Cognitive function and cholinergic neurochemistry in weanling rats exposed to chlorpyrifos.

Previous studies have shown that moderate to high levels of chlorpyrifos (CPF) alter cognitive function in adult and immature rats. In the present study, we tested the hypothesis that lower-level exposure to CPF before or immediately after weaning causes deficits in cognitive function. A total of 78 Long-Evans rats were injected subcutaneously with 0, 0.3 or 7.0 mg/kg CPF every 4 days before or after weaning and were tested with the Morris swim task from postnatal day 24 through 28. Exposure to CPF before weaning did not cause signs of overt cholinergic intoxication or impaired growth nor did the exposures cause significant inhibition of regional brain cholinesterase (ChE) activity or reduction in muscarinic receptors 24 h after the last injection. However, spatial learning was impaired after 5 days of training in the group of weanling rats administered 7.0 mg/kg CPF. Rats administered 0.3 or 7.0 mg/kg CPF after weaning were also impaired in the task, without significant changes in brain ChE activity. These data indicate that low-level exposure to CPF caused deficits in cognitive function in weanling rats, and these effects did not appear to be mediated by the inhibition of brain ChE. It is suggested that the alteration of cognitive function in juvenile rats is an important functional correlate of the cellular and molecular effects of CPF in the immature brain. The mechanisms for CPF-induced cognitive dysfunction are unknown.

In vitro and in vivo effects of chlorpyrifos on glutathione peroxidase and catalase in developing rat brain.

Preliminary findings of a study on the role of oxidative stress in the developmental neurotoxicity of chlorpyrifos (CPF) indicates that in vitro exposure to 1-100 microM CPF or 1-100 nM CPF-oxon had no effect on the activity of glutathione peroxidase (GSHpx) in brain homogenates from postnatal day (PN) 21 rats, or on the activity of purified GSHpx. A single high-dose acute injection of 45 mg/kg CPF to PN19 rats also did not significantly alter GSHpx activity at PN21, in spite of extensive (72%) brain acetylcholinesterase (AChE) inhibition. However, catalase activity was significantly reduced by 28%. PN21 pups exposed maternally to a lower effective dose of CPF throughout development (dams injected with 50 mg/kg every 3 days) also had normal GSHpx activity, but a 30% increase in H2O2-independent NADPH consumption. Brain catalase activity in these rats was significantly increased by 24%. These preliminary data suggest that specific GSHpx activity is not altered by in vitro or in vivo exposures to CPF-oxon or CPF, but catalase and an unknown H2O2-independent NADPH-consuming factor were affected differentially depending on the type and timing of exposure.

Additive inhibitory action of chlorpyrifos and polycyclic aromatic hydrocarbons on acetylcholinesterase activity in vitro.

This study tested the hypothesis that the inhibition of acetylcholinesterase is greater when the insecticide chlorpyrifos (CPF) is in the presence of several polycyclic aromatic hydrocarbons (PAHs) found in house dust. CPF-oxon (CPFO) inhibition curves of purified AChE (electric eel) were generated in the presence or absence of different concentrations of the PAHs pyrene, benzo(a)pyrene, anthracene, and fluoranthene. Without CPF-oxon, all four PAHs themselves inhibited AChE activity with IC50 values in the range 8.2-17 microM. The IC50 for benzo(a)pyrene with human recombinant AChE was 1.5 microM. When AChE was incubated with CPF-oxon together with the PAHs, the inhibitory effect on AChE was additive. This was exemplified by large (60-80%) and significant (P<0.01) inhibition in AChE activity by the PAHs when combined with nanomolar concentrations of CPF-oxon. Kinetic studies indicated that benzo(a)pyrene inhibited AChE in a noncompetitive manner, and the reduction in maximal velocity (Vmax) by benzo(a)pyrene and CPFO together was the sum of the inhibitory effect of the two inhibitors alone, further supporting an additive effect. These data suggest that some PAHs have anticholinesterase activity, and contribute in an additive manner to the inhibitory effect of CPFO on AChE in vitro. Further research is needed to determine the toxicological relevance of these findings.

Intrahippocampal administration of lead (Pb) impairs performance of rats in the Morris water maze.

We examined spatial learning in the Morris water maze after daily acute bilateral micro-injection of 13.9 ng sodium acetate (NaAc) or 37.9 ng lead acetate (PbAc) in 1 microliter volumes into the dorsal hippocampus of normal adult rats. After six days of injections and water maze training, rats injected with NaAc were able to find a hidden platform in 8.3 s, and those injected with PbAc were significantly slower (15.2 s; p < 0.02). In a second experiment, rats were trained to find a hidden platform before injections began and then tested in order to determine if intrahippocampal injections of Pb affected the recall of a previously learned task. The escape latency on the first day after injections began was increased slightly when compared to the last day of training before injections, however the NaAc and PbAc groups were not significantly different over three days of injections. Both treatment groups performed as well as they did before injections began by the second day of injections. These results suggest that the direct injection of Pb into the hippocampus impairs the acquisition but not the recall of the spatial learning task in the Morris water maze.

Age-dependent effects of developmental lead exposure on performance in the Morris water maze.

The neurobehavioral toxicity of developmental exposure to lead (Pb) was investigated by conducting tests of spatial learning in the Morris water maze. Female Long-Evans rats were exposed to 0 or 250 ppm Pb acetate in the diet beginning 10 days prior to breeding and continued throughout gestation and lactation. Pups were weaned onto the same diets as the dams at postnatal day 20 (PN20). Increased levels of Pb were detected in the hippocampus of the 250 ppm Pb acetate group relative to controls. The highest concentration of Pb measured in the hippocampus was at PN21 with decreasing levels at older ages. In the Morris Water Maze, a statistically significant (p < 0.03; female rats) or near significant (p < 0.07; male rats) increase in the time required to find the hidden platform (escape latency) was observed when Pb-treated rats were tested in a reference memory paradigm. This effect was only observed when rats were tested at PN21 and not at older ages. No significant effects of developmental Pb exposure were measured when rats were tested in a working memory paradigm of the Morris water maze at any age. These initial studies indicate an impairment of performance in the swim task in PN21 rats exposed to Pb during development. The age-dependent effect of Pb in this learning paradigm is consistent with previous studies in experimental animals and with the observation that children are more susceptible to Pb-induced cognitive deficits than adults. The Morris water maze may be useful for studying the effects of Pb on learning and memory, and their neurochemical basis.

Biochemical evidence of an interaction of lead at the zinc allosteric sites of the NMDA receptor complex: effects of neuronal development.

Electrophysiological and biochemical studies have shown that Pb2+ inhibits the activation of the N-Methyl-D-Aspartate (NMDA) receptor complex, an excitatory amino acid receptor subtype known to play an important role in neuronal development and cognitive function. In the present study we have provided biochemical evidence that Pb2+ may inhibit NMDA receptor function via an interaction at the Zn2+ allosteric sites. Binding of [3H]-MK-801 to the NMDA receptor ion channel was used as a biochemical indicator of receptor function. The initial experiments indicated that Zn2+ inhibited [3H]-MK-801 binding via high and low affinity sites in PN14 membranes with potencies of 0.77 +/- 0.05 and 57.4 +/- 6.9 microM, respectively. Similar effects were present in neuronal membranes from adult rats, but Zn2+ was significantly less potent (IC50: 153 +/- 21; p < 0.025) in inhibiting [3H]-MK-801 via the low affinity site than in PN14 membranes. The addition of Pb2+ to Zn2+/[3H]-MK-801 displacement curves significantly altered the inhibition of [3H]-MK-801 binding by Zn2+. IN PN14 membranes, Pb2+ increased the potency of Zn2+ inhibition at the high affinity site and decreased the Zn2+ potency at the low affinity site suggestive of a competitive interaction. In adult membranes, Pb2+ did not alter the potency of Zn2+ inhibition of [3H]-MK-801 binding at either site in concentrations up to 40 microM. These findings suggest that the Zn2+ allosteric sites are more sensitive to Pb2+ effects in immature brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental lead exposure alters N-methyl-D-aspartate and muscarinic cholinergic receptors in the rat hippocampus: an autoradiographic study.

We have used quantitative autoradiography of the N-Methyl-D-Aspartate (NMDA) receptor non-competitive antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohep-ten-5,10-imine maleate in the tritiated form ([3H]-MK-801) and the muscarinic cholinergic receptor antagonist [3H]-N-Methylscopolamine ([3H]-NMS) to determine the effects of developmental exposure to lead (Pb) on NMDA and muscarinic receptors in the rat hippocampus. Exposure to Pb during development resulted in age-specific changes in the level of binding of both ligands to their respective receptors. Pb exposure caused significant increases (19-49%) in [3H]-MK-801 binding throughout the hippocampus and entorhinal cortex (ECTX) of postnatal day (PN) 14 rats relative to controls. Small but significant region-specific reductions (14-18%) and increases (12-15%) in [3H]-MK-801 binding were measured in PN28 rats. No significant differences in [3H]-MK-801 binding were observed among any of the groups at PN56. Unlike [3H]-MK-801 binding, the most significant effects of Pb on [3H]-NMS binding were reductions in binding of 10-20% observed in the hippocampus and ECTX of Pb-exposed rats at PN14. Additionally, Pb in vitro had no effect on the binding of [3H]-NMS to brain sections (10 microM Pb acetate) or neuronal membrane preparations (0.1 nM to 0.1 mM Pb acetate) from normal neonatal or adult rats. These findings indicate that there may be a critical developmental window during which Pb effects are most pronounced, and the magnitude and direction of these changes is dependent upon the age of the animal and the neurotransmitter system being examined. It is suggested that NMDA and muscarinic receptors play an important role in the developmental toxicity of Pb.

Differential regulation of muscarinic receptor subtypes in rat brain regions by repeated injections of parathion.

Repeated injections with increasing moderate doses of parathion into adult male rats for 21 days resulted in 84-90% inhibition of acetylcholinesterase in the brain without overt signs of toxicity. Muscarinic acetylcholine receptor (mAChR) affinities for ligands were unaffected, but there was significant down-regulation of the m4 receptor subtype gene product, m1 mRNA and m3 mRNA in the frontal cortex as well as the m4 subtype and m4 mRNA in the striatum. However, in the hippocampus, there were no significant reductions in either the m1 receptor subtype nor its mRNA. The data suggest that the receptor subtype down-regulations in the cortex and striatum are due to reductions in mRNA expression. Since the degrees of inhibition of acetylcholinesterase were similar in the 3 brain regions, it is suggested that the in situ concentrations of paraoxon were also similar. Accordingly, the absence of down-regulation of the m1 receptor in the hippocampus is not due to a lower concentration of paraoxon than in the cortex or striatum. It is possible that injections of higher parathion doses would produce down-regulation of mAChRs in the hippocampus, and that the hippocampus may have differences in the feed-back mechanisms for receptor regulation from those in the frontal cortex and the striatum.

Neurochemical aspects of hippocampal and cortical Pb2+ neurotoxicity.

The amino acid glutamate is the neurotransmitter used by most excitatory synapses in the mammalian brain. Glutamatergic synapses in the hippocampus and cerebral cortex play an important role in synaptic plasticity. In the developing brain, the interaction of neurotoxins with presynaptic and/or postsynaptic sites on glutamatergic neurons could alter synaptic plasticity. Recent studies have shown that chronic lead (Pb2+) exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with the N-Methyl-D-Aspartate (NMDA) glutamate receptor subtype. The NMDA receptor-ion channel complex regulates calcium influx and is involved in the initiation of changes in synaptic plasticity. In vitro and in vivo neurochemical studies have found that Pb2+ has a marked inhibitory effect on the activation of the NMDA receptor-ion channel complex. Evidence indicates that the inhibitory effect of Pb2+ on the NMDA receptor complex may be mediated by its interaction with a zinc regulatory site on the receptor complex. The ability of Pb2+ to inhibit NMDA receptor-ion channel function was shown to be age-dependent and brain region-specific. The age-dependent effects of Pb2+ on the NMDA receptor complex may help explain the selective toxicity of this heavy metal in the developing brain.

Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion.

The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs after dietary exposure for 14 d to sublethal concentrations of parathion in a field rodent species, and that significant though incomplete recovery in AChE and mAChRs occurs in 7 d following termination of exposure.

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells.

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of vocally expressed emotions by mentally retarded and nonretarded individuals.

Nonretarded, TMR, and EMR individuals matched on MA attempted to identify the emotional states (happy, angry, sad, or fearful) of tape-recorded voices speaking in an unfamiliar language by pointing to one of four stick-figure depictions of the emotions. Although analyses indicated that TMR subjects were less accurate overall than were EMR and nonretarded subjects, all three groups were able to identify each of the emotions well above chance expectation. Of the four emotions, fear was judged least accurately by each group. Correlational analyses revealed that accuracy in decoding emotional passages was positively related to IQ and CA. Generally, the experimental and correlational findings indicated few important differences in patterns of emotion identification among the groups and were consistent with results obtained in other studies of nonverbal decoding by nonretarded individuals.