Elsevier

Neurotoxicology and Teratology

Volume 24, Issue 6, November–December 2002, Pages 805-811
Neurotoxicology and Teratology

Brief communication
Increased high-affinity nicotinic receptor-binding in rats exposed to lead during development

https://doi.org/10.1016/S0892-0362(02)00314-8Get rights and content

Abstract

Receptor autoradiography and membrane radioligand-binding assays were used to determine the expression of nicotinic cholinergic receptors in the brains of weanling rats exposed to low-levels of lead (Pb) during development. Nicotinic receptors were identified with the frog toxin epibatidine (EB) that binds with high affinity to a variety of receptors containing α and β subunits. Rat pups were exposed to Pb from their mothers given 750-ppm Pb in the diet beginning on gestational day 0 through postnatal day (PN) 21. Blood Pb levels ranged from 36.5 to 46.5 μg/dl in the PN21 pups, and this exposure did not alter their body weight when compared to control rats. Several brain regions identified by autoradiographic studies as having significant binding of EB were dissected from control and Pb-treated pups and used in saturation-binding experiments with membrane preparations to determine the affinity constant (Kd) and maximal-binding capacity (Bmax) of [3H]EB. Results indicate that the Bmax of [3H]EB was increased in several brain regions in Pb-treated rat pups, without a significant effect on Kd estimates. [3H]EB-binding to membranes from untreated rats was not affected by in vitro exposure to 20-μM Pb, indicating that the effect of Pb on [3H]EB-binding in vivo was not likely due to direct influence of free Pb remaining in the tissue at the time of assay. The data therefore suggest that expression of nicotinic receptors that bind [3H]EB were increased by developmental exposure to Pb. Several possible mechanisms for these effects and the potential toxicological significance are discussed.

Introduction

Inorganic lead (Pb) is pervasive in the environment and continues to be a major public health concern, especially because of its demonstrated effects on cognition in children [26], [39]. Pb targets many sites within the developing nervous system. One such target appears to be voltage- and neurotransmitter-gated ion channels on neurons. For example, Pb is known to inhibit N-methyl-d-aspartate (NMDA) glutamatergic receptor function in vitro [14], [18], and it can alter the expression of these receptors in developing rat brain [15]. Since NMDA receptors are important for the induction of long-term potentiation (LTP), a synaptic model of learning and memory [22], it has been proposed that Pb-induced impairment of NMDA receptor function and expression may represent a molecular mechanism that underlies some of the effects of Pb on cognitive function (see Ref. [27] for review). Another type of ion channel that is important in learning and memory processes and LTP is the nicotinic cholinergic receptor [16]. These receptor-ion channel complexes are found at the neuromuscular junction, and throughout the central and peripheral nervous systems.

The structural and functional heterogeneity of central nicotinic receptors has led to the identification of several subtypes depending on various combinations of α and β subunits that make up the pentameric channel complex [2]. There are 16 individual nicotinic receptor subunit genes that have been identified with molecular cloning techniques in vertebrates [20]. Although many nicotinic receptor subtypes exist in mammalian brain, two of the most abundant and well-characterized subtypes are the heteromeric α4β2 subtypes and homomeric α7-bearing receptors (see [4], [20] for review; Ref. [36]). The α4β2 receptors have a high affinity for acetylcholine (ACh) and ACh agonists, and low affinity for α-bungarotoxin (BTX). These α4β2 receptors can be identified with cytisine [11]. The second population is the α7-bearing receptors that have a high affinity for BTX and have a low affinity for ACh agonists [4], [36]. A population of neuronal nicotinic receptors can be identified with very high-affinity binding of the frog toxin epibatidine (EB) [8], [31]. Physiological and pharmacological characterization of these receptors suggests that EB binds to those receptors labeled with cytisine, with some overlap with other non-α7-bearing receptors such as the α2β3 subtype [31], [38].

Several laboratories have demonstrated that neuronal nicotinic responses are exquisitely sensitive to Pb in vitro [13], [25], [28], [42]. For example, in N1E-115 neuroblastoma cells, 1-nM to 3-μM Pb reduced ACh-induced ion conductances by 26–90% [28]. The mechanism of the inhibitory effect of Pb has been characterized in vitro in hippocampal neurons as noncompetitive, voltage-independent and primarily acting on the fast-desensitizing nicotinic current associated with α7 subunit-bearing receptors [13]. The α7-bearing subtype is believed to be a presynaptic and postsynaptic receptor that is extremely permeable to Ca2+, and may serve to modulate synaptic plasticity and development [33]. The Ca2+ permeability, involvement in plasticity and sensitivity to Pb of the α7 receptor is strikingly similar to that of the NMDA receptor. However, it has become clear that non-α7-bearing receptors are very sensitive to Pb in vitro as well [29], [30], and there are differences in sensitivity to Pb among receptors expressing the α3, α4, β2 and β4 subunits [42]. It is not known whether the inhibitory in vitro effects of Pb on nicotinic receptors occur in vivo, nor is it known if Pb alters the expression or pharmacology of nicotinic receptors in developing brain. The objective of the present study is to test the hypothesis that developmental exposure to Pb alters the expression of neuronal nicotinic receptors that bind with high affinity to EB in young rats. It is hoped that these data will provide some insight as to whether the effects of Pb on nicotinic receptors in vitro are relevant to the effects of Pb in vivo. EB was used in autoradiographic and membrane-binding studies to determine if binding was different between control and Pb-exposed rats. Pregnant rats were exposed to Pb in their diet so that their pups were exposed to Pb in utero and during lactation.

Section snippets

Animals and Pb exposure

Animal protocol was approved by the Animal Care and Use Committee at Johns Hopkins University. Adult male (200–250 g) and female (175–200 g) Long–Evans rats were housed individually in plastic cages at 22±2 °C under a 12L:12D photoperiod. Female rats were assigned to a diet containing 0- or 750-ppm Pb acetate, and food and water intake was allowed ad libitum. Pb acetate was incorporated into the diet (Custom Agway 1000 with Pb acetate and Ca2+ propionate) by the manufacturer (ICN Nutritional

Pb exposure and general toxicity

Exposure of the pups to Pb in utero and during lactation did not result in overt toxicity as judged by signs of intoxication (e.g., altered gait, tremors and hyperactivity). Nor did the exposures cause a significant difference in body weight between treatment groups at PN3 or PN21 (Table 1). Maternal toxicity did not appear to occur. The dams did not exhibit signs of intoxication, and the body weight of Pb-exposed dams was not different than control dams at the time when their pups were weaned.

Discussion

The general pattern of [125I]BTX- and [125I]EB-binding in autoradiographic studies indicated that these two ligands bound to different populations of nicotinic receptors that are regionally segregated in the brains of newly weaned rats (Fig. 1). The data suggest that these two populations may be expressed differentially within specific brain regions, where they may serve different functional roles. This functional diversity is suggested by evidence that electrophysiological responses to

Acknowledgements

We would like to thank K. Rogers and A. Taylor for the technical support.

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