltage clamp analysis of ethanol effects on pacemaker currents of Apl.vsta neurons, Bram Research, 278 (1983) 341-345 56 Seeman, P , C h a n , M , Goldberg, M , Sauks, T and San, L , The binding of Ca ++ to the cell membrane increased by volatile anesthetics (alcohols, acetone, ether) which induce sensitization of nerve or muscle, Btochtm Btophvs Acta, 225 (1971) 185-193 57 Silver, L H and ~Irelstman, S N ,Eftects of alcohols upon pacemaker activity in neurons of Aplysla call]brntca, Cell molec Neurobtol, 2 (1982) 215-226 58 Smith, S J and Zucker, R S , Aequorln response facilitation and intracellular calcium accumulation in molluscan neurones, J Ph)'slol (Lond), 300 (1980) 167-196 59 Smith, T G . J r , Barker, J L and Gainer. H , Requirements for bursting pacemaker potential acnvlty m molluscan neurons, Nature (Lond), 253 (1975) 450-452 60 Smlth, T G , B a r k e r , J L , S m l t h , B M a n d C o l b u r n , T R , Voltage clamping with mlcroelectrodes, J Neuroscl Meth, 3 (1980) 105-128 61 Strumwasser, F , The cellular basis ot behavior in Aplysm, J Psvchlat Res , 8 (1971) 237-257 62 Thompson, S H , Three pharmacologically distinct potassium channels in molluscan neurones, J Phvstol (Lond), 265 (1977) 465-488 63 Til[otson, D , Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Proc nat Acad Set U S A , 76 (1979) 1497-1500 64 Wilson. W A and Wachtel, H . Negat,ve resistance charactenstlc essential for the maintenance of slow oscillations in bursting neurons, Science, 186 (1974) 932-934 65 Yellen, G , Single Ca2+-actlvated nonselective cation channels in neuroblastoma, Nature (Lond), 296 (1982) 357-359