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f Pharmacology and Experimental Therapeutics >76 by The Williams & Wilkins Co. ' ~Vol. 199, No. 1 Printed in U.SA. ODE OF ACTION OF TRICHLOROETHYLENE ON SQUID AXON MEMBRANES1 BRIJ BHUSHAN SHRIVASTAV, TOSHIO NARAHASHI, RICHARD J. KITZ AND JAMES D. ROBERTS tment of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina and Department of Anesthesiology, Massachusetts General Hospital, Boston, Massachusetts Accepted for publication April 27, 1976 ABSTRACT Shrivastav, Bru Bhushan, Toshio Narahashi, Richard J. Kite and James D. Roberts: Mode of action of trichloroethylene on squid axon membranes. J. Pharmacol. Exp. Ther. 199: 179-188, 1976. The mode of action of trichloroethylene on electrical properties of squid giant axons has been studied by means of voltage clamp techniques. Trichloroethylene decreased the resting membrane potential in a manner dependent upon the concentration, the depolarization by 50% saturated trichloroethylene attaining 28.4 and 32.7% of the initial value at 20 and 108C, respectively. Leakage conductance was decreased to 34.6% of the control by 30% saturated trichloroethylene at 10-12C. It appears that the methylene-induced depolarization is at least in part due to a decrease in resting ^ium permeability. Both peak transient and steady-state conductance increases were suppressed by trichloroethylene, and the curve relating the steady-state conduct ance to the membrane potential was shifted in the depolarizing direction while the peak transient conductance curve was not appreciably shifted. The reversal potential for the peak transient current was greatly shifted by trichloroethylene in the direction of hyperpolarization in a manner dependent on the concentration, the maximum shift amounting to 25 mV at 10C. This effect was less pronounced at 20C. The shift in the reversal potential is mostly due to a decrease in selectivity of the peak transient channel and partly due to an accumulation of sodium ions inside. Analyses of dose-response relation^in suppressing peak transient and steady-state conductances show that trichloroethylene interacts with receptor on a one-to-one stoichiometric basis. Steady-state sodium inactivation curve was shifted by trichloroethylene in the direction of hyperpolarization. All of these effects were partially reversed after washing the axon with anesthetic-free media. The accumulation of sodium ions inside would be much more pronounced in small nerve fibers in the brain than in giant axon and, to gether with the observed decrease in the selectivity of peak transient channels, would play a significant role in general anesthesia. ltage clamp techniques have been successused to separate ionic currents into sodium :eived for publication June 23, 1975. nd reprint requests to: Dr. Brij Bhushan astav, Department of Physiology and Pharlogy, Duke University Medical Center, Durham, 27710. and potassium components during nerve excitation (Hodgkin et al,, 1952; Hodgkin and Huxley, 1952a) and to elucidate the mechanism of action of various neuroactive agents in terms of membrane ionic conductances. For example, 'This study was supported by Grants GM 21724 and NS 10823 from the National Institutes of Health. 179 SL 0328^0 *^-5s f** - Sl?! nr *. 5 * : **4 -- * rtr- * * Si; =S 3 180 SHRIVASTAV BT AL. Vol, 199 barbiturates have been shown to decrease peak transient and steady-state conductances (Narahashi et al., 1969; Blaustein, 1968). Procaine, dibucaine, lidocaine and tertiary and quater nary tropine esters also inhibit peak transient and steady-state conductances (Taylor 1959; Shanes e al., 1959; Blaustein and Goldman, 1966; Hille, 1967; Narahashi et al., 1969, 1972; Strichartz, 1973). Similarly alcohols decrease both conductances (Armstrong and Binstock, 1964; Moore e al., 1964); The mechanism of volatile general anesthet ics on neurons remains largely to be explored. There has been no complete study dealing with the ionic mechanism of action of these anesthet ics, We have performed voltage clamp experi ments to study the effects of trichloroethylene on ionic conductances of squid giant axon membranes. The squid axon was chosen as a model because voltage clamp experiments could be performed with a high degree of accuracy and also because a wealth of information was availa ble on its electrical properties. Preliminary accounts of this study have been reported (Shrivastav and Narahashi, 1974). Methods Materials. Experiments were performed at the Marine Biological Laboratory, Woods Hole, Mass., using the giant axons from the squid, Loligo pealei. The diameter of the axons ranged from 325 to 700 p. Isolated axons were cleaned by removing thin nerve fibers and connective tissues. For internal perfusion experiments, the axoplasm was removed by means of a small roller, and the axon was internally perfused with standard internal solution by the method origi nally developed by Baker et al. (1961) and modified by Narahashi and Anderson (1967). The desired rate of flow of the internal perfusate was achieved by adjusting the hydrostatic pressure of the standard internal solution reservoir. Depending on the diame ter of the axon, the flow rate was maintained 30 to 90 *il/min. External solution was perfused continuously. Solutions. Filtered sea water was used as external perfusate in all experiments. Anesthetic was dissolved to saturate sea water at a temperature of 21-23C. The solubility of trichloroethylene in water at 25C is estimated to be 8.4 mM (Adriani, 1972), Thus, when saturated in sea water, the concentration of trichloro ethylene should not exceed 8.4 mM. The anestheticsaturated sea water was then diluted with sea water to give a desired strength just before each change of anesthetic concentration. No correction of the trichlo roethylene concentration in sea water was made to compensate for possible loss by evaporation during external perfusion. The presence of anesthetic in sea water did not affect the pH, which was between 7,9 and 8. The composition of the the standard internal solution was as follows; 350 mM K \ 50 mM Na*, 50 mM F`, 320 mM glutamate , 333 mM sucrose and 15 mM phosphate buffer at pH 7.3. The ionic composi tion of Woods Hole sea water is 459 mM Na% 9.7 mM K+, 535 mM Cl', 10 mM Ca++, and 52 mM Mg+t (P. Mangelsdorf, Jr., personal communication). Electrophysiological measurements. A glass cap illary of 75 a in outer diameter and filled with 0,6 KCI solution was inserted longitudinally into the axon to measure the resting membrane potential. The capillary contained a 25-p platinum wire to reduce high frequency impedance. The reference electrode was made of a glass capillary of about 500 a in diameter. This capillary was filled with 2% agar in sea water. The membrane potential..was continuously recorded on a strip chart recorder. For voltage clamp experi ments, a 75-a platinum wire plated with platinum black was attached "piggy-back" to the internal capillary potential electrode. This wire served as the internal current electrode. External current electrodes consisted of three platinum plates of 4-mm width coated with platinum black. The central plate was connected to an operational amplifier to measure currents, and the adjacent plates were directly grounded to serve as guard electrodes. The voltage clamp circuit developed by Moore and Cole (1963) was used in the form described by Wang et al. (1972). During voltage clamp, the membrane potential was held at a slightly hyperpolarized level to remove sodium inactivation. Command depolarizing pulses were applied and membrane currents associated with these pulses were recorded. Occasionally the mem brane was undamped to record the resting membrane potential and the action potential. The membrane potential of intact axons was not corrected for junc tion potentials. Leakage currents associated with step hyperpolarizations were also recorded. Peak transient and steady-state current were not corrected for leak age current. Membrane potential measurements were not corrected for junction potential of the interna! electrode which was of the order of 10 mV (Curtis and Cole, 1942). Temperature. The experiments were conducted at 20 or 10-12C. Statistical tests. Student's t tests were employed to evaluate the difference between the means. Results Resting membrane potential. Resting mem brane potential decreased after external appli cation of trichloroethylene to the intact axon. The higher the concentration of anesthetic, the greater was the membrane depolarization. Fig ure 1 illustrates the dose-response relationships 0328^ SL TRICHLOROETHYLENE ON AXON MEMBRANES 181 Fig. 1. Effects of external application of different icentrations of trichloroethylene on the resting mbrane potential of squid giant axons at 20 and 3. Measurements were made at the steady state, at 15 minutes for each change of anesthetic jentration. Ordinate represents the percentage of ease in membrane potential (E,,,), and abscissa percentage of saturation of trichloroethylene, h point represents the mean S.E.M. with the iber of axons as indicated. Curves were drawn by tl^^prrease in resting membrane potential duOTiby trichloroethylene. The concentrai of trichloroethylene was increased cumula;ly, and after introduction of a new concen:ion, measurements were made when the mg membrane potential attained a steady :e in about 15 minutes. The percentage of olarizations of the membrane at 20C ged from 10.9 1.4% (mean S.E.M., 5 ns) by 6.5% saturated-trichloroethylene solui (from mean of -57.0 to -50.8 mV) to 28.4 .2% (5 axons) by 50% saturated solution (to ).8 mV). At 10C, the percent decrease in mbrane potential was 5,9 1.2% (4 axons) >m mean value of -67.7 to -63.7 mV) and ) 1.6% (3 axons) (to -46.0 mV) by 5 and s saturated solutions, respectively. Thus, the hloroethylene-induced depolarization was iter at 20 than at 10C at concentrations of J saturation or less, but the reversed effect . seen as concentrations of 30% saturation or re. After washing the nerves for about 1 Vj irs with anesthetic-free sea water, the resting mbrane potential was restored to 85.9 % (5 axons) and 82.9 1.3% (4 axons) of the trol at 20 and 10C, respectively. Action potential. Figure 2 shows the action potentials recorded at 12C before and during external application of 30% saturated trichloro ethylene to an intact axon for 15 minutes, and after washing with anesthetic-free sea water. The threshold membrane potential became less negative after introduction of trichloroethylene, and the amplitude of the action potential was effectively suppressed with both the rising and falling phases slowed. In some cases, the nerve started firing repetitively in the anesthetic. Washing the axon with anesthetic-free sea water for about 1 Vs hours partially restored the amplitude of the action potential. However, the recovery of the action potential was complete if perfused axon was washed internally. The undershoot that immediately followed the fall ing phase of the action potential was almost absent in the trichloroethylene-treated axons. There was no recovery of undershoot on washing the axons either externally or internally, even when the spike amplitude was completely re stored. Leakage conductance. The leakage current was measured by application of hyperpolarizing pulses. The percentage of decrease in leakage conductance in trichloroethylene amounts to 34.6 6,1 (mean =t S.E.M., 5 axons) at 10-12C when the intact axons-were exposed to 30% saturated trichloroethylene sea water. Recovery of leakage conductance after washing was 198.5 70.7% of the control (5 axons). Membrane ionic currents. Figure 3 shows families of membrane currents associated with mV Fig. 2. Action potentials of the squid giant axon before (control) and during external application (15 minutes) of 30% saturated trichloroethylene in sea water and after washing with anesthetic-free sea water at 12C. SL 032842 182 S 16 73 I(20*C) Tricmofthyfane SHRIVASTAV ET AL. Vol. 19 5r C 50% figuration TOwV *50 __ * *10 10 Fic. 3. Membrane currents associated with various step depolarizations from the holding membrane potential of -70 mV before (A, control) and during external application (15 minutes each) of 30% (B) and 50' (C) saturated trichloroethylene in sea water and after washing with anesthetic-free sea water (D). The membrane potential during step depolarization is given at the end of each current record (20C). Fig. 4. Current-voltage relations (Im - Em) for peak transient current (I,,) and steady-state current (I,,) before (control) and during external applications (15 minutes each) of 10.8% and 50% saturated trichlo roethylene and after washing with anesthetic-free sea water. Eftp, holding membrane potential (20C). various magnitudes of step depolarizations ai 20C before and during application of trichloro ethylene to an intact axon for about 15 minutes for each concentration and after washing with anesthetic-free medium. Trichloroethylene re duced inward peak transient (Ip) and outward steady-state (I,,) currents (fig. 3, B and C). Another distinct change was a slight increase in outward peak transient currents. In all of the nine axon preparations used, the recovery ol ionic currents after washing with anestheticfree sea water was incomplete (fig. 3D). The time-to-peak transient current (tP) was not affected by low concentrations of trichloroethyl ene but underwent a small but consistent in crease by higher concentrations. Measured at +20 mV with 30% saturated trichloroethylene, the increase in tp amounted to 8.9 * 3.5% and was not statistically significant (P > .2, 5 axons). The onset of the steady-state potassium current was delayed after application of trichlo roethylene. Current-voltage relations. The amplitudes of peak transient and steady-state currents of the same intact axon as used for figure 3 are plotted against the membrane potentials in figure 4. The following features become immedi ately apparent from the current-voltage curves: 1. Trichloroethylene suppressed peak tran sient currents at the membrane potentials more positive than -40 mV. TRICHLOROETHYLENE ON AXON MEMBRANES 183 . It suppressed steady-state currents at all potentials examined. . iBr reversal potential for peak transient rent was shifted in the direction of hyperpozation. . After external washing with anesthetic-free water, only partial recovery of both ionic rents was observed. , The recovery of the reversal potential after ;rnal washing was poor, he reversal potentials for peak transient rent of intact axons at 10 and 20C are led against the concentration of trichlorovlene in figure 5. The shift in the reversal ential by trichloroethylene was much greater 0 than at 20C.. In the internally perfused axons the reversal potential for peak transient current was esti mated to be 46.0 =t 2.0 mV (mean S.E.M., 4 axons, 10-I2C). When the axons were exter nally exposed to 30% saturated trichloroethyl ene while suspending the internal flow, the re versal potential was changed to 26.5 4.9 mV. Upon resuming the internal flow without remov ing anesthetic from the external perfusate, the reversal potential quickly recovered to a value of 44.2 1.4 mV. Then the anesthetic was removed from the external perfusate solution, and the reversal potential remained almost unchanged at 43.7 1.1 mV. These results suggest the possibility that the drug acts from inside of the axon to shift the reversal potential. Membrane ionic conductances. The membrane conductances for peak transient and steady-state currents were calculated by equations: P " Ie Em -- Ep -- i,, Em - E,, (1) (2) ic. 5. The reversal potential (Ep) for peak tran- t Met at different percentages of saturation of ilc^^wlene at 10 and 20C. Each point repre$ fMKean S.E.M. with the number of axons :ated. where g, I and E with subscript refer to the conductance, current and reversal potential for peak (p) and steady-state (ss) components, respectively, and Em refers to the membrane potential. The steady-state slope conductance, 0 *5 :a.- C. Cr. 3 ig. 6. Peak transient conductance (g,,) (A! and steady-state conductance (g,,) (B) plotted in a logarithmic e as a function of the membrane potential (Em) before control and during external application (15 minutes h) of 10,8 and 50% saturated trichloroethylene and after washing with anesthetic-free sea water (20C). jws in B indicate the membrane potentials where g,, attains a half-maximal value. I I 184 SHRIVASTAV ET AL. Vol. JS.'i g,, (slope), was also calculated from the equa tion g,, (slope) dEm (3) The membrane conductances of the same intact axon as used for figures 3 and 4 are plotted on a logarithmic scale against the mem brane potential in figure 6. After application of trichloroethylene for 15 minutes, the peak tran sient conductance curve was shifted downward along the conductance axis, but was not appre ciably shifted along the potential axis (fig. 6A). The membrane potentials at which the peak transient conductance attained a half-maximal value were estimated to be -37.5 0.9 mV in the control, and -36.2 1.7 and -35.5 2.7 mV after application of 18 and 50% saturated trichloroethylene sea water, respectively (4 axons). Neither of the latter two values is different significantly from the control value (P > .2). The steady-state conductance curve was also suppressed downward and was shifted in the direction of depolarization (fig. 6B). The membrane potentials at which the conductance attained a half-maximal value (arrows, fig. 6B) were estimated as follows (5 axons): control -28.0 3.3 mV, 6.5% saturated trichloroethyl ene -20.6 st 2.9 mV (P > .02), 18% saturated -12.0 2.7 mV (P > .001), 50% saturated -4.0 2.3 mV (P > .001), after washing -17.0 4.9 mV (P > .02). Dose-response relationship. The dose response curves for the suppression of peak conductance of intact axons by trichloroethyl ene at 20 and 10C are illustrated in figure 7A The effect on the peak conductance was no: greatly affected by temperature change, and the small difference at the highest concentration used (50% saturation) was not statistically sig nificant (P > .1). The data on the steady-state conductance are plotted in figure 7B. Again there was no marked difference between 10 and 20C in the percentage of suppression at the highest concentration (P > .1). It can be seen from figure 7, A and B, that trichloroethylene suppressed the peak transient and steady-state conductances to approximately the same extent at either 10 or 20C. Figure 8 illustrates Hill's plots for the data at 20C according to the equation: log ---------- --- n log [S1 - log K Vmi ~ V (41 where V is the response or percentage of inhibi tion of conductance, Vro,, is the maximum response (100% inhibition), (S) is the percent saturation of trichloroethylene, n is the interac tion coefficient and K is the apparent dissocia tion constant. The slopes of the curves provide the value of n, which are estimated to be 0.9 and 1.0 for the maximum peak transient conductance and the steady-state conductance at 60 mV, respec- FlO. 7. Dose-response relations for the action of trichloroethylene in suppressing the maximum peal, C\s uctance (gcim.ii) (A) and the maximum steady-state slope conductance (gw,,,,,,,,,) (B) at lc and 20 C. Measurements were made at the steady state (15 minutes) after application of trichloroethylene Each point represents the mean S.E.M. with the number of axons as indicated. Curves were drawn by eye. SL 032845 TRICHLOROETHYLENE ON AXON MEMBRANES 186 ic. 8. Hill's plots for the action of trichloroethyl' in suppressing die maximum peak transient constance (gp) and the steady-6tate conductance (g,,) -i-60 mV. Each point represents the mean value of e measurements. V, percentage of decrease of iductance; VM,, maximum percentage of dease of conductance (100%); jS], percentage of uration of trichloroethylene. The slopes of the ight lines are 0.9 and 1-0 for gp and gH, respecly (20C). ly. This indicates that trichloroethylene ds to its receptor on a one-to-one stoichio:ric basis thereby blocking the conductances, j apparent dissociation constants for the k transient and steady-state conductances e^^^ted to be 41 and 50% saturation of hlMKhylene, respectively, steady-state sodium inactivation. The ct of trichloroethylene on the steady-state ium inactivation was also examined at 10 C ig intact axons. Prepulses of various amplies and 45-msec duration were followed by a depolarizing pulse of +50 or +60 mV with a isec duration. The amplitude of peak trantt current associated withjhe test pulse was asured and plotted as a ratio to its maximum ue against the membrane potential of the pulse. Figure 9 depicts such steady-state ium inactivation curves before and during ilication of 10% and 20% saturated solutions richloroethylene for 15 minutes in each case, 1 after washing with anesthetic-free solution, e measurements were fitted by the equation h. = (5) ere h* represents the ratio of the peak sorni current- associated with the test pulse to maximum value, Eh represents the mem brane potential for h,, = 0.5 and k represents the slope factor (Hodgkin and Huxley, 1952b). Trichloroethylene shifted the curve in the direc tion of hyperpolarization. The shift of the curve, measured at half-maximal value, was 5.6 + 2.0 mV for 10% and 5.9 2.0 mV for 20% saturated toward the control level, the difference between the recovery and control curves being estimated to be 1.1 1.6 mV (mean S.E.M., 5 axons). Trichloroethylene also increased the slope fac tor k by 1.4 0.6 and 3.1 0.5 in 10 and 20% saturated sea water (mean S.E.M., 5 axons). After washing, the increase in k remained by 1.0 1.1 (mean + S.E.M., 5 axons). Discussion When applied externally to intact axons at 10C, 30% saturated trichloroethylene depolar ized the squid axon membrane from the mean value of -67.7 to -48.0 mV. The observed decrease in leakage conductance by 34.6% is in keeping with the depolarization, for the leakage conductance is largely composed of potassium conductance. Since the depolarization attained a steady-state level relatively rapidly (about 10 minutes), it can be assumed that the change in Fig. 9. Steady-state inactivation curve before () and during external application (15 minutes) of tri chloroethylene at 10% (O) and 20% (#) saturation levels, and after washing with anesthetic-free sea water (A). Ip, peak current associated with test step depolarization; ,, the maximum value of Ip. The abscissa represents the membrane potentials of condi tioning pulses. The measurements were fitted by equation 5 with the aid of Hewlett-Packard 9821A calculator. The E,, and k values in equation 5 for control, 10 and 20% of trichloroethylene saturation and after washing were -46.8, -51.3, -55.1 and -46.5 mV, and 4.3, 7.5, 7.4, 7.2, respectively. Temper ature, 10C. U. 2 3 186 SHRIVASTAV ET AL. Vo!, m internal ionic concentrations is negligible. Thus, if one assumes that the decrease in leakage conductance is entirely due to a decrease in potassium permeability, the change in resting sodium permeability may be calcu* lated from the constant field equation (Hodgkin and Katz, 1949): pK[K]0 + P<..[Naj0 + Pc,[ClJ1 Pk[K], + Pn[Na], + Pc,[Cl]o where P is the permeability to ions in subscript, [] is the concentration of ions outside (o) or inside (i) of the axon, and R, T and F have their usual meaning. Taking [K], - 335 mM, [Na]j = 52 mM and [Cl], - 135 mM (Rosenberg, 1973) and the values for the ionic concentrations in natural sea water as described in "Methods," the sodium permeability after exposure to 30% saturated trichloroethylene at 10C is calcu lated to be 2.55-fold of the initial control value. When applied externally, trichloroethylene caused a shift of the reversal potential for peak transient current in the direction of hyperpolar ization (figs. 4 and 5). The possible causes of the shift are as follows: Increase in internal sodium concentration. The influx of radioactive sodium in normal intact axons was estimated to be 29.0 x 10'11 mol/cm'sec (unpublished data). For an axon with a diameter of 500 n and exposed to trichloroethylene for 10 minutes, the increase in internal sodium concentration due to a 2.55-fold increase in resting sodium permeability is cal culated to be 3.5 mM. This will shift the reversal potential in the direction of hyperpolarization only by 1.6 mV. Change in ionic selectivity of the sodium channel. The ratio Pk/Pn. during peak tran sient current can be calculated by the constant field equation. Since the contribution of chlo ride permeability to peak transient current is negligible, equation 6 can be reduced to de scribe the reversal potential (Ep): E [n (Pk/Pn.)EKIq+ [Na],, F (Pk/P*.) IK J, + [Na], With the values [K), = 335 mM, [Na], = 52 mM, [K]0 * 9.7 mM and [Na],, = 459 mM, the ratio Pk/Pn. for control axons at 10C is esti mated to be 1:12.9 for Ep equal to 43.2 mV (no: corrected for junction potential) and 1:5.1 forE, equal to 33.2 mV (corrected for a 10-mV junction potential). Similarly, taking [Na], = 55.-5 mM and [K],, [KL and [Na], unchanged above for axons treated with 30% saturated trichloroethylene at 10"C for 10 minutes, selec tivity, ratio Pk/Pn. is calculated to be 1:2.0 for Ep equal to 18.5 mV (not corrected for junction potential) and 1:1.2 for Ep equal to 8.5 mV (corrected for a 10-mV junction potential). In both cases, therefore, the selectivity ratio P/ Pn of the peak transient channels is increased by a factor of 4 to 6 as a result of exposure of 30^ saturated trichloroethylene at 10C. Leakage conductance. The leakage conduct ance in the control axons is small and i' decreased by trichloroethylene. However, there is evidence that the leakage current shows a rectification in depolarized membranes (Adelman and Taylor, 1961). Thus, if trichloroethyl ene greatly augmented the rectification, then the reversal potential for peak transient current as estimated without correction for the leakage current would be shifted in the hyperpolarizim: direction. Steady-state potassium current. If the steady-state potassium current started flowing earlier than normal, it would contribute signifi cantly to the total peak transient current, thereby causing a shift in Ep. However, this can be ruled out because trichloroethylene retard.' the onset of the potassium currents rather than accelerates it (fig. 3). Halothane applied externally to squid giam axons in low concentrations also depolarized the membrane and decreased the reversal potential for peak transient current (Shrivastav and Narahashi, 1974). Trichloroethylene possesses some unique fea tures of nerve blocking action which are lacking in pentobarbital and procaine (table 1). They include depolarization of the membrane decrease in leakage conductance, absence of the shift of the curve-relating peak transient con ductance to membrane potential, absence of the change in the time-to-peak current and shift ot the steady-state sodium inactivation curve in the hyperpolarizing direction. It should be noted that trichloroethylene has some effects on the resting membrane characteristics as exem plified by the resting potential and the leakage SL 032847 t u v u u t l * * 1 I t I > I J iM n m TRICHLOROETHYLENE ON AXON MEMBRANES 187 TABLE 1 Effects of trichloroethylene, pentobarbital and procaine on various properties of squid axon membranes 0, no change; 1, decietae; ], increase; --, shift in he depolarizing direction; shift in the hyperpoarizing direction. Trichloro Pento Pro ethylene barbital' caine* testing potential tction potential Vak conductance neadv-fitflte conductance leakage conduct- ance I'eak conductance-mem- brant potential curve Time-to-peak current Reversal potential for peak current -tteady-state Na inac- tivation curve 1 1 1 1 1 O O *** 0O 1 , J 11 11 OI -- 11 0-- O "From Narahaehi et al. (1969). * From Taylor (1959) and Shanes et ol. (1959). onductance, as well on the membrane characaristics during activity. On the other hand, entobarbital and procaine affect primarily the characteristics during activity. Ii^Knan and dog, the blood levels of trichlo- rethylene during surgical anesthesia have been stimated to about 0.8 and 2.0 mM, respectively Adrian), 1972), which correspond to 9.5 and 3.8% saturated trichloroethylene in water at 5C, respectively. Therefore, the trichloroethlene concentrations of 10 to 50% saturation in a water at 10 and 20C used in the present ludy are of the same -order of magnitude as lose in the blood during surgical anesthesia. A nerve fiber with a diameter much smaller ran that of the squid giant axon has a very irge surface 'area/volume ratio. Therefore, rese concentrations of trichloroethylene will tore quickly increase the internal sodium conentration as a result of an increase in resting xiium permeability. If one assumes that the ame degree of the sodium permeability inease occurs in a small fiber as in a squid giant xon, the internal sodium concentration inreases from 52 to 81.6 mM in a fiber with a iameter of 1 p in 10 seconds as against 55.5 iM in a fiber with a diameter of 500 u in 10 linutes. This factor by itself will cause a shift f the reversal potential for peak transient current by as much as 10.9 mV in the small fiber exposed for only 10 seconds as against the shift of only 1.6 mV in the giant axon exposed for 10 minutes. Such a large shift will drastically cur tail the peak of the action potential. In anes thetized cats exposed to high pressures of inert gases, a cation imbalance has also been found in their 6pinal fluids (Bennett and Hayward, 1967). In addition, trichloroethylene decreases the selectivity of the sodium channel during activity, suppresses peak - transient conduct ance and depolarizes the nerve membrane. All these factors together will have a profound de pressing effect on the electrical activity of small nerve fibers and nerve endings which are found in abundance in the brain. Acknowledgments. The authors wish to thank Dr. Mere] H. Harmel for providing tri chloroethylene, Mrs. May Wu and Mrs. Debra Kuhlman for data analysis, Mr. Edward M. Harris for maintenance of electronics equip ment and Mrs. Arlene F. McClenny, Mrs. Gillian C. Cockerill and Mrs. Delilah Munday for secretarial assistance. References Adelman, W. J. and Taylor, R. E.: Leakage current rectification in squid giant axons. Nature (London) 190: 883-885, 1961. Adrian!, J,: The Chemistry and Physics, of Anes thesia, Charles C Thomas, Publisher, Springfield, 111., 1972. Armstrong, C. M. and Binstock, L.: The effect of several alcohols on the properties of the squid giant axons. J. Gen. Physiol. 48: 265-277, 1964. Baker, P. F., Hodgkin, A. L, and Shaw, T. I.: Replacement of the protoplasm of a giant nerve fibre with artificial solutions. Nature (London) 190: 885-887, 1961. Bennett, P. B. and Hayward, A. J.: Electrolyte imbalance as the mechanism for inert gas narcosis and anesthesia. Nature (London) 213 : 938-939, 1967. Blaustein, M. P.: Barbiturates block sodium and potassium conductance increases in voltageclamped lobster axons. J. Gen. 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