Toxicological Sciences 66, 139-147 (2002)
Copyright © 2002 by the Society of Toxicology
NEUROTOXICOLOGY |
Rat Hippocampal Glutamate and GABA Release Exhibit Biphasic Effects as a Function of Chronic Lead Exposure Level
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* Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, P. O. Box 1649, Peoria, Illinois 61656;
Neurotoxicology Division, U. S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and
Department of Psychology, University of North Carolina, Chapel Hill, North Carolina 27599
Received August 2, 2001; accepted November 16, 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous work has suggested that the lead (Pb) exposure-induced decrease in K+-evoked hippocampal glutamate (GLU) release is an important factor in the elevated threshold and diminished magnitude reported for hippocampal long-term potentiation (LTP) in exposed animals. In addition, complex dose-effect relationships between Pb exposure level and LTP have been reported. This investigation was conducted to determine the effects of Pb on hippocampal GLU and GABA release as a function of exposure level. Rats were continuously exposed to 0.1, 0.2, 0.5, or 1.0% Pb in the drinking water beginning at gestational day 15–16. Hippocampal transmitter release was induced in adult males by perfusion of 150 mM K+ in the presence of Ca+2 (total release) through a microdialysis probe in one test session, followed by perfusion through a contralateral probe in the absence of Ca+2 (Ca+2-independent release) in the second session. Chronic exposure produced decreases in total K+-stimulated hippocampal GLU and GABA release at exposure levels of 0.1–0.5% Pb. Maximal effects were seen in the 0.2% group (blood Pb = 40 µg/100 ml), and changes in total release could be directly traced to alterations in the Ca+2-dependent component. However, these effects were less evident in the 0.5% group and were no longer present in the 1.0% Pb group, thus defining U-shaped dose-effect relationships. Moreover, in the absence of Ca+2 in the dialysis perfusate, K+-induced release was elevated in the 2 highest exposure groups, suggesting a Pb+2-induced enhancement in evoked release. This pattern of results indicates the presence of 2 actions of Pb on in vivotransmitter release: a more potent suppression of stimulated release seen at lower exposure levels (27–62 µg/100 ml) combined with Ca+2-mimetic actions to independently induce exocytosis that is exhibited at higher exposure levels (
62 µg/100 ml). Furthermore, significant similarities in the dose-effect relationships uncovered in measures of evoked GLU release and hippocampal LTP (M. E. Gilbertet al., 1999b,
Neurotoxicology20, 71–82) reinforce the conclusion that exposure-related changes in GLU release play a significant role in the Pb-induced effects seen in this model of synaptic plasticity. Key Words: lead; glutamate; GABA; hippocampus; biphasic; long-term potentiation; microdialysis; calcium.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite a long history of scientific investigation lead (Pb) neurotoxicity remains a significant public health concern. The assertion that young children are the population at greatest risk has been reinforced by epidemiological investigations that have repeatedly associated developmental Pb exposure with impairments in cognitive function (e.g., Bellinger et al., 1991
; Needleman and Gatsonis, 1990). Animal studies have also indicated that exposure to Pb beginning in early development and resulting in environmentally relevant blood levels produces learning deficits (e.g., Cohn et al., 1993; Rice, 1993). As a result, these behavioral effects of exposure have prompted experimental studies to determine the mechanisms underlying the actions of Pb on cognitive function.
One prominent approach has involved the use of the widely accepted cellular model of learning and memory, long-term potentiation (LTP; see reviews by Barnes, 1995; Bliss and Collingridge, 1993). Several lines of evidence support the belief that the processes of LTP provide a neurophysiological substrate for learning and information storage. In studies focused on the effects on LTP in hippocampal CA1 and dentate gyrus there has been widespread agreement that chronic Pb exposure diminishes potentiation (Lasley and Gilbert, 2000). Greater insight into the neurobiological bases of LTP and recent findings on the cellular actions of Pb have indicated target sites where exposure may act to interfere with the processes underlying this plasticity. These actions include documented effects of Pb on neurotransmitter release in vivo (Lasley and Gilbert, 1996; Lasley et al., 1999), on N-methyl-D-aspartate (NMDA) receptor function (Alkondon et al., 1990; Cory-Slechta et al., 1997; Nihei et al., 2000), and on protein kinase C (PKC) activity (Long et al., 1994; Tomsig and Suszkiw,1995), a pathway involved in transmitter release and neuroplasticity (Bartschat and Rhodes, 1995; Colley and Routtenberg, 1993; Terrian, 1995).
Measures of presynaptic function at glutamatergic synapses in chronically exposed animals have produced results that can be related to the effects of Pb on LTP. Perforant path stimulation to induce paired-pulse facilitation in dentate gyrus, a measure that is primarily mediated by enhanced GLU release, was reduced in animals exhibiting blood Pb values of 30–40 µg/100 ml (Lasley and Gilbert, 1996; Ruan et al., 1998). It has also been shown that exposure at these blood Pb levels results in diminished depolarization-induced hippocampal GLU release as quantified by microdialysis in awake animals (Lasley and Gilbert, 1996; Lasley et al., 1999). Focal perfusion of high K+ was utilized to measure GLU release and define the Ca+2-dependent and Ca+2-independent components by inclusion or removal of Ca+2 from the perfusion fluid. While chronic exposure to 0.2% Pb diminished the K+-stimulated increase in total (Ca+2-dependent + Ca+2-independent components) extracellular GLU compared to that in control animals, no group differences were observed under Ca+2-free conditions. This observation indicated that the exposure-induced decrease in total GLU release was due to Pb-related decrements in the Ca+2-dependent component, and suggested that this component was most sensitive to exposure effects on Ca+2-dependent synaptic processes.
Earlier studies have also suggested that the exposure-induced decrease in K+-evoked hippocampal GLU release is an important factor in the elevated threshold and diminished magnitude of hippocampal LTP in animals exposed to 0.2% Pb (Gilbert et al., 1996, 1999a; Lasley and Gilbert, 1996). This notion is strengthened by the results of parallel studies on the effects of the same exposure level on hippocampal LTP and GLU release as a function of developmental period (Gilbert et al., 1999a; Lasley et al., 1999). More recently, Gilbert et al., (1999b) examined the effects on LTP as a function of chronic exposure level and observed a U-shaped dose-effect relationship. Induction thresholds were elevated and LTP magnitudes were diminished at intermediate exposure levels (
25–60 µg/100 ml). However, no effect was seen at the highest exposure (118 µg/100 ml), suggesting that Pb was affecting multiple synaptic processes. The following investigation was conducted to determine the effects of Pb on hippocampal GLU and GABA release as a function of exposure level. This design would further elucidate the mechanisms by which Pb alters neurotransmitter release in vivo, and indicate the importance of stimulated GLU release in the biphasic dose-effect relationship observed with Pb and LTP. In this work a U-shaped dose-effect function was observed with significant reductions in K+-stimulated hippocampal GLU and GABA release, but as seen with hippocampal LTP, a reversal of the Pb-induced decrement was also uncovered at the higher exposure levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Pregnant Long-Evans hooded rats (Charles River, Wilmington, MA) were obtained at gestational day 15–16 and placed on NIH-07 laboratory chow (Harlan Teklad, Madison, WI). At parturition independent groups of dams were placed on 0.1% (545 ppm, representing 7 litters), 0.2% (1090 ppm, 8 litters), 0.5% (2725 ppm, 6 litters), and 1.0% (5450 ppm, 6 litters) Pb acetate in the drinking water, and litters culled to 8 pups retaining the maximum number of males. All drinking solutions were adjusted to a pH
6.0 with acetic acid; as a result precipitation of Pb salts was not observed. Exposure was continued throughout lactation, thus exposing the offspring through the maternal milk. Control dams were maintained on distilled drinking water (15 litters) and NIH-07 chow. At day 21 male offspring were weaned to the same drinking water as that given their dam, and were maintained on these regimens with free access to food and water until testing as adults. One to two males were utilized from each litter. Lights in the animal room were set on a 12:12 cycle with temperature maintained at 24 ± 1°C. In order to characterize the exposure protocols blood and brain Pb concentrations were determined in separate groups of identically exposed male animals at 120 days of age (n = 5–6 for each value). Blood samples (1000 µl) were collected in heparinized syringes via cardiac puncture of rats anesthetized with pentobarbital. Blood Pb analyses were performed by anodic stripping voltammetry. Whole brains were removed following blood collection and were analyzed for Pb content by graphite furnace atomic absorption spectrometry by ESA Labs (Chelmsford, MA).
Intracerebral dialysis.
Rats were anesthetized with sodium pentobarbital (60 mg/kg, ip) supplemented with 2–3% halothane in O2, mounted in a stereotaxic frame, and plastic guide cannulae implanted bilaterally into the dorsal hippocampus (3.90 mm posterior to bregma, 1.90 mm lateral to the midline, and 2.60 mm ventral to the skull surface; Paxinos and Watson, 1998). Cannulae were secured with dental acrylic and machine screws.
A CMA/11 dialysis probe (CMA Microdialysis, Acton, MA) with a 2 mm active length (concentric tube design, OD = 0.24 mm, molecular weight cutoff = 20,000 Da) was inserted into the cannula 2–5 days later, and the awake animal immediately placed into a plexiglas chamber that allowed freedom of movement. Experimental sessions were conducted within 5 days of surgery to minimize the influence of gliosis on extracellular concentrations (Gerin and Privat, 1996; Whittle et al., 1998), and test sessions varied randomly across this interval for all experimental groups. This probe location resulted in an area of dialyzed tissue that essentially comprised the dorsal hippocampal CA1 and dentate regions. The probe inlet was connected by FEP tubing to a syringe pump through a liquid switch and dual channel quartz-lined liquid swivel (Instech Labs, Plymouth Meeting, PA). The probe outlet was connected to the swivel by the same tubing and to a collection vial by fused silica tubing (75 µm ID). Before probe insertion for each testing session (no more than 3 test sessions/probe) the dialysis system was perfused with methanol:water (50:50, v/v) or Kathon CG microbiocide (Rohm & Haas) followed by 0.4 mM EDTA and an Ultrex II water (J. T. Baker, Phillipsburg, NJ) flush. All perfusion solutions for dialysis were prepared with the Ultrex II water and chloride salts of the highest quality commercially available.
A modified Ringer's solution (in mM: Na+ 145, K+ 4.0, Ca+2 1.3, Cl- 152) was perfused through the probes at 2.0 µl/min and collected in tubes containing 5 µl of 0.5 M HCl and stored at –75°C until derivatization. Two to three h after probe insertion baseline extracellular fluid concentrations of GLU and GABA were assessed by four 30-min collections prior to switching for 40 min to modified Ringer's with 150 mM K+ (K+ replaced Na+ to maintain isotonicity) containing 400 µM threo-3-hydroxyaspartate (to inhibit glutamate reuptake). Under these flow conditions extraction efficiency of the perfusate from the dialysis probe is approximately 10%, indicating that the maximal extracellular fluid K+ concentration produced in vivo is in the range of 15 mM. The need to employ a reuptake inhibitor in the perfusion medium in order to elicit a quantifiable effect of K+ stimulation on extracellular glutamate has been cited by others (Herrera-Marschitz et al., 1992). Furthermore, this manipulation eliminated the possibility of an exposure effect on release being masked by Pb-induced changes in neuronal or glial reuptake (Herrera-Marschitz et al., 1996). During the period of elevated extracellular fluid transmitter concentrations (80 min) 20-min sample collections were made followed by two 30-min collections to redefine the baseline.
Experimental design.
The hippocampus in one hemisphere was perfused with Ca+2-containing solution on the first test day, while the contralateral hippocampus was perfused the following day with nominally Ca+2-free modified Ringer's (Mg+2 replacing Ca+2) containing 1 mM methoxyverapamil (MVP) to block voltage-sensitive Ca+2 channels (Schneggenburger et al., 1993). When utilized, Ca+2-free solutions were perfused throughout the testing session—including the period of high K+ stimulation with the glutamate reuptake inhibitor—to determine Ca+2-independent release. This design permitted the operational definition of Ca+2-dependent GLU or GABA release as the difference between total release (occurring in the presence of Ca+2) and Ca+2-independent release. The latter release component can be quantified only by isolation as it is also present during Ca+2 perfusion.
Previous work has shown that correction of sample analyte concentrations by recovery measures determined in vitro produces greater variability about the mean than that about uncorrected values (Glick et al., 1994). Thus, analyte levels determined in this study were not corrected for recovery. Probe placement was verified by perfusion of the anesthetized animal with 10% formalin, sectioning of the tissue, and staining of the sections with neutral red. Placements generating experimental data varied < ±1 mm from the identified coordinates.
Amino acid analysis.
Amino acids were quantified as previously described (Lasley, 1991). Methionine sulfone in 0.1 M HCl was added as an internal standard to each fraction and the mixture dried under vacuum prior to derivatization with phenylisothiocyanate and a final drying step. Derivatives were redissolved in the initial mobile phase, and analyzed by binary gradient liquid chromatography with detection by ultraviolet absorbance at 254 nm (Waters Chromatography Div., Milford, MA). Flow rate was 1.0 ml/min. GLU and GABA were quantitated by integration of peak areas with comparison to standards of known concentrations included in each set of samples. Serine was also determined as a control nontransmitter amino acid (Wolf and Xue, 1998). Methionine sulfone and amino acid standards were obtained from Sigma Chemical Co. (St. Louis, MO). Mobile phases for the gradient chromatography were obtained directly from Waters Chromatography Division.
Data analysis.
Data were analyzed by a two-factor analysis of variance for each amino acid in the presence or absence of Ca+2 with exposure level and time after K+ as the main factors with a repeated measures analysis employed for the time factor. The bases of significant main effects for total or Ca+2-independent release were determined by Newman-Keuls post hoc tests comparing individual group means between an exposed and control group. The mean Ca+2-dependent release at each time point was determined as the difference between the total and Ca+2-independent means with the standard deviation based on the summed variance of the total and Ca+2-independent values. Means for an exposed and control group were then compared by Newman-Keuls tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Blood and Brain Pb Concentrations
Blood and brain Pb values from independent groups of identically exposed animals at 120 days of age are shown in Table 1
(n = 5–6 for each value) and are taken from a recent report (Gilbert et al., 1999b). Previous work (Lasley et al., 1999) has indicated that these concentrations achieve steady-state levels by 90–100 days of age. Blood and brain measures clearly increase across groups in a dose-dependent fashion. Moreover, values in the 0.2% Pb group agree well with other reported measures in animals chronically exposed at this level from birth (e.g., Lasley and Gilbert, 1996; Lasley et al., 1999).
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Glutamate Release
With Ca+2 in the perfusion medium, baseline (i.e., prestimulation) extracellular fluid GLU concentrations were not different from control values in any of the exposure groups (Table 2
). However, GLU release evoked by high K+ stimulation in the presence of Ca+2 (a measure of total release) was reduced by low levels of Pb (exposure level x time after K+ interaction F(40,580) = 6.83, p < 0.001). The time courses of the GLU release responses in the control and 0.2% Pb groups are shown in the top panel of Figure 1. The response of this exposure group is displayed as it constitutes the maximal reduction in release and can be directly compared to previously published findings (Lasley and Gilbert, 1996; Lasley et al., 1999). Those studies have also demonstrated that during high K+ stimulation the first 20-min collection period is most sensitive to the effects of chronic Pb exposure. GLU responses during this stimulation interval are thus presented across exposure levels in the bottom panel of Figure 1, which displays a significant reduction in GLU release in the 0.1 and 0.2% groups (Newman-Keuls q = 4.23, p < 0.01, and 9.67, p < 0.001, respectively). In contrast chronic exposure produced no changes in the 0.5 and 1.0% Pb groups. Accordingly the responses of both these latter groups were significantly greater than that in the 0.2% Pb group (Newman-Keuls q = 10.46 and 8.86, respectively, with both p < 0.001), thus establishing a U-shaped dose-effect relationship.
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In the absence of Ca+2 in the perfusion medium, baseline extracellular GLU concentrations were comparable to control levels in all but the 1.0% exposure group, where a significant increase was observed (Table 2
). GLU release evoked by high K+ in the absence of Ca+2 (a measure of Ca+2-independent release) was enhanced at higher exposure levels (exposure level x time after K+ interaction F(40,470) = 8.47, p < 0.001). The top panel of Figure 2 shows the time courses of evoked GLU responses observed under Ca+2-free perfusion conditions in the control and 0.2% groups. The bottom panel of Figure 2 displays stimulated Ca+2-independent release across exposure conditions during the first 20-min stimulation interval where increases are evident at the 0.5 and 1.0% exposure levels (Newman-Keuls q = 6.23, p < 0.001, and 9.42, p < 0.001, respectively). An analogous effect of Pb in the 0.2% group exhibited a trend, but did not attain statistical significance (p = 0.06). For purposes of overall comparison, the elevated baseline has not been removed from the magnitude of the response in the 1.0% group in Figure 2.
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Subtracting the corresponding values for total and Ca+2-independent release provides a more specific index of depolarization-induced GLU release presumably of exocytotic origin. Ca+2-dependent GLU release across time for the control and 0.2% groups is shown in the top panel of Figure 3
. This release component was significantly decreased in the 0.2% Pb group compared to controls after both 20 and 40 min of high K+ stimulation (q = 8.44, p < 0.001, and q = 4.24, p < 0.05, respectively). Data for exposure groups at the 20-min interval are presented in the bottom panel of Figure 3. Furthermore, in this figure the elevated baseline GLU levels observed in the 1.0% group (Table 2) have been removed from the Ca+2-independent component before differences with total release values were calculated to provide a more accurate estimate of the K+-evoked Ca+2-dependent portion. In addition to the decrease in Ca+2-dependent GLU release found in the 0.2% group in the first 20-min interval, an analogous effect of Pb in the 0.1% group exhibited a trend, but did not attain statistical significance (p = 0.07). However, no difference in Ca+2-dependent GLU release was evident in the 0.5 and 1.0% groups relative to controls. The Ca+2-dependent components in these 2 groups were significantly greater than that of the 0.2% group (q = 5.11, p < 0.01, and q = 6.30, p < 0.001, respectively), again establishing a U-shaped dose-effect relationship.
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GABA Release
Pb exposure level exerted a significant effect on baseline extracellular GABA concentrations with Ca+2 present in the perfusion medium: GABA levels were elevated in the 1.0% Pb group compared to control values (Table 2
). Consistent with the findings with GLU, GABA release evoked by high K+ stimulation in the presence of Ca+2 (total release) was reduced by low levels of Pb. The analysis of variance revealed a significant exposure level x time after K+ interaction (F(40,560) = 6.73, p < 0.001). The time courses of the GABA responses in the control and 0.2% Pb groups (Newman-Keuls q = 14.04, 9.18, and 7.99 at 20, 40, and 60 min, respectively, all p < 0.001) are shown in the top panel of Figure 4. GABA responses during the first 20-min stimulation interval are presented across exposure levels in the bottom panel of Figure 4. High K+-evoked GABA release was also significantly decreased in the 0.1% (q = 8.51, p <0.001) and 0.5% (q = 3.44, p < 0.05) groups relative to that of control animals, but was not altered in the 1.0% Pb group during this period. The responses of the 0.5 and 1.0% groups were significantly greater than that of the 0.2% Pb group over this interval (q = 8.30 and 9.88, respectively, both p < 0.001).
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In the absence of Ca+2 in the perfusion medium chronic exposure to 1.0% Pb resulted in a marked elevation in prestimulation extracellular GABA concentrations compared to control values (Table 2
). As with GLU, a significant exposure level x time after K+ interaction (F(40,450) = 2.36, p < 0.001) was revealed with high K+ stimulation in the absence of Ca+2 (Ca+2-independent release). The top panel of Figure 5 displays the time courses of K+-stimulated increases in extracellular GABA concentrations observed under Ca+2-free perfusion conditions in the control and 0.2% groups. Responses during the first 20-min stimulation period are displayed across exposure groups in the bottom panel of Figure 5, which shows significant increases in extracellular GABA in the 0.5 and 1.0% groups (q = 3.65, p < 0.05, and 11.38, p < 0.001, respectively). For purposes of overall comparison, the elevated baseline has not been removed from the magnitude of the response of the 1.0% group in Figure 5.
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Subtracting the corresponding values for total and Ca+2-independent release at each time point in control and Pb-exposed animals results in the measures of Ca+2-dependent GABA release across time for the control and 0.2% groups shown at the top of Figure 6
. Data for all exposure groups are presented across the first 20-min interval at the bottom of Figure 6. In addition, the elevated baseline GABA levels seen with the 1.0% group (Table 2) have been removed from the Ca+2-independent component before differences with total release values were computed to provide a more accurate estimate of the K+-induced exocytotic GABA release. Evoked Ca+2-dependent release was significantly decreased in the 0.2% Pb group relative to controls over the first hour of K+ stimulation (q = 8.21, 7.72, and 6.49 at 20, 40, and 60 min after initiation of high K+, respectively, all p < 0.001). Ca+2-dependent GABA release was also decreased in the 0.1% group compared to controls in the first 20-min interval (q = 4.58, p < 0.05). As a result, the Ca+2-dependent components in the 0.5 and 1.0% Pb groups were significantly greater than that of the 0.2% group (q = 4.39 and 5.03, respectively, both p < 0.01), again establishing a biphasic dose-effect relationship.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic Pb exposure produced decreases in total and Ca+2-dependent K+-stimulated hippocampal GLU and GABA release at exposure levels producing blood Pb values from 27–62 µg/100 ml. However, these effects were less evident at blood Pb concentrations of 62 µg/100 ml and were no longer present in the highest exposure group that produced blood Pb levels of 118 µg/100 ml. The depolarization-evoked responses thus define U-shaped dose-effect relationships, suggesting the presence of two or more synaptic actions of Pb with individual dose-effect curves of varying sensitivity to the metal.
The most prominent effects observed in this study were the Pb-induced decrements in K+-stimulated GLU and GABA release seen at intermediate exposure levels. Maximal effects were consistently observed in the 0.2% Pb animals and are representative of the exposure effects manifested in the other affected groups. The time courses of the GLU responses to high K+ in the 0.2% group are shown at the top of Figures 1–3, and are in agreement with previous reports utilizing continuous exposure from birth at this exposure level (Lasley and Gilbert, 1996; Lasley et al., 1999). These data are consistent with previous observations that the Ca+2-dependent component is most sensitive to treatment effects that are thought to be exerted on Ca+2-mediated synaptic processes (Lasley et al., 1999). However, based on studies involving acute exposure to Pb+2 in vitro (Minnema et al., 1986; Minnema and Michaelson, 1986; Shao and Suszkiw, 1991) and previous work performed in this laboratory (Lasley and Gilbert, 1996), a total release measure that incorporates both Pb+2-induced changes in release and the Ca+2-dependent component likely represents a more reliable indicator of transmitter present in the synaptic cleft upon excitation. With no effect present in the absence of Ca+ in the perfusion fluid (top, Fig. 2), the actions of exposure on total GLU release can be traced directly to the effects on the Ca+2-dependent component.
Qualitatively similar effects were seen at intermediate exposure levels in the time course of the hippocampal GABA response to high K+ in the 0.2% Pb group (top halves of Figs. 4–6) compared to that of controls in agreement with the results of Lasley et al. (1999). An exposure effect on total GABA release was not found in an earlier report (Lasley and Gilbert, 1996), but a decrease in the Ca+2-dependent component is common to all 3 studies. This could occur because of the utilization of different approaches to estimate Ca+2-independent release in Lasley et al. (1999) and in the current work.
The mechanisms underlying these Pb-induced decreases in stimulated transmitter release have not been defined, though plausible candidates have been identified. A portion of the K+-evoked release may be glial in origin (e.g., Rutledge and Kimelberg, 1996), but evidence reported by Longuemare and Swanson (1997) indicates that glial glutamate release is not induced by brain K+ concentrations achievable in vivo. Employing the whole-cell patch clamp approach, Braga et al. (1999a) reported that nominal concentrations of Pb+2 acutely applied to cultured hippocampal cells blocked the tetrodotoxin-sensitive release of GLU and GABA. This effect was manifested by reduction of the amplitude and frequency of GLU- and GABA-mediated postsynaptic currents evoked by spontaneous neuronal firing, and exhibited an IC50 of 68 nM. The authors proposed that this effect is mediated via the binding of Pb+2 to voltage-sensitive Ca+2 channels. Another potential target for Pb+2-induced impairment of transmitter release is protein kinase C (PKC). Activation of this kinase is known to have a stimulatory effect on release (Dekker et al., 1991), possibly by increasing the conductance of voltage-sensitive Ca+2 channels near synaptic release sites (Bartschat and Rhodes, 1995; Terrian, 1995). Acute exposure to free Pb+2 in vitro stimulates PKC activity in the picomolar range (Long et al., 1994; Markovac and Goldstein, 1988; Tomsig and Suszkiw, 1995), a potency much greater than that of Ca+2. However, Pb+2 displays much less efficacy than Ca+2, resulting in the characterization of Pb+2 as a partial agonist of the kinase (Tomsig and Suszkiw, 1995). Thus, in the presence of physiological concentrations of Ca+2, Pb+2 in the low nanomolar range can prevent maximal activation of PKC and may impair transmitter release in animals exposed to the metal. While data supporting both of these mechanisms are compelling and based on Pb+2 concentrations likely achieved in vivo from environmental exposure, neither effect has been demonstrated in tissue from chronically exposed animals. In addition, there is evidence that Pb exposure confined to early development diminishes K+-stimulated transmitter release (Lasley et al., 1999) and EPSP slope potentiation in the dentate gyrus of adult animals (Gilbert et al., 1999a), suggesting that a developmental aberration may also be a factor in the effect on release.
Changes in the components of K+-stimulated GLU and GABA release as a function of exposure level were also notable. K+-evoked total GLU and GABA release (bottom, Figs. 1 and 4) as well as the associated Ca+2-dependent components (bottom, Figs. 3 and 6) were diminished over the first 20-min high K+ perfusion period in the 0.1% Pb group (27 µg/100 ml blood) compared to controls. This extends to a lower exposure level previous observations of decreased evoked transmitter release (Lasley and Gilbert, 1996; Lasley et al., 1999). In contrast, the total and Ca+2-dependent GLU and GABA responses in the 0.5 and 1.0% Pb groups were all greater than those in the 0.2% group, thereby establishing biphasic dose-effect relationships.
Changes in the Ca+2-independent component of K+-induced GLU and GABA release as a function of exposure level also were apparent (bottom, Figs. 2 and 5). For each transmitter a dose-dependent increase occurred in the absence of Ca+2 until significance was achieved in the 0.5 and 1.0% Pb groups. This observation suggests a significant contribution to extracellular transmitter levels from Pb+2-induced release that occurs independently of depolarization-induced Ca+2-dependent release, and that is unmasked in the relative absence of Ca+2. A similar mechanism may account for the elevated baseline extracellular concentrations of GLU and GABA in the 1.0% Pb group prior to high K+ perfusion (Table 2). Increased extracellular transmitter may derive from the Ca+2-mimetic effects of Pb+2 and other metals to induce exocytosis (Tomsig and Suszkiw, 1996). Consistent with this proposal, perfusion of 1 mM Cd+2 through a hippocampal dialysis probe increases baseline extracellular GLU severalfold (Lasley and Gilbert, unpublished observations).
The findings from the current investigation cannot be accounted for by increased excretion or altered tissue disposition of Pb, as both blood and brain Pb concentrations were increased across exposure levels in a dose-dependent manner. U-shaped dose-effect functions with Pb exposure have been reported with various experimental paradigms including maze performance (Geist et al., 1985), operant conditioning and drug discrimination in rodents (Cory-Slechta, 1984), and in tests of simple reaction time in populations of exposed humans (Bleeker et al., 1997). From the evidence presented herein, a plausible basis for the reversal at higher exposure levels of the Pb-induced diminution of the K+-stimulated transmitter responses can be proposed. In the exposure groups in which the Pb effect is partially or fully reversed the Ca+2-independent component of release is elevated, suggesting a compensation for the deleterious effects of exposure on K+-stimulated GLU and GABA release evident at lower exposure levels. However, this release component is less sensitive to the actions of Pb than the Ca+2-dependent component, which exhibits a reduction in response at the lowest exposure level utilized. A differential sensitivity of these two mechanisms has also been noted with acute application of Pb+2 to cultured hippocampal neurons (Braga et al., 1999a,b). The combination of these individual dose-effect relationships results in the biphasic curve seen in the measures of total GLU and GABA release. The reversal of the decrease in the Ca+2-dependent components of release observed at higher exposure levels (bottom, Figs. 3 and 6) and the elevated baseline extracellular transmitter levels in the 1.0% group (Table 2) are observations also consistent with the ability of Pb+2 to independently drive exocytosis. That is, at blood Pb values 62 µg/100 ml the Ca+2-mimetic properties of Pb+2 can be discerned whether Ca+2 is present or not. These studies represent the first dose-effect analysis of developmental Pb exposure and in vivo hippocampal GLU and GABA release whereby a clear and consistent biphasic pattern of effects was evident across multiple measures.
Biphasic dose-effect relationships have been observed in other studies employing analogous experimental designs. Significant similarities are evident in the effects of chronic Pb on measures of hippocampal LTP (Gilbert et al., 1999b) and K+-stimulated hippocampal GLU release as a function of chronic exposure level. An impairment of LTP magnitude was reported in the 0.1, 0.2, and 0.5% Pb groups, but no effect was observable in the 1.0% group. A similar pattern of results was seen across groups in LTP induction thresholds: chronic exposure to 0.1% and 0.2% Pb increased the induction thresholds for LTP, a Pb effect that was not present in the higher exposure level groups. These observations lead to the conclusion that the Pb-induced decreases in depolarization-evoked hippocampal GLU release may be a significant contributing factor to the exposure-related changes observed in the measures of LTP.
In summary, K+-stimulated hippocampal GLU and GABA release were diminished in comparison to control animals with a maximal effect at the 0.2% exposure level. Biphasic dose-effect relationships were observed on the basis of decreased responses to high K+ in the 0.1–0.5% groups but in the presence of partially or fully reversed exposure effects in the 0.5–1.0% groups. This pattern of results indicates the presence of a more potent suppression of stimulated release seen at lower exposure levels combined with Ca+2-mimetic actions to independently induce exocytosis that is exhibited at higher exposure levels. Furthermore, significant similarities in the dose-effect relationships uncovered in measures of evoked GLU release and hippocampal LTP (Gilbert et al., 1999b) reinforce the belief that exposure-related changes in GLU release may be a significant factor in the Pb-induced effects seen in this model of synaptic plasticity. Subsequent studies should focus on the biochemical mechanisms underlying LTP that are disturbed by chronic Pb exposure.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant ES06253, the National Research Council, and the U. S. Environmental Protection Agency.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (309) 671-8403. E-mail: sml{at}uic.edu.
This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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REFERENCES |
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