Ethanol effects on synaptic neurotransmission and tetanus-induced synaptic plasticity in hippocampal slices of chronic in vivo lead-exposed adult rats

BRAIN RESEARCH ELSEVIER

Brain Research 734 (] 996) 61-71

Research report

Ethanol effects on synaptic neurotransmission and tetanus-induced synaptic plasticity in hippocampal slices of chronic in vivo lead-exposed adult rats Cathy A. Grover *, Gerald D. Frye Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A & M University, Health Sciences Center, College Station, TX 77843-1114, USA

Accepted 27 February 1996

Abstract The interaction of chronic in vivo lead exposure and acute in vitro ethanol treatment on synaptic neurotransmission and plasticity were studied using extracellular electrophysiological techniques in CA1 region of hippocampal brain slices from adult rats. Neither chronic lead exposure nor acute ethanol treatment had any significant effect on field excitatory postsynaptic potentials (EPSPs). In vivo lead exposure enhanced short-term potentiation (STP, potentiation that decays within 30 min) by 21% shortly after 'weak' tetanus, but had no effect on long-term potentiation (LTP, sustained at least 1 h). In vitro bath application of 60 mM ethanol inhibited STP by 35% and blocked LTP induced by 'weak' tetanus in slices from Pb-exposed rats (500 ppm lead acetate, 56-70 days), while having no effect on STP or LTP in slices from control counterpart Na-exposed rats (pair-fed 216 ppm sodium acetate). In contrast, 'strong'-tetanus-induced LTP was abolished in Pb-exposed slices, and 60 mM ethanol slightly inhibited STP and blocked LTP in slices from Na-exposed rats. These differences could not be explained by differences in ethanol inhibition of NMDA-mediated field EPSPs because they were similarly reduced in slices from Na-exposed (30%) and Pb-exposed (25%) rats. These findings suggest that the strength of the tetanus used determines whether or not synaptic plasticity is blocked by either chronic lead exposure or acute ethanol treatment, and that even in adult rats, hippocampal synaptic LTP can be compromised by combined exposure to ethanol and lead. More importantly, these findings suggest the consequences of combined lead exposure and alcohol abuse in the adult human population may not be fully recognized yet. Keywords: Short-term potentiation; Long-term potentiation; Lead; NMDA; Excitatory postsynaptic potential; Electrophysiology; Extracellular recording; Neurotoxicity

1. Introduction Environmental pollution and alcohol abuse pose significant public health problems for our society. Alcohol consumption correlates with increased blood-lead levels in humans [26,53], and alcoholics have been reported to have moderate blood-lead levels (28-72.5 t x g / 1 0 0 ml) [21]. Animal studies suggest there may be a direct impact of lead on ethanol consumption. For example, lead-exposed adult rats increased consumption of ethanol in a free-access situation [45,46] but decreased operant responding for ethanol [47]. And, recent animal studies suggest that chronic lead exposure may actually blunt several behavioral responses to ethanol rather than potentiating ethanol sensitivity (e.g., antipunishment [48], rate depressant [31], and hypoalgesic [14] effects of ethanol). However, the

* Corresponding author. Fax: [email protected]

+ 1 (409) 845-0699; e-mail:

interactions of lead and ethanol at the cellular level have yet to be determined. Lead and ethanol appear to exert many similar neurotoxic actions. At a cellular level both agents exert inhibitory effects on excitatory synaptic transmission. For example, evoked dendritic field excitatory postsynaptic potentials (EPSPs) have been reported to be inhibited by both lead [5] and ethanol [54], as have pharmacologically isolated N M D A - r e c e p t o r - m e d i a t e d synaptic potentials [30,41] and N M D A - a c t i v a t e d whole-cell currents [2,49]. Possibly related to the inhibition of N M D A receptor activity, as well as the impairment of learning and memory by these substances [24,25,50,56,60], both lead [3,5,6] and ethanol [12,55] block the induction of long-term potentiation (LTP). LTP of synaptic potentials and other forms of synaptic plasticity are considered likely cellular substrates of learning and m e m o r y [11]. Whether ethanol or lead enhance the neurotoxic actions of one another when they are present together is not yet clear. Hippocampal function

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006-8993(96)00300-9

62

C.A. Grover, G.D. F~e / Brain Research 734 (1996)61-71

is a likely target for the combined effects of these two agents because the hippocampus, which is important for learning and memory, is a central nervous system site of either ethanol [62] or lead [1,20,22] sequestration a n d / o r action, The purpose of the present study was to determine whether chronic in vivo lead exposure increases the inhibitory actions of ethanol at the cellular level. The interaction of lead and ethanol was studied by examining changes in evoked field EPSPs and tetanus-induced synaptic plasticity including, short-term potentiation (STP) which decays within 30 rain and long-term potentiation (LTP) which is sustained at least 1 h, in the CA1 region of hippocampal slices. First, the present study supports the idea of some similarities between cellular actions of lead and ethanol by showing that the inhibition of LTP by either chronic lead exposure or acute ethanol treatment depends on the tetanus used to induce synaptic plasticity. Secondly, and more importantly, these experiments show an additive effect of combined lead exposure and ethanol treatment on synaptic plasticity under conditions which neither agent by itself is inhibitory. Furthermore, because there was no additional ethanol-mediated inhibition of NMDA-mediated field EPSPs in slices from chronic leadexposed rats, the additive effects on LTP likely were not via actions at NMDA-receptors. Portions of this data appear in abstract form [29].

2. Materials and methods 2.1. Animals and chronic Pb e + exposure

The 70 male Sprague-Dawley rats (Timco-Harlan Industries, Houston, TX) used here had a mean body weight of 102.8 _+ 2.37 g (standard error) at the beginning of the study. Throughout the metal exposure period of 56-70 days half of the rats (Pb-exposed) were presented ad libitum distilled water containing 1.3 mM lead acetate (500 ppm). Weight-matched control (Na-exposed) rats were individually pair-fed a control solution containing essentially the same amount of acetate daily (distilled water containing 2.4 mM sodium acetate (216 ppm)) such that each of a Pb-exposed - Na-exposed pair received the same volume of drinking solution during the exposure period. Lead concentrations of trunk blood collected at decapitation were determined via dry ashing and atomic absorption spectrophotometry as described in detail previously [13]. 2.2. Slice preparation

Standard techniques were used to obtain transverse slices from the middle third of the hippocampus of Na-exposed or Pb-exposed rats [30]. Briefly, after decapitation the brain was rapidly removed, cooled to ~ 4°C, the hippocampus dissected free of surrounding tissue, and

transverse slices (400-500 Ixm thick) were cut on a McI1wain Tissue Chopper (Mickle Lab. Engineering, Surrey, UK). After incubating in a standard physiological solution (in mM: NaCI 120, KCI 3, CaC12 2.5, MgC12 1.2, NaHzPO 4 1.2, NaHCO 3 22.6, glucose 11.1; continually gassed 95% 0 2, 5% CO 2) for 1 h in a tissue chamber, slices were transferred to a plexiglas 2 ml recording chamber, completely submerged, and continually perfused (4 ml/min, 30°C). Slices were acclimated to the recording chamber for at least 30 min before beginning extracellular recording. The number of slices used per rat for each experimental condition are indicated in the text or figures below. 2.3. Extracellular recordings

Standard extracellular recording techniques were used to obtain dendritic field excitatory postsynaptic potentials (EPSPs) from the CA1 region of the hippocampus [23,30]. Glass recording electrodes (1.0 mm OD, resistances of 4-15 M fL filled with 1 M NaC1) connected to an Axoclamp-2 amplifier (Axon Instr., Foster City, CA) were placed in the stratum radiatum of the CA1 region of the hippocampus. A bipolar stimulating electrode was positioned in the stratum radiatum of the C A 3 / C A 2 region of the Schaffer collateral/commissural pathway. Initially, for each slice, graded field EPSPs were evoked by increasing stimulus intensities (0.14-0.50 mA) to determine which current produced the maximum (no evidence of a population spike) and half-maximum field EPSP. 'Weak' tetanus: For the duration of all 'weak' tetanus experiments, four individual field EPSPs were monitored at stimulus intensities (0.16-0.28 mA, 0.25 Hz) that elicited half-maximum responses, digitized using P-Clamp software (Axon Instr.) and averaged. After stable voltage trace recordings were acquired with test stimuli each minute for 20 min, a brief 'weak' tetanus (train of four pulses, each pulse 0.2 ms on/9.8 ms off, train repeated four times, inter-train interval of 196 ms, stimulus intensity set at half-maximum) was applied. Voltage trace recordings were obtained every minute for 60 min after tetanus, and again at 90 min post-tetanus. 'Strong' tetanus: For 'strong' tetanus (train of 100 pulses at 100 Hz, each pulse 0.2 ms on/9.8 ms off, train repeated two times, inter-train interval of 20 s, stimulus intensity set at half-maximum) experiments. Input/output curves were generated (six graded evoked field EPSPs) by increasing test stimulus intensities (0.14-0.50 mA), were repeated four times at 21, 16, 11, and 6 min prior to tetanus (often denoted as - 2 1 , - 16, - 11, or - 6 ) , and at 5, 10, 15, 30, 60, and 90 min post-tetanus, digitized and averaged at each test stimulus intensity. Therefore, a mean trace for each of the six graded stimuli was available for analysis at each pre- and post-tetanus time recorded. The important difference between 'weak' and 'strong' tetanus was the number of pulses applied. During both

C.A. Grover, G.D. Frye~Brain Research 734 (1996)61-71 types of tetanus, ' w e a k ' and 'strong', the stimulus intensity was set to evoke a half-maximum response. Amplitude of the peak (mV; A M P L I T U D E ) for each averaged voltage trace recording was measured using P-Clamp (Clampfit) software. 2.4. Drugs Lead acetate and sodium acetate were purchased from Sigma (St. Louis, MO), absolute ethanol from the Warner-Graham (Cockeysville, MD). Both 6,7-dinitroquinoxaline-2,3-dione (DNQX) and DL-2-amino-5phosphonopentanoic acid (AP5) were purchased from Tocris Neuramin (Bristol, UK). Ethanol was applied via bath perfusion. Five min of bath perfusion is sufficient to detect significant effects of ethanol with our delivery system [30]. To pharmacologically isolate NMDA-mediated field EPSPs, slices were continually perfused for 1 h prior to extracellular recording in a low Mg 2+ physiological solution (replace 1.2 mM MgC12 with 0.1 mM) containing the non-NMDA antagonist D N Q X (10 IxM) [30]. After ethanol treatment the N M D A antagonist AP5 (50 IxM) was used to verify that field EPSPs were NMDA-mediated.

63

separate groups of Pb-exposed rats used in the various experiments discussed below (EPSPs, Weak, and Strong). Hippocampal slices from some of the rats used in Weak experiments were also used in the N M D A experiments. Total fluid consumption also was comparable for Na-exposed (3307.71 _ 129.28 g) and Pb-exposed (3162.23 + 110.77 g) rats, P = 0.39. Lead residues in trunk blood for Pb-exposed rats (24.1 + 1.2 ~ g / d l ) were significantly ( P < 0.001) greater than for Na-exposed control rats (1.6 _ 0.2 t~g/dl). The blood lead levels in these rats are consistent with levels reported in previous studies of chronic lead/ethanol interactions [14,31,45-48]. 3.2. In vivo effects of Pb 2+ and in vitro effects of ethanol on field EPSPs

Pb 2+ that has accumulated in the brains of the chronically exposed animals might be expected to interfere with synaptic potentials as does acute lead treatment [5]. However, in vivo chronic lead exposure had no significant effect on synaptic potentials. We compared amplitudes (mV) of half-maximum and maximum field EPSPs evoked with 0.15-0.19 mA and 0.18-0.25 mA current, respectively, in hippocampal slices during baseline recording of tests discussed below. As can be seen in Fig. 1A (repre-

2.5. Statistical analyses Baseline values were determined for each individual slice by averaging the responses recorded during the first 15 min of the experiment (min - 2 0 to - 6 for LTP experiments; min 0 - 1 5 for synaptic potential experiments). This window of time was selected because it was the pre-tetanus period just prior to the addition of ethanol. All subsequent field EPSPs are expressed as a percentage of baseline. Where appropriate, analysis of variance ( A N O V A ) or repeated measures A N O V A tests were performed with SPSS for Windows (SPSS, Chicago, IL). Significant interactions were further examined by analyzing Simple Effects. The Tukey test was used for all post-hoc individual pair-wise comparisons. For all analyses P < 0.05 was accepted as evidence of significance. Data are expressed as mean + standard error of the mean (S.E.M.).

3. Results

3.1. Body weights, fluid intake and blood lead residues Neither initial body weights ( 1 0 4 . 2 4 + 3 . 6 5 g and 1 0 1 . 6 2 + 2 . 9 8 g; P = 0 . 5 8 ) , nor final body weights (350.40 __+4.10 g and 363.22 ___6.11 g; P = 0.09) for Naexposed (n = 35) and Pb-exposed (n = 35) rats, respectively, used in all experiments were significantly different. More importantly, initial ( P = 0.11) and final ( P = 0.22) body weights were not statistically different between the

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Field EPSPs Fig. 1. In vivo lead had no effect on evoked field synaptic potentials. (A) Individual voltage traces of evoked half-maximum and maximum field EPSPs recorded in CAl region of hippocampal slices from Na-exposed and Pb-exposed rats. (B) Evoked field EPSPs in slices are insensitive to chronic in vivo lead exposure. Symbols and bars represent the mean + S.E.M. amplitudes of half maximum (experiments EPSPs and Weak) or maximum (experiments EPSPs and Strong) evoked field EPSPs in slices from Na-exposed and Pb-exposed rats recorded extracellularly during Baseline in separate experiments discussed below, n refers to the number of slices used in each condition.

64

C.A. Grover, G.D. Frye~Brain Research 734 (1996) 61-71

sentative individual traces) and Fig. 1B (mean data) halfm a x i m u m or maximum field EPSPs were similar in slices from Na-exposed and Pb-exposed rats. Some [54], but not all [17], have reported ethanol inhibition of synaptic potentials. A 10-min treatment with ethanol had no statistically significant effect on evoked half-maximum ( n = 5; 1 slice per rat per group) or maxim u m ( n = 5; 1 slice per rat per group) field EPSPs in slices from rats in groups Na-exposed ( P > 0.05) or Pbexposed ( P > 0.05). More importantly, for slices in each group, field EPSPs after 5 and 6 min of bath perfusion with ethanol were similar to pre-ethanol field EPSPs. Therefore, in subsequent experiments a 6-min period of ethanol treatment was used to study the effects of ethanol on the induction of synaptic plasticity (see Section 3.3 and Section 3.4). This finding suggests that during this brief period of treatment 60 mM ethanol does not b l u n t / b l o c k transmitter mechanisms (primarily AMPA mediated) involved in the generation of the synaptic potentials used to evaluate synaptic plasticity in the following studies, while saying nothing about ethanol effects on mechanisms involved in the induction of LTP (e.g., NMDA, GABA, voltage-gated calcium channels). 3.3. In ui~'o Pb 2 + and in ~,itro ethanol effects on 'weak'tetanus-induced S T P and L T P

Individual voltage traces shown in Fig. 2A are representative of half-maximal field EPSPs evoked with 0.17-0.28 mA current 6 min before (a), 10 rain after (b), and 60 rain after ' w e a k ' tetanus (c), and reflect time points used to assess Baseline, STP and LTP, respectively. These times were selected for the following reasons: 6 min before tetanus was the last baseline recording prior to the addition of ethanol; 10 min after tetanus allows sufficient time for ethanol to wash from our recording chamber [30] while representing the early stages of STP; 60 rain is the standard time used to define in vitro slice LTP [11]. Although the peak amplitudes of these individual pre-tetanus baseline (a) traces appear different, the group means were not statistically different. These traces were selected to represent the range of half-maximum field EPSPs observed in hippocampal slices from both Na-exposed and Pb-exposed rats, while the changes in amplitude after tetanus relative to pre-tetanus in these individual traces are representative of group mean effects. Slices were continuously perfused with physiological solution or solution containing 60 mM ethanol 5 rain before through 1 min after ' w e a k ' tetanus. In contrast to ethanol not having an effect on evoked field EPSPs in our experiments discussed above (see Section 3.2 above), after 5 rain of treatment, immediately before ' w e a k ' tetanus (time - 1 min) ethanol significantly depressed field EPSPs by 19 + 6.7% in slices from Pb-exposed rats ( P < 0 . 0 0 1 ) , see Fig. 2B. This inconsistency can not be easily explained because up through the 5 rain of ethanol treatment, other than recording 15 min rather

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Time (min) Fig. 2. Combined in vivo lead and in vitro ethanol (60 mM) inhibition and block of 'weak'-tetanus induced STP and LTP, respectively. (A) Individual voltage traces, evoked by half-maximal test stimuli, from Na-exposed and Pb-exposed ethanol-treatedand untreated slices showing short- and long-termeffects of 'weak' tetanus. The - 6 rain (a) voltage trace represents the last baseline recording, while the 10 (b) and 60 min (c) traces represent responses obtainedat time points used to test for STP and LTP, respectively.Even though the peak amplitudesof these individual pre-tetanusbaseline (a) traces appear differentthe group means were not statistically different. These traces were selected to represent the range of half-maximumfield EPSPs observed in hippocampal slices from both Na-exposed and Pb-exposed rats, while the changes in amplitude after tetanus relativeto pre-tetanusin these individualtraces are representative of group mean effects. (B) Symbolsand bars represent the mean_+ S.E.M. effects of ethanol on normalized field EPSPs evoked by the half-maximum test stimulus across the time course of 'weak' tetanus experiments in slices from Na-exposed and Pb-exposed rats. Slices were either continuouslyperfused with physiologicalsolutionalone or solution containing 60 mM ethanol 5 rain before through 1 min after (time between dashed lines) ' weak' tetanus(arrow). Italicizedlower case letters refer to the time during the experimentalprotocol at which each trace in A was recorded, n refers to the number of slices (one per rat) used in each condition.

than 10 min of baseline, the protocol was the same for the two experiments. Field EPSPs were 58 + 3.9% (Na-exposed Control), 53 _+ 2.4% (Na-exposed EtOH), 72 _+ 7.8% (Pb-exposed Control), and 33 -t- 5.6% (Pb-exposed EtOH) larger immediately after (time 0) ' w e a k ' tetanus compared to immediately before (time - 1 ) , suggesting post-tetanic potentiation (PTP) occurred for each of the conditions. However, PTP was significantly reduced in Pb-exposed EtOH, and completely absent by 1 min after tetanus (see filled triangles in Fig. 2B).

C.A. Grover, G.D. Frye/Brain Research 734 (1996) 61-71 Table 1 The magnitude of STP (10 min) and LTP (60 min) after 'weak' tetanus in CAI area of hippocampal slices from adult Na-exposed and Pb-exposed rats Group

Time after tetanus 10 min

60 min

Na-exposed Control (n = 12) Na-exposed EtOH (n = 7)

33.94 + 5.33 27.11 ___3.38 FI.17 = 0.83, P = 0.37 a 51.63 ____4.45 _ F1,22 = 6.50, P = 0.02 a 17.39 + 3.28 F],]8 = 31.52, P < 0.001 b

18.59 + 5.9 28.07 + 11.73 FI.17 = 0.64, P = 0.43 a 27.71 + 5.09 FI,22 = 1.35, P = 0.26 a - 5.89 _+4.90 FI,18 = 20.43, P < 0.001 b

Pb-exposed Control (n = 12)

Pb-exposed EtOH (n = 8)

Values are mean (% of B a s e l i n e - 1 0 0 ) + S.E.M. a Compared to Na-exposed Control same time. b Compared to Pb-exposed Control same time. n refers to the number of slices tested.

R e g a r d i n g STP, h a v i n g a l l o w e d sufficient time for solution e x c h a n g e , field E P S P s were significantly potentiated at 10 min after ' w e a k ' tetanus (see b in Fig. 2B) c o m p a r e d to 6 min before (see a in Fig. 2B) for each o f the groups, all P < 0.05. A l t h o u g h S T P at 10 min after tetanus was apparent for all treatments, it was significantly larger in slices f r o m P b - e x p o s e d c o m p a r e d to N a - e x p o s e d rats. At 10 min after tetanus the m a g n i t u d e (Post-tetanus % o f B a s e l i n e - 100%; i.e., the a m o u n t o f change) o f S T P was significantly reduced in P b - e x p o s e d E t O H relative to Pbe x p o s e d slices, but was the s a m e for N a - e x p o s e d E t O H and N a - e x p o s e d slices (see T a b l e 1). Furthermore, as can be seen in Fig. 2B, P T P a n d / o r S T P w e r e transiently inhibited in P b - e x p o s e d and N a - e x p o s e d slices while ethanol was w a s h i n g out o f the r e c o r d i n g c h a m b e r ( c o m pare the first seven open and closed triangles after second dashed line to those at 10 min in Fig. 2B). Statistical tests indicated that relative to 10 min post-tetanus field E P S P s w e r e inhibited at 2 through 5 m i n post-tetanus in Pb-exposed E t O H slices. A l t h o u g h field E P S P s were not statistically different across the 2 through 10 m i n post-tetanus in N a - e x p o s e d E t O H slices, 2 through 8 rain after tetanus they were statistically smaller than those o f N a - e x p o s e d . T h e transient nature o f the e t h a n o l - i n d u c e d inhibition was p r e s u m a b l y due to the r e m o v a l of ethanol. As for L T P , field E P S P s w e r e significantly potentiated 60 min after ' w e a k ' tetanus c o m p a r e d to 6 min before in N a - e x p o s e d Control, N a - e x p o s e d E t O H , and P b - e x p o s e d Control (all P < 0.05), but not P b - e x p o s e d E t O H slices ( P = 0.269). The m a g n i t u d e (Post-tetanus % o f B a s e l i n e - 100%; i.e., the a m o u n t o f change) o f L T P was c o m p a r a ble for N a - e x p o s e d Control and N a - e x p o s e d E t O H slices (see T a b l e 1). T h e s e findings suggest that chronic lead treatment increases the sensitivity o f both S T P and L T P to inhibition by ethanol under ' w e a k ' tetanus conditions. In

65

the absence o f chronic lead e x p o s u r e these m e a s u r e s appear to be resistant to ethanol. 3.4. In vivo P b 2 + and in vitro ethanol effects on 'strong'tetanus-induced S T P and L T P A l t h o u g h the failure of ethanol to b l o c k L T P in the a b o v e study under control conditions was s o m e w h a t surprising, B e n t z and B r o w n i n g [10] h a v e recently reported that 50 but not 25 m M ethanol inhibited L T P induced by h i g h - f r e q u e n c y tetanus (one train o f 100 stimuli d e l i v e r e d at 100 Hz), whereas 100 but not 50 m M b l o c k e d L T P induced by theta burst stimulation (10 bursts at 5 H z with four pulses within each burst). Together, these findings could suggest that the strength o f the tetanus stimulation

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Time (min) Fig. 3. In vivo lead and in vitro ethanol (60 mM) inhibition of 'strong' tetanus-induced LTP at the highest intensity test stimulus used. (A) Individual voltage traces, evoked by the highest test stimuli used, from Na-exposed and Pb-exposed ethanol-treated and untreated slices showing short- and long-term effects of 'strong' tetanus. The - 6 min (a) voltage trace represents the last baseline recording, while the 10 (b) and 60 min (c) traces represent responses obtained at time points used to test for STP and LTP, respectively. (B) Symbols and bars represent the mean + S.E.M. effects of ethanol on normalized field EPSPs evoked by the highest test stimulus used across the time course of 'strong' tetanus experiments in slices from Na-exposed and Pb-exposed rats. Slices were either continuously perfused with physiological solution alone or solution containing 60 mM ethanol 5 min before through 1 min after (time between dashed lines) 'weak' tetanus (arrow). Field EPSPs were not evoked between 60 and 90 min post-tetanus (i.e., during break in X-axis). Italicized lower case letters refer to the time during the experimental protocol at which each trace in A was recorded, n refers to the number of slices (one per rat) used in each condition.

C.A. Grocer, G.D. F ~ e / Brain Research 734 (1996) 61-71

66

used to induce LTP is a factor in ethanol inhibition of synaptic plasticity. Therefore, in the next set of experiments a stronger tetanus was used in an attempt to unmask ethanol inhibition of LTP in Na-exposed slices, and to determine the impact of lead. In addition, six graded field EPSPs evoked by increasing stimulus intensities (input/output curves) were used to determine whether the effects of ethanol on LTP depend on the voltage size of the test response. For each slice given a 'strong' tetanus with half-maximum stimulation (0.14-0.19 mA current), the maximum voltage response obtained with the highest relative stimulus intensity (0.16-0.29 mA current) at 21, 16, 11, and 6 min before tetanus were averaged and used as the baseline. Na-exposed EtOH and Pb-exposed EtOH slices were continuously perfused with physiological solution containing 60 mM ethanol for 5 rain before through 1 rain after 'strong' tetanus. Individual voltage traces shown in Fig. 3A are examples of maximum field EPSPs evoked 6 rain before (a), 10 rain after (b), and again at 60 rain after tetanus (c), and reflect time points used to assess Baseline, STP and LTP, respectively. Maximum field EPSPs for all groups were significantly potentiated at 10 min after 'strong' tetanus, all P < 0.05. Additionally, significant interactions and subsequent post-hoc tests indicated that STP was apparent for all groups at all but the lowest stimulus intensity, all P < 0.05. The magnitude of 'strong'-tetanus-induced STP was significantly reduced for Na-exposed EtOH and Pbexposed Control compared to Na-exposed Control slices (all P < 0.05), see b in Fig. 3B. The magnitude (Posttetanus % of Baseline - 100%; i.e., the amount of change) of STP was not significantly different for Pb-exposed Control and Pb-exposed EtOH slices except at the fourth stimulus intensity. Thus, ethanol consistently reduced 'strong'-tetanus-induced STP in hippocampal slices from control animals, but had little effect on STP in slices already inhibited by chronic in vivo lead exposure. Clearly shown in Fig. 3B, ' strong' -tetanus induced LTP in Na-exposed slices. A repeated measures ANOVA on field EPSPs acquired at the highest test stimulus revealed a significant group × treatment × time interaction ( P < 0.04). Field EPSPs were potentiated at 60 min after 'strong'

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Relative Stimulus Intensity Fig. 4. In vivo lead and in vitro ethanol (60 m M ) inhibition of L T P across all test stimulus intensities 60 rain after ' s t r o n g ' tetanus. Points on i n p u t / o u t p u t curves represent mean_+ S.E.M. effects of ethanol a n d / o r lead on field E P S P s (normalized to a) across the six test stimulus intensities before (Pre-tetanus) a n d 60 min after ' s t r o n g ' tetanus (Posttetanus). Italicized lower case letters refer to c o r r e s p o n d i n g means depicted in Fig. 3B, and n is the same as in Fig. 3B. Individual voltage traces (insets) represent the last baseline field E P S P (1) and the field E P S P recorded 60 min (2) after ' s t r o n g ' tetanus at the third test stimulus intensity (approximately half m a x i m u m baseline responses, similar to ' w e a k ' tetanus experiments).

tetanus only in slices from Na-exposed Control (see c in Fig. 3B; P = 0.001), and Pb-exposed EtOH (see Fig. 4: P = 0.04) rats. It is difficult to see in Fig. 3B that Pb-exposed EtOH at 60 min after tetanus (c) is slightly larger than its own pre-tetanus baseline (a) because of the overlapping symbols and error bars. As can be seen in Fig. 4 the variance at especially the highest stimulus intensity was reduced in the Pb-exposed EtOH group relative to the

Table 2 Effects of in vitro ethanol on the m a g n i t u d e of L T P 60 min after ' s t r o n g ' tetanus in C A I area of h i p p o c a m p a l slices f r o m adult N a - e x p o s e d and P b - e x p o s e d rats Group

Relative stimulus intensity 1

2

3

4

5

6

N a - e x p o s e d Control (n = 8) N a - e x p o s e d E t O H ( n = 8) P b - e x p o s e d Control (n = 7) P b - e x p o s e d E t O H (n = 8)

7.97+3.55 a 0.34.+0.90 * 1.31 _+ 1.45 0.31 _+0.63 . a

16.966.53 ~b 2.92_+5.73 * 7.49 _+ 3.81 * 5 . 2 7 _ + 3 . 5 0 *a~,

2 7 . 3 7 _ + 3 . 9 3 b~ 4.74.+6.34 ~ 10.26 _+ 5.58 * 1 0 . 0 8 - + 4 . 7 7 *b

3 3 . 1 3 _ + 5 . 6 9 ~d 9.89+7.31 * 9.13 +_ 6.44 * 1 4 . 3 0 - + 6 . 1 9 *b

4 2 . 4 0 ± 7.89 d 8.29-+8.08 * 10.32 +_ 8.07 ~ 1 4 . 8 8 - + 5 . 2 4 ~b

41.5+_6.95 d 5.61 -+9.19 * 11.57 + 8.96 * 12.71 .+3.88 *b

Values are m e a n (% of Baseline - 100) ± S.E.M. * P < 0.05 c o m p a r e d to same relative stimulus intensity of N a - e x p o s e d Control. ~.b.~.d p < 0.05 within the same g r o u p w h e n letters are different. n refers to the n u m b e r of slices tested.

C.A. Grover, G.D. Frye/Brain Research 734 (1996) 61-71

Pb-exposed and Na-exposed EtOH groups which probably accounts for why potentiation was statistically significant in the combined but not the individual conditions. In fact, magnitude of potentiation for Pb-exposed EtOH was significantly less than for Na-exposed Controls slices ( P < 0.05), and not statistically different than the post-tetanus potential changes for Pb-exposed and Na-exposed EtOH slices (see Table 2). The results of all individual mean comparisons across the six graded stimulations and between groups at 60 min after tetanus are indicated in Table 2. Together these findings suggest that 'strong'-tetanus-induced LTP was abolished in slices from rats chronically exposed to lead, and completely blocked by ethanol in Na-exposed slices regardless of the test stimulus intensity. Thus, under these conditions of maximal inhibition with

A

67

either lead or ethanol alone, the presence of an additive interaction could not be evaluated. The inhibition of LTP in Pb-exposed Control and Naexposed EtOH slices seen in Fig. 3A and B probably was not due to the presence of population spikes (i.e., a population of somatic action potentials; which could interfere with measurements of peak amplitude) because inhibition also occurred with lower test stimulus intensities that lacked contamination of population spikes or having already reached the maximum AMPLITUDE prior to 'strong' tetanus (e.g., see insets in Fig. 4). Fig. 4 shows individual voltage traces (insets) obtained with the third stimulus at 60 rain after 'strong' tetanus for all treatment groups. Note that the third relative stimulus intensity evoked approximately a half-maximum field EPSP, and thus is similar to

. Na-exposed

. Pb-exposed

~

10 ms

B

2015lO-

o

C o =

14o-

iu~ ~

Na-expo,~A

Baseline ', EtOH

Pb-exposed

Wash

AP5

i(60mM)

Wash

(50 ~tM)

,

~

40I0

' I |

---0-- Na-exposed (n=8) = Pb.-exposed(n=8)

,~ M

I N

L U

/ M

Time (min) Fig. 5. In vivo lead and in vitro ethanol (60 mM) effects on pharmacologically isolated NMDA-mediated field EPSPs. (A) Individual voltage traces from Na-exposed and Pb-exposed hippocampal slices showing approximate ethanol inhibition of NMDA-mediated field EPSPs. Voltage traces a, b and c represent the last baseline recording and after 5 min bath perfusion with ethanol, respectively. Although the shape of the field EPSPs depicted here appears different, these shapes occurred in both groups, and the integrated AREA and % inhibition by ethanol were similar in these two slices. (B) Evoked NMDA-mediated field EPSPs in slices are insensitive to chronic in vivo lead exposure. Symbols and bars represent the mean _+ S.E.M. AREA of evoked NMDA-mediated field EPSPs in slices from Na-exposed and Pb-exposed rats during baseline period of the experiment depicted in B below. (C) Evoked NMDA-mediated field EPSPs in slices from Na-exposed and Pb-exposed rats are inhibited similarly by ethanol. Symbols and bars represent the mean + S.E.M. effects of ethanol and AP5 on evoked pharmacologically isolated NMDA-mediated field EPSPs in slices from Na-exposed and Pb-exposed rats. n refers to the number of slices (one per rat) used in each condition.

68

C.A. GroL,er, G.D. Frye~Brain Research 734 (1996)61-71

the test stimulus used in the 'weak' tetanus experiments. Repeated measures ANOVAs revealed significant time × relative stimulus intensities interactions, and subsequent post-hoc tests indicated that LTP was apparent at all but the two lowest stimulus intensities in slices from Na-exposed Control and Pb-exposed EtOH (all P < 0.05).

Clearly, ethanol had no additional inhibitory effects on pharmacologically isolated NMDA-mediated field EPSPs in hippocampal slices from rats chronically exposed to lead relative to control rats.

4. Discussion 3.5. In vivo Pb 2 + and in vitro ethanol effects on N M D A mediated field EPS P s

NMDA-receptor activity is needed for the induction of LTP in CA1 hippocampus [11], and both ethanol and lead specifically reduce NMDA-mediated activity in cells from this region [40,63]. We tested the effects of 60 mM ethanol on pharmacologically isolated NMDA-mediated field EPSPs to determine whether differential effects here might account for the differences in ethanol inhibition of 'weak'and 'strong'-tetanus-induced LTP observed above. AREA rather than AMPLITUDE measures were used to quantirate responses for these experiments since we have previously found this to be a more consistent index for NMDA-mediated events [30]. Like unisolated synaptic potentials (see Fig. 1A), NMDA-mediated field EPSPs in slices from Na-exposed rats were not significantly different ( P > 0.05) from those of Pb-exposed rats (see Fig. 5B), suggesting that chronic lead exposure did not reduce/blunt evoked NMDA-mediated field EPSPs under these conditions. Depicted in Fig. 5A are individual voltage traces of pharmacologically isolated NMDA-mediated field EPSPs recorded from Na-exposed and Pb-exposed hippocampal slices before and after 5 min bath perfusion with ethanol. Even though the shape of the field EPSPs depicted here appears different for the two groups, these shapes occurred in both groups, and the integrated AREA and % inhibition by ethanol were similar in these two slices. Fig. 5C shows the mean normalized AREA of field EPSPs for slices in low Mg 2+ and 10 IxM DNQX alone (Baseline), followed by addition of 60 mM ethanol (EtOH) and after washout (Wash), and followed by addition of 50 IxM AP5 and a final washout. NMDAmediated field EPSPs were similarly reduced 30 + 6.4% and 25 ___3.6% by ethanol 5 min after perfusion (20 min) in Na-exposed and Pb-exposed slices, respectively. This reduction in field EPSP size was clearly due to ethanol because when ethanol was removed (Wash) the field EPSPs returned to baseline size. It is worth noting that by 15 min of continuous ethanol exposure (at 30 min) acute tolerance to ethanol inhibition [30] occurred in 2 / 8 and 0,/8 of the slices from Na-exposed and Pb-exposed rats, respectively. However, acute tolerance to ethanol inhibition of NMDA-mediated field EPSPs was probably not important in the LTP experiments discussed above because it occurred at a point in time after which ethanol treatment was terminated in all LTP experiments. Finally, AP5 nearly abolished voltage responses in slices from both groups confirming that these field EPSPs were NMDA-mediated.

Two important implications come from the present experiments. First, exposure to lead and ethanol can have significant neurotoxic consequences when encountered simultaneously. This is suggested by the surprisingly complete antagonism of synaptic plasticity due to 'weak' tetanus by combined ethanol and lead exposure that were innocuous when tested alone. Second, that the magnitude of lead a n d / o r ethanol interaction in a particular neurophysiological system may be dependent on the level of system activity. This conclusion is based on the finding that 'strong' tetanus which would be expected to generate a more robust plasticity than 'weak' tetanus never the less showed greater vulnerability to both lead and ethanol. To the best of our knowledge, this is the first report of additive or synergistic effects of chronic in vivo lead exposure and acute in vitro ethanol treatment at the neuronal level. The present findings, by showing that the susceptibility of CNS function is compounded by combined chronic lead exposure and a single exposure to ethanol, are an extension of others that have shown chronic coadministration of ethanol and lead potentiate some of each others cellular effects [58]. We studied the individual and combined effects of chronic lead exposure and acute ethanol treatment on synaptic plasticity induced by two intensities of tetanus. The unexpected complexity of the effects of these two neurotoxicants by themselves and together suggests that evaluating the additivity of chronic lead exposure and acute ethanol treatment with models of synaptic plasticity may be difficult, in this regard, 'weak'tetanus-induced LTP, which was unaffected by either chronic in vivo lead exposure alone or acute in vitro ethanol treatment alone, was completely blocked in ethanol-treated hippocampal slices from chronic lead-exposed rats, suggesting additivity, or even synergism of these two neurotoxicants. However, 'strong'-tetanus-induced LTP was maximally inhibited by either chronic lead exposure or acute ethanol treatment alone. Therefore, as would be expected, the post-'strong'-tetanus responses in the combined neurotoxicant condition were not different from either the chronic lead exposure alone or acute ethanol alone conditions, rendering this model of LTP uninformative about the additivity of the two substances. Little is known about the neurotoxicant effects of lead a n d / o r ethanol on multiple forms of LTP. We extend reports of others that either lead [3,6,36] or ethanol [12,44,55] alone inhibit synaptic plasticity, by showing that blockade of LTP by either neurotoxicant alone depends on the intensity of the tetanus used to induce potentiation.

C.A. Grover, G.D. Frye~Brain Research 734 (1996) 61-71

Although others have reported that ethanol inhibition of LTP depends on the model of LTP used, by showing that LTP induced by different intensities of tetanus is differentially concentration dependent [10], to our knowledge this had not yet been reported for lead. We found that either chronic lead exposure alone or acute ethanol treatment alone completely blocked LTP induced by a 'strong' tetanus without having any effect on LTP induced by a 'weak' tetanus. Our findings suggests that mechanisms impaired by either of these neurotoxicants may be responsible for the differences between some of the various models of LTP. An awareness of the differences between various models of the same general phenomenon is essential for understanding how the same level of exposure to a neurotoxicant can produce different, perhaps even opposing findings. Multiple forms of LTP, induced by different frequencies of stimulation a n d / o r pharmacological manipulations, exist and probably involve different caicium-dependent mechanisms/pathways [7,32]. One form of LTP is generated by low-frequency tetanus and requires activation of the ligand-gated and voltage-sensitive NMDA receptor which admits Ca 2÷ into the postsynaptic cell initiating a cascade of calcium-mediated events responsible for LTP (NMDA LTP) [11,19]. A second form of LTP induction requires high-frequency tetanus and activates both NMDA receptors and voltage-gated calcium (VGCC) channels [32]. While both NMDA LTP and VGCC LTP promote postsynaptic influx of Ca 2÷ it is believed that the cascade of events triggered by calcium influx may differ because of where and how much calcium enters the cell [32]. Recently it was shown that the strength of the tetanus used determines the involvement of internal calcium stores in LTP [9], and that NMDA LTP, but not VGCC LTP, is blocked by the PKC inhibitor H-7. Both lead and ethanol are known to have inhibitory effects on NMDA receptor activity [2,12,16,30,34,39,41,44,55,63] and VGCC currents [8,15,16,37,38,61]. Pb 2+ also competes with Ca 2÷ entry via VGCCs [52,59] making it possible for Pb 2÷ to interfere with second-messenger functions of calcium. Understanding the cellular mechanisms involved in the different models of LTP led us to further explore the effects of these neurotoxicants on some of those mechanisms specific for the induction of LTP. The present study, like that of Hori et al. [33], does not pinpoint NMDA receptor mechanisms as being involved in Pb 2÷ block of LTP. Hori et al. showed that an acutely applied Pb 2+ concentration that completely blocked LTP in brain slice had no effect on responses induced by ionophoretic application of NMDA [33]. Likewise, we showed that chronic lead exposure that resulted in total block of our 'strong' tetanus model of LTP had no effect on pharmacologically isolated NMDA-mediated field EPSPs (prior to ethanol exposure). Similarly, the present study suggests, like Bentz and Browning [10], that ethanol effects on LTP may not be due exclusively to effects on NMDA receptor function.

69

Here ethanol did inhibit pharmacologically isolated NMDA-mediated field EPSPs, but did not inhibit 'weak'tetanus-induced LTP. Furthermore, the additivity of ethanol and lead on 'weak'-tetanus-induced LTP seen in the present study likely was not due to lead/ethanol interactions at NMDA receptors because the ethanol sensitivity of pharmacologically isolated NMDA-mediated field EPSPs were not different in slices from Na-exposed and Pb-exposed rats. While there are many models of LTP, LTP is only one form of synaptic plasticity susceptible to neurotoxicity. In addition to LTP, in vitro tetanic stimulation in the hippocampus can result in the temporally distinct components of post-tetanic potentiation (PTP) [27], which decays within a few minutes after tetanus, and STP which typically decays within 30 min but always before 60 min [11]. Whereas pre- and postsynaptic mechanisms are involved in LTP, PTP is the result of residual calcium in the presynaptic terminal and therefore lasts only as long as excess calcium is being cleared from the terminal [64]. The mechanisms responsible for STP are thought to involve interactions between pre- and postsynaptic factors [51 ], but unlike LTP persists in the presence of protein kinase inhibitors [42]. The extent to which the short-term changes of STP equate to PTP and LTP are not fully understood [11]. Little has been reported regarding the effects of Pb 2÷ on STP, however, the present finding of enhanced STP following 'weak' tetanus in slices from lead rats is reminiscent of the report of acute pb2+-induced increase in somatic short-term potentiation following paired-pulse stimulation [4]. Potentiation following paired-pulse stimulation, like PTP involves residual presynaptic calcium and is NMDA-receptor independent. Whether it relates to the presynaptic mechanisms involved in STP is not known. Likewise, what relationship the enhanced STP seen in the present set of 'weak' tetanus experiments in slices from lead-exposed rats has to LTP is not known. Finally, little is known concerning what level of Pb 2+ is present at the extracellular level to exert its neurotoxic effects such as those tested in the present study. Blood lead concentrations of chronic lead-exposed adult rats in the present study are equivalent to those considered 'medium' for adult humans [18]. Brain Pb 2+ concentrations could not be obtained here because the tissue was needed for electrophysiological recording, however, rats of similar age and exposure had whole brain levels of 0.4 ppm (400 ~g p b 2 + / g brain) [28]. Elimination of Pb 2+ in brain is known to be very slow [18]. For adult mice given a single i.v. injection of [21°Pb]acetate the half life of 2~°pb in brain was 49 days [35]. Our findings that chronic lead exposure blocked LTP but not NMDA receptor activity are similar to those of acutely applied 10-100 IxM Pb 2+ reported by Hori et al., suggesting that Pb 2+ concentrations of our slices may have been within a reasonable range of their actual measured Pb 2+ concentrations (2-7 txM Pb 2+) [33]. However, a clear concern is that the

70

C.A. Groc, er, G.D. F ~ e / Brain Research 734 (1996)61-71

concentration of Pb 2+ in h i p p o c a m p a l slices m i g h t h a v e b e e n d i m i n i s h e d due to leaching during incubation and e l e c t r o p h y s i o l o g i c a l recording. Others h a v e s h o w n that ~gF-nuclear m a g n e t i c r e s o n a n c e - m e a s u r e d free Pb 2+ in cerebral cortical slices is very small c o m p a r e d to the total brain Pb 2+ m e a s u r e d by atomic absorption spectrophotometry, c o n c l u d i n g that either a v e r y small portion of the w h o l e brain Pb 2+ is free Pb 2+, or that free Pb 2+ is extruded f r o m the extracellular fluids to the perfusion solution [57]. But species of Pb 2+ that are b o u n d to cellular c o m p o n e n t s m a y h a v e direct neurotoxic actions a n d / o r m a y be c h a n g e d to free Pb 2+ [43]. If b o u n d and free Pb 2+ are c o m p l e t e l y leached f r o m our h i p p o c a m p a l slices prior to e l e c t r o p h y s i o l o g i c a l recording, then the o b s e r v e d n e u r o t o x i c effects o f chronic lead exposure m a y be due to neuroadaptation. In s u m m a r y , the procedures used in this study effectively resulted in m o d e r a t e blood lead concentrations in l e a d - e x p o s e d adult rats that w e r e significantly higher than those in control rats. A l t h o u g h , e v o k e d field E P S P s after ' w e a k ' tetanus were potentiated in both the short- and l o n g - t e r m in slices f r o m both control and l e a d - e x p o s e d rats, S T P was e n h a n c e d in l e a d - e x p o s e d slices. S T P was reduced and L T P was o c c l u d e d f o l l o w i n g ' s t r o n g ' tetanus in l e a d - e x p o s e d slices. N e i t h e r chronic lead exposure or acute ethanol treatment had any o b s e r v a b l e effect on ' w e a k ' - t e t a n u s - i n d u c e d synaptic plasticity, but w h e n c o m bined b l o c k e d LTP, suggesting additivity. Furthermore, both chronic lead e x p o s u r e and acute ethanol b l o c k of L T P w e r e tetanus dependent, adding to the k n o w n similarities o f these neurotoxicants. Our findings o f n e u r o t o x i c a n t additivity suggest the c o n s e q u e n c e s of ' m e d i u m ' b l o o d lead levels m a y h a v e m o r e of an impact on C N S functions than are e x p e c t e d w h e n w e restrict ourselves to concerns o f these agents in isolation.

Acknowledgements This p r o j e c t was s u p p o r t e d in part by grants 1 F 3 2 E S 0 5 6 3 9 f r o m N I E H S ( C . A . G . ) and by A A 0 6 3 2 2 f r o m N I A A A (G.D.F.). The authors wish to thank Dr. W i l l i a m H. Griffith for a careful perusal of the manuscript, and Dr. Gerald Bratton and G i n g e r Griffin for analysis of b l o o d lead concentrations.

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