Brain Research, 507 (1990) 17-22

17

Elsevier B R E S 15112

Effect of temperature on the inhibition of the GABA-gated response by intracellular calcium T o o r u M a r u y a m a ~, Y o s h l m l I k e m o t o -~ a n d N o r l o A k a l k e ~ 1Department of Neurophvstolog~, Toho]tu Untverstt7 School of Medtcme, Sendat (Japan) and 2Department o¢ Dental Anesthesiology, Faculty of Dentistry, Kyushu University, Ful~uol~a (Japan) (Accepted 13 June 1989)

Key ~ords Frog sensory neuron Internal perfusmn, 7 - A m m o b u t y n c amd Chloride current, T e m p e r a t u r e InhiNtton

T h e effects of the voltage-dependent Ca2~mward current (I(~) on 7 - a m m o b u t y n c acid ( G A B A ) - m d u c e d CI current ( I t 0 in isolated frog sensory n e u r o n s were examined at different temperatures and with different c o n c e n t r a n o n s of external Ca 2+ using the concentration-clamp t e c h m q u e T h e total a m o u n t of m h l b m o n of G A B A - m d u c e d I o by a preceding Ic~ increased in a hvperbohc m a n n e r with increasing Ca '~' reflux T h e time course of recovery of the G A B A response after inhibition by Ca TM influx followed a single e x p o n e n n a l and was faclhtated by warming but slowed dramatmally by a shght coohng from 20 to 15 °C in spite of a decrease in Ca e+ reflux It is discussed that the e n e r g y - d e p e n d e n t temperature-sens~twe m m c p u m p and exchange systems at the surface m e m b r a n e and Intracellular organelles regulate the cytoplasmic free Ca 2+ thus explaining the quantltatwe effects of Ca z+ reflux and temperature on the m h l b m o n of the G A B A - g a t e d CI channel

INTRODUCTION

MATERIALS AND METttODS

Intracellular-free Ca 2+ ([Ca2+],) has a pwotal role m the transductlon of extracellular s~gnals and the regulan o n and modulatmn of fundamental cellular functmns ~2 T h e [Ca2+], is profoundly influenced by some physlolog-

tcal conditions, e g temperature and extracellular concentratton of Ca2+([CaZ-]o ) whmh act on Ca 2+ extruding and sequestering mechamsms located at the surface

m e m b r a n e and mtracellular organelles, respectively 25 28 3~ Several types of voltage-dependent mmc channels have been found to be highly sensitive t o [Ca2+], Ca2+-actwated

K + conductances in neurons Z5 e029 ~s

Ca2+-actwated non-selectwe cation conductances m cultured heart cells 14 and neuroblastoma cells 39, and Ca e+actwated CI- conductances m mouse spinal cord neurons ~3 and rat and frog sensory neurons 22 27 Recently, tt has been reported that drug-gated ionic conductances are also affected by a transient change m

[Ca2+], An increase of [Ca2+], inhibited the AChreduced mcotlntc CI response m Lymnaea stagnahs neurons ~ and the G A B A - l n d u c e d CI responses m frog sensory neurons 22 and pltmtary lntermedmte lobe cells ~7, and famhtated the onset of desensmzat~on of the mcotmm A C h responses m muscle endplate ~° In the present experiments therefore, we e x a m m e d the effects of temperature and [Ca2+],, on the [Ca2+],-sensmve G A B A reduced chloride current ( l c 0 in frog sensory neurons

Preparation Dorsal root gangha dissected from decapitated American bull-frog (Rana catesbiana) were used throughout the experiments After removal of the calcified tissue surrounding the gangha the capsules enveloping the ganglion masses were digested m Ringer s solution containing 0 3% collagcnase and 0 05% trypsin at p H 7 4 for about 18 m m at 37 °C e C o n t i n u o u s gentle shaking ot the gangha by bubbling the bathing m e d m m ~lth 99% O: facdltated the enzymatic digestion Thereafter, single neurons were isolated mechanically from the g a n g h o n by using finely polished pins under binocular observation The cells were left overnight at 10 °C m culture m c d m m containing equal parts of Ringer s solution and Eagle s m i n i m u m essential m e d i u m (Nlssm, Japan) Sohmons To separate CI and Ca: ~ currents from all other m m c currents Na* and K ~ in the external and internal test solutions v, ere s u b s n t u t e d with T n s (hydroxvmethyl) a m m o m e t h a n e (Tns ~) and Cs + respectively q h e internal and external test solutuuons contained 120 m M C1 and the external test solunons had various concentrations of Ca-" ~ depending upon the experimental purpose 1 h e ~omc composition ol the standard internal and external solutions ~ e r e as follows (in raM) internal CsCI 95 Cs-aspartate 10 TEA-CI 25 E G T A - C a > buffer ([Ca -'+1, = ~ x 10 s M), external Tns-CI 89, C s ( I 2 CaCI. 2 TEA-CI 25, glucose 5 Increases in [Ca > ],, v, ere made bv lsotomc s u b s n t u t m n of T n s - ( I by ( a C I . The [Ca "-* l, v, as calculated by the following equation add Ca = l + K ' { [ E G ' I A 1 + [Ca]} ICal 1 + [Cal K where [Ca] represents the desired free ( a 2+ c o n c e n t r a n o n , [EGTA] represents the c o n c c n t r a n o n of E G q A , and K Is an apparent assoclanon constant for Ca 2' - E G T A of 1117 ~ at p H 7 2 and 22 °C ~4 T h u s , m standard internal solution a free Ca 2+ concentration of 3 x 10 ~ M was obtained using 1 m M E G T A and 0 28 m M CaCI. The

Correspondence N Akalke D e p a r t m e n t of Neurophyslology, Tohoku Umversltv School of Medicine Sendat 980 Japan

18 pH of external and internal test solutions were adjusted to 7 4 and 7 2 respectively using Tris-base and N-2-hvdroxvethvlplperazmeN -[2-ethanesulfomc acid] (HEPES) Suction ptpette Several ~solated neurons were aspirated into a tapered capdlary tube having a fire-pohshed tip with interior dmmeter of about 120 itm and were then transferred to a culture dish with dmmetcr of 3 cm (Falcon) These cells had a dmmeter of approxlmately 30-40 1~m and were clearly visible under binocular magmfication (x80) A suction pipette techmque was applied to these neurons ~ J9_,4 using Pyrex glass with ~ mm outer dmmeter pulled to a shank length of 2 5-3 0 ram, cut at an outer diameter of about 40 /.tin, then fire-pohshed to give an tuner dmmeter of approximately 7 ¢tm The part ot the soma membrane aspirated to the suction pipette tip by a negatwe pressure of about 3 cm Hg was ruptured spontaneousl'v or b~ applying a 5-20 nA square wa~e pulse ol depolarizing (10-50 ms) Fhcreafter, the suction pipette electrode with the atttached neuron was internally perfused at a constant flow rate of 1 ml/mm The success of the internal perfus~on with this sucnon pipette tcchmque was evaluated by testing how close the reversal potential for the GABA-mduced CI response (Ec~AB~,) was to the CI eqmhbrmm potential (Ec~) ol about +4 mV as calculated from the Nernst equation based on the CI activmes In the external and internal solutions Electrtcal measurements The membrane potential was measured through an Ag-AgCI w~re m a Rmger-agar plug mounted in the suctmn pipette holder The reference electrode was also an Ag-AgCI wire m a Rlnger-agar plug mounted in the plastic tube used for exchanging the external solution (see below) The resistance between the suction p~pette filled with standard internal solution and the relercnce electrode was 200-300 kf2 Both electrodes were led to a voltage-clamp clrcmt and membrane potentml was controlled by a single-electrode voltage-clamp system switching at a frequency of 10 kHz and passing current for 36% of the cycle "04 Clamp currents were measured as the voltage drop across a 10-M.O resister in the feedback path of a headstage amphfier In this system, the suctmn electrode could carry t~meaveraged currents exceeding 100 nA at a switching frequency of 10 kHz, without showing any s~gns of polanzatmn or other artifacts Both current and voltage were monitored on a &gltal storage oscdloscope (National, VP-5730A), and were s~multaneously recorded on an mk recorder (R~kadenki, R-22) and stored on a magnenc data recorder (TEAC MR-30) Results were expressed as mean 4- S E M Apphcatton oJ external soluttons The concentration-clamp techtuque was used for extremely rapid (within 2 ms) changes of the external solutmn ~ The cell-attached tip of the suction electrode was inserted into a plastic tube through a c~rcular hole of approximately 500/xm in dmmeter The lower end of th~s tube could be exposed directly to external solutions contained in culture d~shcs which were placed on a turntable The suction (-3 cm Hg) apphed to the upper end of the tube was controlled by an electromagnetic valve driven by 24 V D C The power supply was switched on for the desired duratmns by a stimulator (N~hon Kohden, SEN-7103) All expertments were done at room temperature (20-22 °C)

A GABA

CH -

10-6M

[r I

"~

[ _ _

[Ca

v C (mY ~ - 5 (a) t ~ - 1 0 tb) -15 {e)

< 100 msec

Z

o_

10=

~

0

~ 1 3 T 9(sec) 4

-r

56 C

-

\116

01

_/ r

T I M E (see)

Fig 1 A suppressive action of Ic.. evoked by a depolanzmg command pulse from -50 mV to 0 mV of 300 ms duraUon, on the GABA-mduced Icl The tnangular shaded area m the upper graph ~s the integrated charge earned by CI- and inhibited by a preceding Ica, forming an outward hump during the G A B A response ( - S I o , nano coulomb, nC) The shaded area m the lower graph is the total amount of charge due to Ca 2+ entry (J'Ic. , nC) ([Ca2+]o = 2 mM) B suppressmn of 3 x 10-6 M GABA-mduced Ict by Ic~ under vanous [Ca2+]o V H = -50 mV Command pulse (Vc) was m&cated in figure Note the different time scales in upper and lower panels m A and B C semdoganthmlc plots of the recovery t~me courses of GABA-mduced Icl after mhlbmon by preceding Ic. evoked at various [Ca2+]o Data were obtained from B Note the respectwe time constants corresponding to 3 different [Ca" ],,

x 10 -° M G A B A , level

with

httle

I o reaches a steady-state (plateau) desensmzatton

During

the

plateau

c u r r e n t l e v e l , Ica c a n b e e v o k e d b y 300 m s d e p o l a r i z i n g pulses

Fig

1A dlustrates a typical transient I c l a f t e r i n d u c t i o n o f Ica

A f t e r c e s s a t i o n o f lea, t h e G A B A

Ic.

5see

bc

teal o (raM)

suppressmn of GABA-mduced

Suppresston of Icl by

rn b

300 msec

9 rnV 300 msec

--50 m V ~

50

Imln a

command RESULTS

B

3x

response gradually

r e t u r n s to Its o r i g i n a l p l a t e a u c u r r e n t l e v e l w i t h a single

The mhzbttlon of the GABA-mduced CI- current (Ic0 c a u s e d b y a n i n c r e a s e in [Ca2+], f o l l o w i n g a c t i v a t i o n o f

exponentml time course

voltage-dependent

c u r r e n t ( - J ' I o ) c a n b e c a l c u l a t e d T h e r e f o r e , !t ts p o s s i b l e to a n a l y z e t h e k i n e t i c s o f t h e I a l n h i b m o n a n d t h e

C a 2+ c u r r e n t s ( l e a ) h a s a l r e a d y b e e n

q u a n t i f i e d in t s o l a t e d s e n s o r y n e u r o n s i m m e r s e d

in a

N a ÷- a n d K + - f r e e R i n g e r s o l u t i o n c o n t a i n i n g 2 m M C a 2+ (refs

22,23)

With this protocol, the total

a m o u n t o f C a 2+ influx ( [ I c ~ ) a n d t h e r e d u c t i o n m CI-

kinetics ot Io recovery

A s p r e v i o u s l y d e s c r i b e d , w h e n cells a r e

A n i n c r e a s e in [Ca2+]o f r o m 2 to 5, 10 o r 20 m M

v o l t a g e - c l a m p e d at a h o l d i n g p o t e n t i a l (VH) o f - 5 0 m V

i n c r e a s e d t h e a m p l i t u d e o f t h e v o l t a g e - d e p e n d e n t Ic~ a n d

a n d let is e h c l t e d b y a r e l a t i v e l y low c o n c e n t r a t i o n o f 3

s h i f t e d t h e t h r e s h o l d a n d m a x i m u m p e a k v o l t a g e s o f Ic

19

IlCa 0

IICa

(nC)

2

4.

6

2

3

4

5

I

I

L

,

I

C-'-~ •

S

2

2

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