Triethyltin exposure suppresses synaptic transmission in area CA1 of the rat hippocampal slice

Neurotoxicology and Teratology, Vol. 10, pp. 539-548.©PergamonPress plc, 1989. Printedin the U.S.A.

0892-0362/88$3.00 + .00

Triethyltin Exposure Suppresses Synaptic Transmission in Area CA1 of the Rat Hippocampal Slice S T E P H E N B. F O U N T A I N , ~ Y E N - L I N G T. T I N G , S T E V E N K. H E N N E S A N D T I M O T H Y J. T E Y L E R

D e p a r t m e n t o f Neurobiology, Northeastern Ohio Universities College o f Medicine, Rootstown, O H 44272 R e c e i v e d 16 D e c e m b e r 1987 FOUNTAIN, S. B., Y.-L. T. TING, S. K. HENNES AND T. J. TEYLER. Triethyltin exposure suppresses synaptic transmission in area CAI of the rat hippocampal slice. NEUROTOXICOL TERATOL 10(6)539--548, 1988.--To examine the effects of TET on the electrophysiology of area CA1 of hippocampus, hippocampal slices were obtained from adult hooded rats and were maintained in vitro using standard techniques. Stimulating and recording electrodes were placed in the Schaffer collaterals and CA1 pyramidal cell body layer, respectively. Following baseline measurements, slices were exposed to either 0, 1, 3, 6, or 10 /xM TET in the incubating medium. Both pyramidal cell excitability and recurrent/feedforward inhibition were suppressed in a dose-dependent manner within 3 hr postexposure. The evoked population spike and population excitatory postsynaptic potential (EPSP) were suppressed significantly by 2 hr postexposure for 1 and 3 p.M TET exposures, and by 45 min postexposure for 6 and 10/xM exposures. A similar dose-dependency was observed for the suppression of recurrent/feedforward inhibition in hippocampal CAl. A second procedure tested the specificity of TET effects to axonal conduction of Schaffer collaterals. Both the stimulating and recording electrode were placed in the Schaffer collaterals so that both the Schaffer collateral population fiber volley and the CA1 pyramidal cell population EPSP could be recorded. TET exposure suppressed pyramidal cell EPSPs without significantly affecting the amplitude of Schaffer collateral fiber volleys. The results support the view that acute TET exposure suppresses synaptic transmission in area CA1 of hippocampus. Triethyltin Fiber volley

Hippocampal slice CAI pyramidal cell layer Synaptic transmission

Neuronal excitability

Recurrent inhibition

real electroshock seizure response (10, 11, 15, 16), and alterations in rats' response to schedules of reinforcement (25,31), among others. The present experiment was designed to investigate the effects of TET exposure on the electrophysiology of area CA1 of hippocampus. Although little direct evidence could be marshaled to predict that hippocampal electrophysiology would be affected by TET exposure, the relatively widespread distribution of TET effects on myelin and peripheral neurotransmission were suggestive. In addition, the putative relevance of hippocampus to a variety of behaviors-extending from seizure susceptibility to learning and memory processes---suggested that an exploration of TET effects on hippocampal electrophysiology could potentially provide new information relevant to understanding the behavioral effects of TET. Finally, the suggestion has been made that the in vitro hippocampal slice preparation, which preserves the major circuitry of the intact hippocampus, might profitably be used as a general assay for neurotoxicity (14,22). Such an assay would, of course, be required to detect the

T R I E T H Y L T I N (TET) is a neurotoxic organometal currently used as an industrial plasticizer and biocide. The most prominent and best-studied of the neurotoxic effects of TET is reversible cerebral edema attributed to transient swelling, vacuolization, and splitting of myelin (28, 36, 37). Biochemical and morphological studies have tended to support the view that the myelin sheath should be considered the primary target of TET neurotoxicity (19,24), though a somewhat different pattern of effects---including neuronal necrosis and derangement of hippocampal circuitry--results from neonatal exposure to TET (39). In adult rats, TET exposure also results in decreased noradrenaline, serotonin, and dopamine concentrations in several brain areas in addition to cerebral edema (4), though the relationship of these changes to cerebral edema is not clear. One neurophysiological result of TET exposure is that the latency of pattern reversal evoked potentials is delayed, a result also common in cases of multiple sclerosis (7). The behavioral effects of TET intoxication in rats include effects resulting from neuromuscular impairments (6,12), reduction in the maxi-

1This work was reported at the annual meeting of the Society for Neuroscience, New Orleans, LA, 1987. 2Requests for reprints should be addressed to Stephen B. Fountain, Department of Psychology, Kent State University, Kent, OH 44242.

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FOUNTAIN,

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neurotoxicity of agents such as TET. On these grounds the present experiment was undertaken. Hippocampal slices were obtained from adult hooded rats and were maintained in vitro using standard techniques (35). Tests of pyramidal cell excitability and recurrentlfeedforward inhibitory processes in area CA1 were conducted by stimulating the Schaffer collaterals of stratum radiatum while recording from the pyramidal cell body layer of area CAl. Tests were administered before and over the 3 hr following exposing the slices to either 0, 1, 3, 6, or 10 PM TET in the incubating medium. Similar methods have been used to assess the electrophysiological consequences of hippocampal slice exposure to trimethyltin (14) and aspartame (13). A second test assessed the effects of TET exposure on fiber volley conduction in the Schaffer collaterals of stratum radiatum, the primary afferent fibers for hippocampal CA1 pyramidal cells. This test assessed the effects of TET on both fiber volley amplitude and conduction velocity relative to changes in CA1 pyramidal cell excitability. We anticipated, based on TET’s myelinotoxic properties, that TET might indirectly reduce pyramidal cell excitability by disrupting fiber volley conduction in Schaffer collaterals through some mechanism related to its myelinotoxicity. However, we could not rule out the possibility that TET might have direct effects on other neurophysiological processes, for example, on neurotransmission (4,6). METHOD

Assessment

of Excitatory

and Inhibitory Systems

Using the method of Teyler (35) hippocampal slices -450 Frn thick were obtained from female Long-Evans derived hooded rats that were 60-90 days of age. Slices were obtained from the middle third of the hippocampal formation (with reference to the septotemporal extent of the structure). The hippocampal slices were then placed in a chamber (35) designed to provide an environment capable of sustaining the living tissue for 8-10 hours or longer. The slices were placed on nylon nets, which suspended the tissue at the interface of a pool of artificial cerebrospinal fluid incubating medium and an atmosphere of adequate oxygen content (95% O,/% CO,). The medium consisted of a Ringer solution of 124 mM NaCl, 3.3 mM KCl, 1.25 mM NaH2P04, 1.2 mM MgS04, 2.4 mM CaCl,, 25 mM NaHCO,, and 10 mM glucose. Slices were allowed a 1-hr equilibration period before stimulation or recording was attempted. A concentric microbipolar stimulating electrode (9) (approximately 100 pm in diameter) was positioned in the Schaffer collaterals of stratum radiatum. A glass micropipette (5-20 pm tip diameter) filled with 2 M NaCl(2-4 Ma resistance) was used to record extracellularly from the CA1 pyramidal cell body layer of the hippocampus (see Fig. 1 for electrode placements). Stimulus pulses used to produce evoked potentials in CA1 ranged from O-10 V with a duration of 0.1 msec. Monosynaptically driven field potentials were amplified, filtered (1 Hz-3 kHz), and displayed on a storage oscilloscope. Waveforms were digitized (5 kHz sampling rate), recorded permanently on 5.25-m floppy disks, then later analyzed using an Apple II microcomputer equipped with an analog to digital converter (A/D+D/A Board, Mountain Computer Inc., Scotts Valley, CA) and a software system developed in-house (LabMan). Prior to exposing the slice to TET, a 15-min preexposure baseline was obtained by recording evoked responses at I-min intervals using a stimulus intensity sufficient to produce a I-mV population spike (designated the “standard

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FIG. 1. Approximate locations of stimulating electrodes in the Schaffer collaterals (SCH) of stratum radiatum, and extracellular recording electrodes in the CA1 pyramidal cell body field (for tests of excitatory and inhibitory systems) and in the Schaffer collaterals (for fiber volley tests) of the hippocampal slice. Only one recording electrode was introduced into each slice.

stimulus”). Stability of the evoked response was deemed acceptable if the amplitude of the population spike varied less than 1% over 15 min; slices that failed to meet this criterion were rejected from the experiment. Following the 15-min baseline, the status of local inhibitory systems was assessed by a paired-pulse “conditioning/test” (C/T) procedure using the standard stimulus and an interpulse delay of 20 msec. Slices that failed to demonstrate at least 7% inhibition of the population spike evoked by the test stimulus, i.e., the second stimulus of the pair, were likewise rejected from the experiment. If the foregoing baseline criteria were met, the experiment proper was begun. Prior to TET exposure, an input/output (I/O) profile was obtained; the I/O profile consisted of CA1 responses recorded for increasing stimulus intensities in the range producing O-2 mV population field potentials. Samples for the I/O profile were obtained at 30-set intervals. After the I/O profile, the stimulus intensity eliciting a 1-mV population spike was used in a C/T test as before to examine the status of inhibitory systems. Next, 3-5 baseline evoked waveforms, obtained at I-min intervals, were recorded prior to TET exposure using the standard stimulus. TET exposure was then accomplished using a push-pull pump that simultaneously emptied the medium pool and replenished it with TETbearing medium. The push-pull pump was composed of two identical glass syringes mounted with their plungers held end-to-end and their barrels fmed some distance apart (35). Following medium exchange, final TET concentrations in the medium pool were either 0, 1, 3, 6, or 10 PM TET-Br (Alpha Products, Danvers, MA). Changes in exictability due to TET exposure were monitored by recording field potentials elicited by the standard stimulus at 5-min intervals following the onset of exposure. Monitoring was interrupted at 60, 120, and 180 minutes postexposure (i.e., postexchange) to obtain an I/O profile and to administer a C/T test. For the I/O profile, stimulus intensities were increased where necessary until a I-mV population spike was obtained. However, stimulus intensities were never increased beyond twice that of the standard stimulus to prevent stimulus-induced potentiation of the pyramidal cell population response. The C/T test was administered using a stimulus sufficient to produce a

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TIME (min) FIG. 2. Timecourse of changes in mean population EPSP slope (as percent of baseline) for 180 rain following either 0, 1, 3, 6, or 10/zM TET exposure. Population EPSPs were recorded extracellularly in the hippocampal CA1 pyramidal cell body layer. TET 0/.tM (--), TET 1/xM (---), TET 3/.~M ('--.), TET 6/zM (- - - ), TET 10/zM (. . . . ).

l-mV population spike in the I/O profile. If such a stimulus was not observed during the I/O profile due to TET exposure, the C/T test was not administered. Following the I/O profile and C/T test, monitoring using the standard stimulus was resumed so as to correspond to 5-min intervals postexposure. Monitoring continued for 3 hr postexposure. No washout procedure was employed; that is, slices were exposed to the appropriate dose of TET throughout the 3-hr monitoring period. Each rat contributed only 1 hippocampal slice to the study. Each slice was exposed to only 1 dose of TET, either 0, 1, 3, 6, or 10 /zM TET, and 4 slices meeting baseline criteria were studied at each TET dose. Dosing was randomized and the experimenter was blind with regard to the dose being studied until the procedure was completed. Population EPSP slope data were obtained using the standard technique of calculating the slope of the initial positive-going portion of the population EPSP waveform in the most linear area encompassing the inflection point of the curve. This widely-accepted measure is highly correlated with population EPSP amplitude, and has the additional advantage that it remains an accurate measure of population EPSP amplitude when, at higher stimulus intensities, later portions of the population EPSP waveform are partially obscured by the population spike. Population spike amplitude data were obtained by averaging the highest positivegoing peaks of the EPSP immediately preceding and following the negative-going population spike, then finding the difference between this value and the lowest point in the population spike waveform. Analysis of variance (ANOVA) was employed for statistical evaluation of these data. Separate univariate A N O V A s with repeated measures (BMDP Statistical Software, Inc.,

1987) were performed for the population EPSP data and for the population spike data, respectively, collected at 5-rain intervals over the 3-hr monitoring period following exposure onset. Thus the effect of TET dose (the single factor) was evaluated across time following exposure. Dunnett's t statistics were used for comparing treatment means to control means. O n l y p values less than 0.05 were considered statistically significant.

Fiber Volley Assessment A second test was used to determine the effects of acute TET exposure on fiber volley conduction in the Schaffer collaterals of stratum radiatum. Slices were prepared as described above with the exception that both stimulating and recording electrodes were positioned in the Schaffer collaterals of stratum radiatum, as shown in Fig. 1. Recording electrodes were positioned so that evoked population fiber volleys of the Schaffer collaterals could be distinguished from evoked population excitatory postsynaptic potentials (EPSPs) recorded in the CA1 synaptic field of stratum radiatum. A standard stimulus was chosen that produced a population fiber volley amplitude somewhat less than the asymptotic amplitude. Once the population fiber volley and EPSP were found to be stable, the slice was exposed to 10 /zM TET using the push-pull pump previously described. TET effects on fiber volleys and EPSPs were monitored by recording waveforms evoked by the standard stimulus every 5 rain. When the evoked population EPSP had declined to asymptotic levels, an I/O profile was obtained. In obtaining this profde, stimulus intensity was increased in an effort to recover the preexposure baseline population EPSP.

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RESULTS TET Effects on Excitatory Systems A comparison of pre- and postexposure I/O and monitoring data indicates that TET suppressed excitatory systems in hippocampal CA1 in a time- and dose-dependent manner. Figures 2 and 3 depict the timecourse of TET-induced suppression of CA1 pyramidal cell population EPSP and population spike, respectively. Figure 2 shows the amplitude of the population EPSP, depicted as percent of baseline population EPSP slope, evoked by a constant stimulus (viz., the standard stimulus) over the 3-hr monitoring period commencing with the onset of TET exposure. The ANOVA performed on these data indicated a significant main effect for dose, F(4,15)=7.61, p =0.0018, and time postexposure, F(35,525)=31.89, p<0.001, and a significant dose x time interaction, F(140,525)=4.42, p<0.001. TET significantly suppressed the evoked population EPSP by 45 min following 10/~M TET exposure, by 35 min postexposure for the 6/~M TET exposure, by 65 min for the 3 /~M TET exposure, and by 75 min following 1/~M TET exposure relative to controls (ps<0.05). Beginning at 165 min postexposure, all TET group population EPSP slopes differed significantly both from each other and from baseline (,o<0.05). Figure 3 shows the timecourse of changes in the amplitude of the population spike (depicted as percent of baseline) evoked by the standard stimulus over the 3-hr monitoring period commencing with the onset of TET exposure. The ANOVA performed on these data indicated a significant main effect for dose, F(4,15)=6.22, p=0.004, and time postexposure, F(35,525)=40.23, p<0.001, and a significant dose × time interaction, F(140, 525)=3.41, p<0.001. TET significantly suppressed the evoked population spike by 15 and 40 min following 10 and 6/~M TET exposures, re-

spectively, relative to controls (ps<0.05), though differences between these two groups disappeared by 30 min postexposure (o>0.05). TET also significantly suppressed the evoked population spike by 120 and 125 min following 3 and 1/~M TET exposures, respectively, relative to controls (ps<0.05); differences between these two dose conditions were never significant (p >0.05). The mean population spike amplitudes of the I and 3 /~M TET conditions were significantly decreased relative to those of controls beginning 125 min postexposure, and those of the 6 and 10/~M.TET conditions were decreased relative to those of all other conditions by 30 min postexposure (ps<0.05). In addition, 3 ~M TET exposure significantly increased population spike amplitude for waveforms sampled at 5-25 min postexposure (p<0.05). A similar, but nonsignificant, effect was observed for the 1 /~M TET dose (p >0.05). Figure 4 shows representative I/O profiles that depict the relationship between stimulus intensity and either population EPSP slope (left panels) or population spike amplitude (right panels). Best-fit lines were derived from linear regression analysis. The I/O profiles obtained preexposure (PRE) and 1, 2, and 3 hr postexposure show dose-dependent suppression of the population EPSP and population spike over time. Whereas population EPSP I/O profiles remained stable over time for control slices, population EPSP I/O profiles shifted to the right as a function of TET dose. A shift to the right by the I/O profile reflects the fact that higher stimulus intensities were required to produce an equivalent evoked response, that is, suppression of excitability. Population EPSP I/O functions shifted to the right by 3 hr following 1 and 3/xM TET exposures and by 1 hr following 6 and 10 /~M TET exposures (top to bottom panels, respectively) as expected from the foregoing analysis of the timecourse of TET sup-

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Recurrent/feedforward inhibitory systems were also suppressed by TET exposure. Figure 5 shows the results of the C/T tests of inhibition conducted preexposure (PRE) and 1, 2, and 3 hr following TET exposure. When the Schaffer collaterals were stimulated twice with a 20-msec delay be-

tween stimulus pulses, the population spike produced by the test pulse (i.e., the second pulse) was suppressed by recurrent/feedforward inhibition both before and for 3 hr after control exposure. This result is reflected in the fact that the group mean amplitude of the suppressed population spike evoked by the test pulse, depicted in Fig. 5, did not increase over time following control exposure. In contrast, both 6 and 10 /xM TET exposure suppressed inhibition by 1 hr postexposure, i.e., these doses produced an increase in the amplitude of the population spike elicited by the test pulse by 1-hr postexposure. Similarly, TET exposure produced suppression of inhibition by 2 hr following 3/zM TET exposure• A trend toward this dose-dependent decrease in inhibition can be seen as early as 1 hr following TET exposure. It should be noted that all tests of inhibition were conducted

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tion of Schaffer collaterals produced both a negative-going population fiber volley waveform followed by a large negative-going population EPSP emanating from CA1 pyramidal cells. Waveforms obtained at 30-min intervals following 10 /zM TET exposure show progressively smaller population EPSP waveforms, but no change in population fiber volley amplitude. In addition, TET did not appear to affect fiber volley conduction velocity as measured from the onset of the stimulus artifact to the peak of the population fiber volley waveform; no change in this latency was observed even when the pyramidal cell population EPSP was completely suppressed. Figure 7 shows I/O functions for population fiber volley amplitude as a function of stimulus intensity (left panel) and for pyramidal cell population EPSP slope as a function of stimulus intensity (right panel) obtained at 90 min following TET exposure for the representative slice whose results are depicted in Fig. 6. Also shown for comparison are preexposure (PRE) baseline results obtained at the monitoring stimulus intensity. (Although some population EPSP waveforms were truncated due to the high gain required to record population fiber volleys, accurate measures of population EPSP amplitude were obtained by recording population EPSP slope using the same methods described above for assessing the effects of TET on pyramidal cell excitability.) The results indicate that TET did not suppress fiber volleys, and when stimulus intensity was increased postexposure, correspondingly larger fiber volleys were evoked. In contrast, TET suppressed pyramidal cell EPSPs postexposure, and the preexposure population EPSP waveform could not be recovered by increasing the stimulus intensity beyond that used for monitoring. Results comparable to those shown in Fig. 7 were obtained in each of 4 hippocampal slices tested.

DISCUSSION

In contrast to the results of numerous earlier studies suggesting that the primary effect of TET is myelinotoxicity (19, 23, 24), our results show that TET also produces relatively rapid changes in excitatory and inhibitory systems in area CA1 of the hippocampal slice. In the present study, the primary effect of TET was to suppress excitability in CA1 pyramidal cells evoked by Schaffer collateral stimulation. At lower doses TET produced a transient increase in excitability prior to suppression. However, TET produced no detectable effect on evoked fiber volleys in the fibers afferent to CA 1 pyramidal cells, namely, the Schaffer collateral fibers of stratum radiatum. Fiber volleys were not affected despite concurrent suppression of postsynaptic EPSPs of CA1 pyramidal cells. In general, these results support the view that TET produced suppression of synaptic transmission; the fact that the Schaffer collateral fiber volley was not affected by TET and the fact that the population EPSP was suppressed by TET together suggest that suppression of excitability was likely due to suppression of synaptic mechanisms, not simply due to generalized deleterious effects on neuronal viability. The results indicate that suppression was especially profound at doses greater than 3 /zM TET. Previous work has shown that TET-induced vacuolization of myelin in vivo does not begin to become apparent until approximately 3-7 hr postexposure (20,23), and that decreases in motor nerve conduction velocity require at least 5 days to develop (18). In comparison to the myelinotoxic effects of TET observed in vivo, which develop over a period of hours to days, the synaptic effects we observed in vitro developed relatively rapidly, within 15 min postexposure for the 10 /xM TET dose, without accompanying changes in fiber volley conduction. The mechanism of action responsible for TET's

TET N E U R O T O X I C I T Y IN H I P P O C A M P A L CA1

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suppression o f synaptic transmission remains to be determined. Future studies employing washout techniques and antidromic firing of hippocampal CA1 pyramidal cells should provide further information regarding the presynaptic versus postsynaptic effects of TET. TET produced no detectable changes in Schaffer collateral fiber volley amplitude or conduction velocity in the fiber volley assessment study. One potential explanation of this result suggested above is that the timecourse of myelinotoxic effects is significantly different from the timecourse of synaptic effects. This hypothesis supposes that had it been possible to monitor TET effects on Schaffer collateral fiber volleys for periods up to several days, changes in fiber volley conduction would have been observed. A second hypothesis is that the classical myelinotoxic effects of TET might not be observed in Schaffer collateral fibers using any methodology. This hypothesis is based on the fact that the majority of Schaffer collateral fibers are unmyelinated or only weakly myelinated (2). Results showing TET-induced behavioral decrements at doses and timepoints where no myelin swelling is observed also appear to be relevant to the issue of distinguishing the synaptic versus myelinotoxic effects of TET (15, 33, 38). For example, Wenger et al. (38) demonstrated that the maximum behavioral effect of TET on the response of mice to a multiple schedule of reward occurred approximately 3 hr following 5-10 mg/kg TET exposures, but no neuropathology was observed at the light microscopic level at that time. These results are consistent with the view that there are dissociable synaptic and myelinotoxic effects of TET. Another important result of the present study was that TET suppressed local inhibitory systems that normally modulate pyramidal cell excitability in area CA1 of hippocampus. Because we equated the pyramidal cell evoked response across C/T tests, the suppression of inhibitory systems we observed can be said to be independent of the suppression of neurotransmission observed at the synapses of Schaffer collateral fibers with CA I pyramidal cell dendrites. Inhibition in hippocampal CAI is produced by local inhibitory basket cells (recurrent inhibition) and feedforward processes (1,8). Further work is required to determine whether TET suppresses both inhibitory systems or whether it acts more selectively on only one of these systems. The exact concentration of TET in brain tissue required to produce neuropathology and behavioral effects in vivo is not known. The TET concentrations used in the present in vitro experiment were chosen based on estimates of TET concentration in brain for behaviorally relevant doses assuming uniform distribution of TET among body f l u i d compartments. The dose range of 1 to 10/zM TET used in the present experiment corresponds to dosed 1-10 times greater than the 1/zM dose of trimethyltin we used in an earlier in vitro study (14). This dose range encompasses the lowest

concentration of TET required to suppress in vitro taurine transport in glioma cells [i.e., 2.5/xM TET (29)], but is lower than the minimum concentration of TET required to suppress in vitro glutamate or lysine transport in glioma ceils [i.e., 135 and 110/xM TET, respectively (29)]. A variety of hypotheses can be advanced to account for TET-induced suppression of hippocampal CA1 excitatory and inhibitory systems. The mechanism o f action might involve TET-induced depletion of excitatory and inhibitory neurotransmitters. Previous work has demonstrated that a single exposure to 5 mg/kg TET depletes whole brain norepinephrine by 21 days postexposure (30). Similar work has shown that TET depletes brain 5-hydroxytryptamine (4, 10, 34) and dopamine levels (4). Behavioral work indicates that TET interacts with adrenergic and GABAergic systems to produce anticonvulsant effects (15). Thus, suppression could be the result of TET-induced neurotransmitter depletion in hippocampal area CA1. Our results suggest that the glutamatergic and GABAergic neurotransmitter s y s t e m s - the primary excitatory and inhibitory neurotransmitter systems of hippocampal C A l - - a r e affected by TET. An alternative hypothesis is that suppression of excitatory and inhibitory systems in hippocampal area CA1 resulted from TET-induced suppression of taurine uptake systems (29). Taurine has widespread depressant effects in the CNS (17,21) and, like TET, has anticonvulsant effects (3,5). Other candidate mechanisms of TET suppression might involve TET effects on brain electrolytes (32) or cAMPphosphodiesterase activity (27). As yet, no study has directly tested any of these hypotheses. In conclusion, our results are consistent with the notion that TET has widespread effects on neurotransmission in the CNS (4, 26, 30). The novel finding from our work is that at least some of the central effects of TET are direct effects on neurotransmission; suppression of neurotransmission in area CA1 of the hippocampal slice was not simply a secondary consequence of myelinotoxic effects. The relationships between TET-induced suppression of neurotransmitter systems, myelin edema, and behavioral changes remain unclear and deserve further scrutiny. ACKNOWLEDGEMENTS This work was supported by the United States Environmental Protection Agency. Although the research described in this article has been funded wholly or in part by the Health Effects Research Laboratory, U.S. Environmental Protection Agency through cooperative agreement No. 813394 to Northeastern Ohio Universities College of Medicine, it has not been subjected to the Agency's peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Additional support was provided by NIH grant DA03755 and ONR grant 86K0664. We thank Jean Zuga for assistance in data collection, Nick LeCursi for technical assistance, and David Jarjoura and Gary McCord for assistance in data analysis.

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