BRES 15114

Mechanisms concerned in the direct effect of isoflurane on rat hippocampal and human neocortical neurons J

Berg-Johnsen and I A Langmoen

Institute for Experimental Medical Research, Umverstty of Oslo, Department of Neurosurgery, Ullevaal Hospital and Department oJ Neuro~urgerv, National Hospital Oslo (Norway) (Accepted 13 June 1989)

Ke) words Anesthesia, Inhalation, Isoflurane, Hlppocampus, Membrane potential, Ion channel

The effect of lSOflurane on postsynaptic neurons was studied by mtracellular recordings from rat h~ppocampus and human neocortex tn vitro Isoflurane caused a hyperpolarlzation of the cell membrane The hyperpolarlzatlon was reversed (although incompletely in some neurons) by mcreasmg the membrane potenttal The reversal potential was -80 _+ 12 mV (mean + S D ) or 12 _+ 6 mV negative to the resting membrane potentml Potassium channel blockers reduced the lSOflurane-lnduced hyperpolarlzation, while chloride loading was without effect The transient depolarization preceding the hyperpolarlzation in some of the neurons was not reversed by hyperpolanzatlon The action potenttal was prolonged by 19 _+ 3% due to a slower rate of rise The rise ttme was almost doubled Finng threshold was increased by 4 +- 3 mV (relanve to the reference electrode) Subthreshold inward recnficatlon was reduced or abolished Some cells showed subthreshold outward recttficatton during isoflurane administration These results suggest that lSOflurane depressed neuronal excitability by (1) hyperpolarlzmg the cell membrane, at least partly by an increase m potassium conductance, (2) slowing the rate of rise of the acuon potentml, presumably due to interference with the fast sodium channel, (3) decreasing subthreshold inward rectification and (4) increasing firing threshold

INTRODUCTION

i s o m e r of e n f l u r a n e It has a d v a n t a g e s o v e r the o l d e r volattle a n e s t h e t i c s 9 to and is t h e r e f o r e b e c o m i n g o n e of

Volatile anesthettcs have a well-known depressant e f f e c t on e x c i t a t o r y synapses 27 32-35 M o r e r e c e n t l y tt has

the m o s t w i d e l y u s e d agents

b e e n s h o w n t h a t b o t h h a l o t h a n e , e t h e r 31 and lSOflurane 5

r e v e r s e d w h e n the m e m b r a n e p o t e n t i a l Is m c r e a s e d (Erev = - 8 0 m V ) and r e d u c e d by p o t a s s i u m c h a n n e l blockers,

h y p e r p o l a r t z e t h e p o s t s y n a p t l c n e u r o n s and that (at least) lSOflurane has an e f f e c t on t h m , u n m y e h n a t e d i n t r a c o r tlcal fibers 3 The hyperpolartzatton

perststs b o t h tn t e t r o d o t o x m

In the p r e s e n t p a p e r we

r e p o r t that t h e h y p e r p o l a n z a t t o n c a u s e d by tsoflttrane is

but not a f f e c t e d by c h l o r t d e l o a d i n g o f t h e n e u r o n I s o f l u r a n e d e p r e s s e d b o t h a n o m a l o u s recttficatlon and the

rismg p h a s e

of the

action

potential,

and

firmg

( T I ' X ) and a f t e r synaptlc transmtsslon is b l o c k e d in low

t h r e s h o l d was i n c r e a s e d

C a e + / h l g h M g ~+, m d t c a t l n g a direct action on postsyn-

afterhyperpolarlzatlons (AHPs) were reduced m amph-

apttc n e u r o n s

tude

This effect s e e m s to be just as strong as the

B o t h t h e m e d m m and the slow

effect on the e x c i t a t o r y synapses 4 To f u r t h e r c h a r a c t e r t z e the h y p e r p o l a n z a t l o n , reversal potentml

tt ~s n e c e s s a r y to d e t e r m m e the

MATERIALS AND METHODS

and to e x a m i n e the effect of ~omc

channel blockers T h e r e is s o m e e v i d e n c e , b o t h f r o m i n v e r t e b r a t e ~7 and v e r t e b r a t e n e u r o n s z3` that addtt~onal m e c h a m s m s m a y c o n t r t b u t e to the postsynapt~c effect of volatile a n e s t h e t ics It ~s t h e r e f o r e e s s e n t m l to e x a m i n e possible effects on o t h e r p a r a m e t e r s ~mportant m c o n t r o l of n e u r o n a l e x o t ability and t l r m g p a t t e r n T h e s e include a n o m a l o u s r e c t i f i c a t i o n , h y p e r p o l a n z m g a f t e r p o t e n t m l s and a c n o n p o t e n t i a l r u i n a t i o n , u p s t r o k e and r e p o l a n z a n o n T h e r e l a t i v e l y n e w v o l a t i l e a n e s t h e t i c ~soflurane ~s an

The hlppocampal slice technique has been described in detail previously24 and will be discussed only briefly Young Wtstar rats (100-200 g) were decapitated under light ketamlne anesthesia, the brain removed and the hippocampus from one hemisphere carefully dissected free Thin transverse slices (0 4-0 6 ram) were cut by hand and transferred to an incubation chamber of the Haas type as The slices were superfused by a gas mixture (95% 0 2, 5% CO2) and bathed in artificial cerebrospmal fluid (ACSF) of the following composition (in raM) NaC1 124, KCI 2, KHzPO 4 1 25, NaHCO 3 26 MgSO 4 2, CaCI 2 2, dextrose 9 The temperature was maintained at ~2 _+ 2 °C Afferent fibers were activated using a bipolar platinum iridium electrode placed in the stratum radlatum Recording electrodes

Correspondence J Berg-Johnsen, Institute for Experimental Me&cal Research Ullevaal Hospital 0407 Oslo 4, Norway 0006-8993/90/$03 50 © 199(I Elsevier Soence Publishers B V (Biomedical Division)

29 were pulled from thin borosihcate glass tubing (1 0 m m o d , 0 6 m m i d ) and filled with 3 M p o t a s s m m acetate, 3 M potassium chloride or 4 M tetraeth3l a m m o n i u m (TEA)/2 M caesium chloride (CsCI) T h e mlcroplpettes had up resistance ol 50-150 Ms'2 The electrodes were placed u n d e r visual control using a stereomicroscope Hippocampal n e u r o n s were penetrated m the CA1 stratum pyramndale and showed electrophyslologlcal properties typical for pyramidal cells 24 ~, ~v T h e m e m b r a n e potential was monitored by a conventmnal D C amplifier with an active bridge circuit for passing current through the recording electrode The input resistance was calculated from deflections in m e m b r a n e potential in response to hyperpolanzing current pulses (100 ms) Simultaneous write-out was provided bv a m i c r o c o m p u t e r or a chart recorder Isoflurane (Abbott) was added to the gas mixture superfusing the slices by a vaporizer (Isoflurane Vapor 19 1) which was carefull) calibrated H u m a n neocortex removed In assocmtlon with temporal Iobectomies was sliced (about 0 5 m m thick) perpendicularly to the surface The stimulating electrode was positioned m the white substance and cells penetrated by the recording electrode m the overlying gray substance

RESULTS

Every cell tested (n = 54) was stable for at least 10 mln following penetration before the tests started, had action potential amplitude exceeding 70 mV (mean 84 mV) and restmg membrane potential of at least -60 mV (mean -65 mV) Mean input resLstance was 37 MI2 Isoflurane was given in two different doses 1 5 and 3 0% Minimum alveolar concentration (MAC), that is the dose glvtng surgical anesthesia m 50% of tested rats, is 1 3842 The two doses caused quahtattvely identical changes Isoflurane hyperpolarized the cell membrane of all neurons tested In rat hippocampal neurons I 5 and 3% lSOflurane caused a 4 + 1 (mean + S D ) and 6 _+ 2 mV hyperpolarization respectwely The average hyperpolarizatton in human neocortlcal neurons (Fig 1) was 5 + 1 mV (3% lsoflurane, n = 5) The hyperpolarizatton was dose-dependent and reversible and was associated with a decrease in input resistance (18 + 3% reduction by 3% lsoflurane) In 44% of the neurons a transient depolarization ( f - 2 mV for less than 2 rain) preceded the hyperpolarlzation The depolarization was usually associated with an increase in input resistance and increased spontaneous activity in spontaneously active cells

Reversal potential Hyperpolarlzation of the cell membrane by 25-40 mV reversed the lsoflurane-mduced hyperpolartzatlon to a depolarizing potential (Fig 2A), although reversal was incomplete in some neurons In neurons with an initial depolarization (Fig 2B), the hyperpolanzmg part of the lsoflurane response reversed without any definite effect on the tsoflurane-mduced depolarization Isoflurane reduced the mput resistance of the neurons also when the hyperpolarization was reversed (Fig 2C) The reversal potential for the lsoflurane-potential was calculated from

current voltage plots (Fig 2D) On average reversal occurred 12 mV negative to restmg membrane potential both in human neocortical and in rat hlppocampal neurons The reversal potential was -80 +_ 12 mV

Ionic rnechanlsrn underlvmg the lsoflurane hvperpolartzatlon

The results of the experiments presented m the preceding section suggest that lsoflurane acts by opening either chloride or potassium channels In order to test these two posslblhties different neurons were either loaded with chloride to change the chlorLde equlhbrmm potential from negative to positive with respect to the resting potential or loaded with potassium channel blockers ( ' I E A and Cs +) In neurons loaded with chloride the nsoflurane (3%) hyperpolarizatlon was 6 +_ 3 mV, which was identical to control In neurons loaded with potassium channel blockers, the isoflurane hyperpolarlzatLon averaged 3 mV compared to 6 mV in control neurons (difference significant at P ~-. 0 01, Wilcoxon two-sample)

The effect on the action potenual Action potentials recorded during control and in the presence of the anesthetic trom the same neuron are superimposed in Fig 3A During lsoflurane admlnistra-

A

-781 °-°-°-°-°-".... \ i

\o\ -83

:~ -95

o-°

o-o o - o - o - o - o - o - o

B 0-O=O=O-O-O-0=O=O(O,

321

\o

o

24

Isoflurane 3%

I

f

1

o

5

lO

MLnutes

Fig 1 A the m e m b r a n e potential ot a h u m a n neuron a,~eraged each minute ( m e m b r a n e potential -78 mV) U p p e r part is control response to 3c'~ isoflurane (filled symbols) Following rucoverv the m e m b r a n e was hyperpol,irized to -95 m V b,¢ current injection and exposed to lSOflurane again (open symbols) B the input resistance of the same neuron at resting m e m b r a n e potential (filled symbols) and at hvperpolanzed level (open svmbolsl

3()

A -50 -

Isoflurane

o -80-

D

Isoflurane

Membrane potent)al (mV

E -110

• Control

2ram

/

/

o Isoflurane 3%

B

E -70- o.o44.o_e),'°°"o.o

..j~..¢..o4

c-

v"

J

o

-80

-

,.0

E ®

C

/

O~O'O-OK)~o/O~o

,301

0 0 ~° 0

-90

°e'o'oe"°°~4-~\ o-o-~

,~

. ° / """ -70

..o" P

-80

,/o-oo-o /• o

/

25

D 1:3.

-90

Is o 3 ~ I

I

I

I

0

10 Minutes

20

-1 2

|

I

-08 -04 Current (nA)

1

0

Fig 2 A chart recording of the membrane potentml of a neuron exposed to 3% lsoflurane (resting membrane potentm1-60 mV) Upper part ~s control response Following recovery the cell was hyperpolanzed to -95 mV by current mjectmn and exposed to lsoflurane again (lower part) B and C the membrane potentml (B) and input resistance (C) m another neuron averaged over l-ram intervals Resting membrane potentml -70 mV The control response (closed orcles) consists of an mmal depolarization followed by hyperpolanzatlon Following hyperpolanzaUon to -90 mV, the hyperpolanzmg part of the lsoflurane response was reversed (open circles) D current voltage plot dunng control (dosed circles) and lsoflurane 3% (open circles) and regressmn hnes drawn (regression coefficient control = 0 82, lsoflurane = 0 57, correlatmn coefficient control = 0 997, isofiurane = 0 993) Restmg membrane potentm1-61 mV Membrane potentml dunng lsoflurane -68 mV Reversal potentml for the tsoflurane-potentml was -86 mV m this cell

tion the orthodromlc input/membrane potential was adjusted to evoke an EPSP with the same time course/ amplitude as control The inset (right) shows the measurements The spike latency increased during isoflurane anesthesia (Fig 3C) The spike amplitude increased initially In some experiments, but decreased again before firing ceased (Fig 3B) The increased amplitude coincided w~th the transient depolarization and mcrease in Input resistance Spike duration measured at half maximum amphtude was prolonged by 19 + 3% (Fig 3D) due to a slower rate of rise The rise time increased dunng lsoflurane (Fig 3F), whereas the fall time remained unchanged (Fig 3E) As the upstroke constitutes the shortest part of the sp~ke, a major effect on the rising phase (87% increase) only results in a modest increase in spike duration The firing threshold for the action potential was increased dunng lSOflurane anesthesia During 3% lSOflurane the firing threshold became 4 + 3 mV more

positive relative to the extracellular reference potential

The effect on mward recttftcatton The input resistance of hlppocampal pyramidal cells Increases as the membrane potential approaches threshold This phenomenon is referred to as anomalous or Inward rectification 19 Twenty-three out of 24 neurons tested by series of depolarizing and hyperpolarlzlng current pulses (Fig 4A) showed subthreshold inward rectification The slope of the pulse amphtude vs current plot in Fig 4B reflects the input resistance The control plot reveals inward rectification Isoflurane (3%) reduced the response to depolanzmg pulses by more than 50%, whereas hyperpolarlzmg pulses were reduced by about 20% Sixty-six per cent of the neurons tested this way showed decreased input resistance In the subthreshold area during lSOflurane In the remaining neurons reward rectification was reversed or abolished Fig 4C displays data from an experiment where a

3l constant depolarizing current pulse (50 ms, 0 3 nA) was superimposed on a longer (150 ms) variable pulse (inset) While the short test pulse amplitude was kept constant, the membrane potential was manipulated by the long pulse The input resistance (as calculated from the amphtude of the constant pulse) is plotted vs the membrane potenttal The control plot shows that input resistance increased as the membrane potential approached threshold (arrow), l e subthreshold inward rectification Following ~soflurane administration the input resistance was reduced from 20 to 16 MQ Close to threshold, input resistance decreased rather than mcreased upon depolarlzatnon (outward rectfflcatton) Fifteen neurons were tested this way All showed subthreshold reward rectification before ~soflurane During

]soflurane administration, 3 cells displayed subthreshold outward rectification, 5 cells no subthreshold rectification, while 7 cells showed a reduced degree ot inward rectification The voltage deflections caused by depolarizing and hyperpolarlzmg pulses were reduced by 40 +_ 14% and 16 4- 9% respectlvel~ Fig 5 shows the response ot a neuron to a sub-

A

Con

Is• 3%

Current

I

(nA)

' - - - J ' ~

-0 3 -0 6

___J 20my 50ms

B

Amplitude

(mv)

J

RIse tume

20mV

2ms

E~

105

L_

|

.

-08 O-O-O--O Oo4~•• /