Chronic Exposure to Lead Causes Persistent Alterations in the Electric Membrane Properties of Neurons in Cell Culture

BRIAN SCOTT and JAMES LEW

Departments of Biology and Psychology, University of Windsor, Windsor, Ontario, Canada, and Surrey Place Centre, 2 Surrey Place, Toronto, Ontario, Canada, M5S 2C2

Received August 17, 1984; revised April 29, 1985

SUMMARY

The effects of chronic lead (Pb) exposure on neuronal electric membrane properties (EMP) were determined using neural cell cultures of adult mouse dorsal root ganglia (DRG). Cultures were exposed to Pb concentrations ranging from 0 to 100 pM for 12 days (8 DIV to 20 DIV). EMP were determined in Pb-free medium either immediately after withdrawal (IWD), or 6 days after withdrawal (6WD) from Pb.

For IWD, regression analysis indicated that a number of EMP varied significantly with increasing Pb concentration. The largest such change occurred for electrical excitability which decreased significantly with in- creasing Pb (P = O.OOO), being reduced by approximately two-thirds for neurons exposed to 100 pM Pb; resting membrane potential increased with Pb (P = 0.000); membrane time constant decreased with Pb (P = 0.007); action potential afterhyperpolarization decreased with Pb (P = 0.023). There was also evidence that the time course of action potentials was accelerated with increasing Pb concentrations, the rate of fall of neurons with biphasic falling phases being particularly increased (P = 0.047). This general pattern of altered EMP was observed for the 6WD condition also, indicating that chronic exposure to Pb caused persistent abnormalities in neuronal membranes even after 6 days of cultivation in Pb-free medium.

The patterns of alterations in EMP suggested that chronic Pb exposure caused a prolonged increase in potassium permeability. It was proposed that the latter was mediated through a Pb-induced increase in intracellular ionic calcium and the associated disruption of calcium homeostasis.

INTRODUCTION

Chronic exposure to relatively high levels of lead (Pb) has long been known to cause serious neurological problems such as peripheral neuropathy and in some cases encephalopathy. Recently, considerable evidence has been obtained indicating that exposure to much lower Pb levels can have subtle sequelae such as deficient long-term memory and reduced psychomotor speed (for review, see Petit and Alfano, 1983). Considering these well- documented deleterious effects of Pb exposure and the numerous sources of Pb in the contemporary environment, there have been relatively few basic electrophysiological studies of either acute or chronic Pb toxicity.

Journal of Neurobiology, Vol. 16, No. 6, pp. 425-433 (1985) 0 1985 John Wiley & Sons, Inc. CCC 0022-3034/85/0600425-09$04.00

426 SCOTT AND LEW

A few studies have obtained evidence of a decrease in peripheral nerve conduction velocity in humans exposed to Pb even in the absence of clinical symptoms (for review see Seppalainen, 1982). For example, Buchthal and Behse (1979) found evidence of slight decreases in conduction velocity even though there was no evidence of the axonal degeneration and demyelination characteristically found in peripheral neuropathy. Therefore, the authors suggested that the change in conduction velocity might be due to a direct effect of Pb on neuronal membranes.

Some work has been carried out investigating the acute effects of Pb on synaptic transmission in vitro (for reviews see Cooper and Sigworth (1979) and Petit and Alfano (1984)). These studies have demonstrated a competitive interaction between Pb and calcium (Ca) a t presynaptic endings, which resulted in decreased Ca influx and therefore decreased transmitter release (e.g., in bullfrog sympathetic ganglion, Kober and Cooper, 1976).

In summary, relatively little work has been done on the electrophysiological effects of chronic Pb exposure and no studies could be found in the literature which utilized intracellular recording to investigate Pb's effect on the electrical properties and functioning of neuronal membranes. Consequently there is little understanding presently of the basic cellular mechanisms by which Pb exerts its toxic effects.

The purpose of the present study was to conduct an electrophysiological investigation of the effects of chronic Pb exposure on the electric membrane properties (EMP) of individual adult mouse DRG neurons in cell culture. A previous study has determined quantitatively the effects of Pb toxicity on neuronal and non-neuronal cell survival (Scott and Lew, 1985). In the present study it has been demonstrated that chronic Pb exposure produced prolonged (6 days or longer) alterations in a number of EMP, including reduced electrical excitability and hyperpolarization of the resting membrane.

MATERIALS AND METHODS

The method for preparing neural cell cultures of adult mouse DRG was identical to that described previously (Scott, 1977a; Scott and Lew, 1985). About 60 DRG were dissected from two adult mice, incubated for 2-3 h in 0.25% collagenase at 36"C, rinsed with phosphate buffered saline (PBS), and triturated into a cell suspension. The cells were plated on collagen- coated coverslips.

Cultures were incubated at 36°C with the carbon dioxide concentration adjusted to produce a pH of 7.4. They were fed daily with 0.3 ml of medium for the first 2 days. On day 3, the cultures were flooded with 2.0 ml of medium and fed three times per week thereafter.

Culture medium consisted of 10% fetal calf serum (GIBCO, Grand Island, NY) in CMRL- 1415 (Connaught Medical Research Laboratory, Toronto), with penicillin and streptomycin present a t 50 units/ml and 50 pg/ml, respectively. Osmotic pressure was 295 mOsm throughout the experiment.

On day 8, four cultures continued to be maintained in Pb-free medium while groups of four cultures each were changed to medium with the following Pb concentrations: 12.5, 25, 50, and 100 p M . The 100-p.M solution was prepared by addition to the culture medium of the appropriate quantity of a 20-mM solution of Pb acetate in water. The intermediate concentrations of Pb medium were prepared by mixing appropriate volumes of the zero and 100-mM Pb medium.

Pb concentrations were checked routinely by atomic absorption spectroscopy (AAS) and found to agree with the values calculated on the basis of the dilution of the stock Pb acetate solution to within ? 10% (n = 10). There is evidence that ionized Pb may be only two-thirds of the calculated concentrations (Scott and Lew, 1985). However, it seems quite likely that the cells are affected by total Pb (i.e., ionized Pb, Pb bound to protein, and Pb in the colloidal form). Also blood Pb levels quoted in the literature generally are total Pb concentrations.

ELECTROPHYSIOLOGY OF CHRONIC Pb 42 7

Therefore, Pb concentrations referred to hereafter represent total Pb as calculated from dilution of the stock solution.

EMP were determined either immediately after withdrawal from the Pb medium (IWD condition) or after 6 days of further cultivation in zero Pb culture medium (6WD condition). For IWD, two experiments were carried out and in each experiment EMP were determined for a t least 40 neurons for a total of 490 neurons. For 6WD, only one experiment was performed and EMP were determined for 52 control neurons not exposed to Pb and for 23 neurons which had previously been exposed to 100 ELM Pb.

For the electrophysiological work, the coverslip culture was rinsed free of Pb by several rinses in PBS followed by a 5-min equilibration in PBS. The latter also removed bicarbonate and brought the pH to 7.4. The culture was then mounted in a special recording chamber attached to an inverted phase contrast microscope. The culture was bathed in a Pb-free solution identical to the usual culture medium but without sodium bicarbonate and with pH adjusted to be 7.4 in air. Temperature was maintained at 36°C.

Glass microelectrodes were filled with 3 M KC1 and had initial resistances of 40-60 Meg Ohms but were routinely broken to 30-50 Meg Ohms by gentle stroking on the collagen substrate. EMP were measured using an intracellular amplifier with bridge circuity for impedance measurements (model N-950, Mentor Corp., Minneapolis, MN). Details of the specific procedure and calculations are given elsewhere (Scott et al., 1979). The following EMP were determined: (1) resting membrane potential (VM); (2) specific membrane resistance (RM); (3) membrane time constant (T), obtained from the approximation T = ZS/IRH where ZS is the threshold current for short-duration pulses (duration < 1/20 TI, and IRH is rheobasic current; (4) specific membrane capacitance (CM) calculated from T = RM x CM; (5) duration (DT) of action potential (AP); (6) AP overshoot (0s); (7) AP afterhyperpolarization (AHP); (8) type of action potential (MB): for APs with a monophasic falling phase, MB = 1; for APs with a biphasic falling phase, MB = 3; (9) rates of rise (RISE) and fall of the AP; for MB = 1, there was only FALLB; for MB = 3, there was FALL1 and FALL2; (10) rheobasic threshold depolarization (VRH) determined from VRH = RI x IRH where RI is cell input resistance; (1 1) cell surface area (AREA) calculated using the appropriate formula for either a sphere or a prolate spheroid.

Appropriate three- and one-way analyses of variance and linear regression analyses were carried out using the Statistical Package for the Social Sciences (SPSS).

RESULTS

Immediate Withdrawal Case (IWD)

Previous investigations of the EMP of neurons in culture (Scott and Edwards, 1981; Scott, Petit and Becker, 1981) had shown that the EMP were significantly affected by both cell size (AREA) and type of action potential (monophasic or biphasic falling phase, MB). Because of these secondary effects, one way analyses of variance were carried out for each EMP by Pb both without and with MB and AREA as covariates (Table 1). Moreover, the results of a preliminary three-way analysis of variance for each EMP by Pb by MB by AREA indicated that of the two- and three- way interactions, only that of T and VRH by AREA were significant. Consequently, the one-way analyses of variance for T and VRH were per- formed separately for small (AREA < 0.5 x lop4) and large (AREA > 0.5 x l op4) cells (rows a and b, respectively, in Table 1).

Of all the EMP measured only VM, T, VRH were significantly affected by Pb exposure (P < 0.05) both with and without MB and AREA as covariates (Table 1). DT was significantly affected by previous Pb exposure only when covariates were not used. The Pb effect was significant for T only for the large cells while the Pb effect on VRH was significant for both large and small cells.

VM increased from a mean of 60.8 mV for the control cellsonot exposed to Pb to 65.2 mV for 100-puM Pb exposed cells, a highly significant (P =

428 SCOTT AND LEW

TABLE 1 Effect of Chronic Lead Exposure on EMP of Neurons in Culture, Immediately

after Withdrawal

ANOVA Lead Concentration (pM) Without ~ ~

~ ~-

Covariates 0 12.5 25 50 100 Maximum EMP ( P ) ( n = 115) ( n = 87) ( n = 86) ( n = 89) ( n = 113) Q Change P

VM 0.000 60.8 62.4 63.0 63.8 65.2 + 7.2% 0.000 RM 0.543 802. 766. 784. 767. 738. - 7.99 0.527 Ta.k 0.455 1.68 1.68 1.52 1.52 1.56 - 7.1% 0.546

0.000 1.80 1.39 1.58 1.25 1.15 -36.14 0.000 CM 0.202 2.30 2.15 2.17 1.95 2.10 - 8.7% 0.222 VRH" I> 0.000 8.09 9.79 9.98 9.94 12.6 +55.8% 0.000

0.000 10.1 8.65 10.4 14.1 16.8 +66.3% 0.000 DT 0.037 1.90 1.71 1.78 1.72 1.76 - 7.4% 0.063 RISE 0.067 200. 211. 194. 218. 203. + 1.5%' 0.079 FALL1' 0.86 42.0 44.2 44.0 49.9 47.4 +12.9% 0.076 FALL2 0.502 84.8 88.4 88.2 90.9 88.5 + 4.4% 0.738 0s 0.879 21.9 22.1 21.0 22.9 21.1 - 3.6% 0.843 AHP 0.229 10.2 10.5 9.65 9.21 9.08 -11.0% 0.218 AREAd 0.000 5760. 6221. 6127. 5839. 5542. - 3.8% 0.000 M B 0.419 2.20 2.08 2.28 2.13 2.03 - 7.7% 0.000

'I AREA < 0.5 x 10-'cm'. I ' AREA ,' 0.5 x 10-'cm2. ' FALLl occurs for B-type neurons only, therefore this analysis has only AREA as covariate.

MB only as covariate. ' AREA only as covariate.

0.000) increase of 4.4 mV or 7.2%. For the larger neurons, T decreased 36.1%. For small and large neurons exposed to 100 p M Pb, VRH increased by 4.5 and 6.7 mV, respectively, both increases being highly significant statistically (P = 0.000). When calculated as a % of the control value, these increases in VRH were greater than those observed for any other EMP-55.8% for the smaller neurons and 66.3% for the larger neurons. Also MB, although not decreasing when the analysis of variance was carried out without AREA as a covariate, did decrease with high significance (P = 0.000) when this covariate was included. This decrease in MB indicated that the relative frequency of neurons with biphasic falling phases to their APs decreased with increasing Pb concentration.

A regression analysis was performed for each EMP as a function of Pb concentration (Table 2). Six EMP, VM, T, VRH, FALL1, AHP, and AREA were found to have significant linear trends with respect to Pb concentration. Graphs were constructed of the six EMP having significant linearity using values obtained from the analysis of variance with MB and AREA as covariates (Fig. 1). VM increased most dramatically between 0 and 12.5 p M Pb, thereafter increasing at a slower rate (Fig. 1A). Here, as for a number of other EMP, there appears to be a nonlinearity a t 12.5 p M Pb. For example, VRH (large cells) dipped at 12.5 p M and then increased in an approximately linear fashion (Fig. 1B). Similarly, T (large cells) dipped dramatically a t 12.5 p M and then increased and finally decreased (Fig. 1C). FALLl increased with increasing Pb but with a number of obvious irregularities probably due to measurment errors (Fig. 1D). AHP decreased in a much more regular fashion with increasing Pb (Fig.lE). AREA once again showed the irregularity a t 12.5 p M Pb since it increased between 0

TABLE 2 Regression Analysis EMP as a Function of Lead Concentration (n = 4901, Immediately

after Withdrawal

12

a 11 E 1 0 -

E 9 -

3 8 -

7 -

0

VM RM T CM VRH DT RISE

- -

-\ ~ t" E 5 - 55 4

.50 - I I I I 1 1 3 I

Intercept

61.7 788.

1.63 2.21 8.84 1.83

203.

Slope

0.0351 - 0.482 - 0.00231 - 0.00178

0.0483

0.0433 - 0,00138

Correlation Coefficient P

0.281 - 0.0649 - 0.122 - 0.0633

0.299 - 0.0784

0.0265

0.000 0.151 0.007 0.162 0.000 0.083 0.559

FALL1 43.3 0.0528 0.119 0.047 FALL2 85.8 0.0556 0.0570 0.208 0s 22.02 - 0.00688 - 0.0182 0.687 AHP 10.3 - 0.0148 - 0.102 0.023 AREA 6030. - 4.098 -0.169 0.000 MB 2.20 - 0.00160 - 0.0602 0.183

E

I , I I l I, I I I

C D

430 SCOTT AND LEW

and 12.5 Pb and then decreased in an approximately linear fashion at higher Pb concentrations (Fig. 1F).

Six Days after Withdrawal Condition (6WD)

For the 6WD neurons, an analysis of variance with MB and AREA as covariates indicated that VM, RM, T, DT and FALL1 were significantly (P < 0.05) affected by the previous Pb exposure. In order to facilitate a comparison between the immediate (IWD) and six-day (6WD) withdrawal results, changes in EMP for each schedule were computed as percentage of the control values and plotted (Fig. 2). It should be noted that with the exception of CM and AREA, the direction of the changes in EMP were the same for both conditions (Fig. 2). However, for 6WD, the increases in CM and AREA both failed to reach statistical significance and therefore may not be very meaningful.

For the 6WD neurons, VM was still elevated, with high significance (P = O.OOO), above that of the control neurons which had not been exposed to Pb. In comparison with the IWD neurons, the degree of hyperpolarization in VM had decreased only slightly (Fig. 2).

RM was decreased by nearly a third compared to controls and the decrease

'Or VRH

401 50

Fig. 2. Comparison of the effects of previous exposure to 100 pM Pb for neurons immediately after withdrawal from Pb (-) and after being cultured for six further days in Pb free medium (- - - -). Vertical bars represent the changes in EMP expressed as a percent of the value obtained for control neurons not exposed to Pb. Statistical significance (P < 0.05) is indicated by _ . Up indicates an increase and down a decrease from control values. Note the general similarity for the two withdrawal schedules indicating that chronic Pb exposure caused relatively persistent alterations in EMP.

ELECTROPHYSIOLOGY OF CHRONIC Pb 431

was now highly significant; this decrease was much greater than that exhibited immediately after withdrawal from Pb. T was decreased as it had been for the IWD condition but it now was highly significant (P = 0.013). For the 6WD neurons, VRH although still somewhat increased failed to reach statistical significance. The only other statistically significant changes for the 6WD condition was an 18.3% decrease in DT and a 23% increase in FALL2. These changes along with the statistically nonsignificant increases in RISE and FALL1 all indicated that the temporal course of the action potential was considerably speeded up by the previous exposure to Pb. This accelerated action potential had also been suggested in the IWD situation by increases in RISE, FALL1, and FALL2 and a decrease in DT although the changes had not quite reached statistical significance (Table 1).

DISCUSSION

A consideration of the pattern of changes in EMP observed in the present study suggested that chronic exposure to Pb produced an increased potassium permeability (Pk). The most important observation in this regard was the Pb-induced increase in VM, since it has been well established that one important mechanism for hyperpolarization is an increase in Pk (Hodgkin and Huxley, 1952).

Also the dramatic increase in VRH (the amount of depolarization required for excitation) could be the result of an increase in Pk (Hodgkin and Huxley, 1952). The Pb-induced decrease in RM (specific membrane resistance) also supports the PI, hypothesis since RM is inversely related to potassium permeability. The increased rates of falling phases (FALL1 and FALL21 of the action potential is another piece of evidence. Finally, the decreased afterhyperpolarization (AHP) (Table 2) supports the Pk hypothesis since the AHP reflects the difference between the resting membrane potential (VM) and the potassium equilibrium potential (EK), and as the Pk increases VM approaches EK and hence the AHP is reduced.

Thus there is considerable evidence that chronic exposure to Pb caused an increase in Pk which in turn altered a number of EMP. There also is some indication that Pb caused this increase in Pk by increasing intracellular calcium (Ca). Probably the most important observation in this regard was the greatly reduced electrical excitability (increased VRH). Krnjevic and Lisiewicz (1972) showed that injecting Ca into the cat spinal motor neuron decreased excitability by causing an increase in membrane conductance especially for potassium. Also, the number of action potentials with a de- tectable biphasic falling phase decreased significantly in the Pb-exposed neurons (i.e., decreased MB1. It may be that with increased intracellular Ca, the biphasicity was diminished due to a Ca induced larger pk increase during the repolarization phase of the AP.

One difficulty with the hypothesized Pb-induced increase in intracellular Ca is that the amplitude of the AHP decreased with increasing Pb although the decrease was small and statistically insignificant. Since the AHP is due to a prolonged increase in Pk, one would have expected it to increase with the increase in intracellular Ca. However, there is evidence that the late Ca dependent potassium current associated with the AHP is quite independent of resting membrane Pk (for review, see Marty, 1983). Morever,

432 SCOTT AND LEW

the amplitude of the AHP measured from the zero potential instead of the resting membrane potential value did increase by approximately 3.3 mV in 100 pM Pb.

It is possible that chronic Pb exposure caused a disturbance in intracellular calcium homeostasis resulting in increased intracellular calcium and the altered EMP as discussed above. Decreased Na’,K’-ATPase activity has been reported in red blood cells of humans with signs of Pb toxicity (Hasan and Vihko, 1967) and in renal membranes of adult rats exposed chronically to low Pb levels (Suketa, Ujiie, and Okada, 1982). It may be that in a similar way Pb exposure reduces the activity of the Na-K ATPase-linked Ca extrusion system and hence disturbs calcium homeostasis. Also, Goldstein (1977) obtained evidence that Pb inhibited Ca uptake by rat brain motochondria and concluded that “sequestration of lead in brain mitochondria may lead to abnormal increases in the intracellular level of Ca in brain.”

The results of the present study have demonstrated that chronic exposure to Pb caused decreased electrical excitability, increased resting membrane potential, and decreased membrane time constant and afterhyperpolarization. While determined here for the membrane of the neuron cell body, it is possible that similar changes occur for the membrane of DRG neural processes. Assuming this is the case and substituting the anomalous values into an equation (7.2.10) provided by Kunov (19661, one finds that conduction velocity would be reduced by approximately 56% for unmyelinated nerves exposed chronically to 100 pM Pb. This calculated change when extrapolated to lower Pb concentrations agrees both in direction and approximate magnitude with that observed in humans chronically exposed to Pb (see Introduction).

The Pb effects observed here could be due to a direct effect of Pb on the cell membrane, e.g., by Pb competing with Ca at membrane channels. However, it is also possible that more indirect effects are responsible. The Pb concentrations used here cause a dramatic decrease in nonneuronal cell density (Scott and Lew 1985) and this could affect EMP, perhaps by altering the supply of trophic agents. Pb is a well known inhibitor of mitochondria1 respiration, as well; it is possible that the resulting decrease in oxidative metabolism could alter the EMP.

This study has clearly demonstrated the usefulness of neural cell cultures in the investigation of the effects of chronic Pb exposure on neuronal EMP. Combined with information gained in a previous study of Pb’s toxic effect on cell survival (Scott and Lew, 19851, the culture technique has provided a broad base of fundamental information about the cellular effects of Pb neurotoxicity. Previous studies have demonstrated the feasibility of con- ducting similar investigations using human nervous tissue (Scott et al., 1979, 1982). Investigation of Pb‘s effects on human neural cell cultures would provide information relevant to human Pb neurotoxicity. This knowl- edge should be valuable in achieving a better understanding of the cellular basis of the various clinical manifestations of Pb neurotoxicity, and in designing programs to minimize the effects of environmental Pb.

The authors wish to thank the Ontario Ministry of Labour and the Natural Sciences and Engineering Council of Canada for the financial assistance which made this study possible. They also thank Ms. Judy Jones for technical assistance and Mr. Patrick McQuarrie of the Great Lakes Institute for carrying out AAS Pb determinations.

ELECTROPHYSIOLOGY OF CHRONIC Pb 433

REFERENCES

BUCHTHAL, F., and BEHSE, F. (1979). Electrophysiology and nerve biopsy in men exposed to lead. Br. J . Znd. Med. 36 135-147.

CAMPBELL, A. M. C., WILLIAMS, E. R., and BARLTROP, D. (1970). Motor neuron disease and exposure to lead. J . Neurol. Neurosurg. Psychiat. 33: 877-885.

COOPER, G. P., and SIGWART, C. D. (1979). Neurophysiological effect of lead. In Lead toxicity, R. L. Singhai and J . A. Thomas, Eds. Urban and Schwartzenberg, Baltimore, MD.

GOLDSTEIN, G. W. (1977). Lead encephalopathy: The significance of lead inhibition of calcium uptake by brain mitochondria. Brain Res. 136: 185-188.

HASAN, J., and VIHKO, V. (1967). Deficient red cell membrane Na+ , K + - ATPase in lead poisoning. Arch. Enuiron. Health 14: 313-318.

HODGKIN, A. L., and HUXLEY, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J . Physiol. (Londi 117: 500-544.

JACK, J . J. B., NOBLE, D., and TSIEN, R. W. (1975). Electric current flow in excitable cells. Clarendon Press, Oxford, p. 37.

KOBER, T. E., and COOPER, G. P. (1976). Lead competitively inhibits calcium-dependent synaptic transmission in the bullfrog sympathetic ganglion. Nature 262: 704-705.

KUNOV, H. (1966). Nonlinear transmission lines simulating nerve axons, Ph. D. thesis, Technical University of Denmark KGS, Lyngbry, Denmark.

MARTY, A. (1983). Calcium'--dependent K' channels with large unitary conductance. Trends in Neurosci. 6: 262-265.

PETIT, T. L., and ALFANO, D. P. (1983). Neurobiological and behavioral effects of lead. In Neurobzology of the trace elements, I. E. Dreosti, R. M. Smith, Eds. Human Press, Clifton.

SCOTT, B. S. (1977a). The effect of elevated potassium on the time course of neuron survival in cultures of dissociated dorsal root ganglia. J . Cell Physiol. 91: 305-316.

SCOTT, B. S. (1977b). Adult mouse dorsal root ganglia neurons in cell culture. J . Neurobiol.

SCOTT, B. S., and EDWARDS, B. A. V. (1980). Electric membrane properties of adult mouse DRG neurons and the effect of culture duration. J . Neurobiol. 11: 291-301.

SCOTT, B. S., and EDWARDS, B. A. V. (1981). The effect of chronic ethanol exposure on the electric membrane properties of DRG neurons in cell culture. J . Neurobiol. 12: 379-390.

SCOTT, B. S., and LEW, J . (1985). Lead neurotoxicity: neuronal and non-neuronal cell survival in fetal and adult DRG cell cultures. J . Neurotox., accepted for publication.

SCOTT, B. S., PETIT, T. L., BECKER, L. E., and EDWARDS, B. A. V. (1979). Electric membrane properties of human DRG neurons in cell culture and the effect of potassium high medium. Brain Res. 178: 529-544.

SCOTT, B. S., PETIT, T. L., BECKER, L. E., and EDWARDS, B. A. V. (1981). Abnormal electric membrane properties of Down's syndrome DRG neurons in cell cultures. Deuelop. Bram Res. 2: 257-270.

SEPPALAINEN, A. M. (1982). Lead poisoning: neurophysiological aspects. Acta Neurol Scand

SILBERGELD, E. K., and ALDER, H. S. (1978). Subcellular mechanisms of lead neurotoxicity. Brain Res. 1 4 8 451-467.

SIMPSON, J. A,, SEATON, D. A,, and ADAMS, R. (1964). Response to treatment with chelating agents of anemia chronic encephalopathy, and myelopathy due to lead poisoning. J . Neurol. Neurosurg. Psychiat. 27: 536-541.

SINGER, R., VALCIUKAS, J. A,, and LILAS, R. (1983). Lead exposure and nerve conduction velocity: the differential time course of sensory and motor nerve effects. Neurotoxicology 4: 193-203.

SUKETA, Y., UJIIE, M., and OKADA, S . (1982). Alteration of sodium and potassium mobilization and of adrenal function by long term ingestion of lead. Biochem. Pharmacol. 31: 2913- 2919.

8: 417-427.

(Suppli 92: 177-184.