Early-life lead exposure and neurodevelopmental disorders.

Lead (Pb2+) exposure is a global public health problem of major proportion with an alarming number of children with blood Pb2+ levels > 10 >g/dL, twice the current CDC reference level for Pb2+ exposure. Mounting evidence from population-based studies suggests an association between chronic early life Pb2+ exposure (CELLE) and psychiatric disorders, specifically schizophrenia (SZ). Preclinical studies suggest a common mechanism in the pathophysiology of CELLE and SZ, NMDA receptor hypofunction. Here we describe human and experimental animal studies providing the evidence for such an association. Further, recent preclinical studies indicate that Pb2+-induced changes in neurotransmitter receptors that mediate the action(s) of drugs of abuse are increased in brain regions associated with addiction circuits in adolescence, a period of increased susceptibility to drug use and abuse and expression of psychiatric disease in humans. In summary, the relationship between the global burden of childhood Pb2+ exposure and the latent onset of psychiatric disorders and predisposition to drug use requires further investigations in human populations.

Effects of chronic manganese exposure on attention and working memory in non-human primates.

Manganese (Mn) is essential for a variety of physiological processes, but at elevated levels, can be neurotoxic. While cognitive dysfunction has been recently appreciated to occur as a result of chronic Mn exposures, it is still unclear as to which cognitive domains are most susceptible to disruption by Mn exposure. We previously described early appearing Mn-induced changes in performance on a paired associate learning task in monkeys chronically exposed to Mn and suggested that performance of this task might be a sensitive tool for detecting cognitive dysfunction resulting from Mn exposure. As chronic Mn exposure has been suggested to be associated with attention, working memory and executive function deficits, the present study was conducted to assess the extent to which detrimental effects of chronic Mn exposure could be detected using tasks specifically designed to preferentially assess attention, working memory, and executive function. Six cynomolgus monkeys received Mn exposure over an approximate 12 month period and three served as control animals. All animals were trained to perform a self-ordered spatial search (SOSS) task and a five choice serial reaction time (5-CSRT) task. Deficits in performance of the SOSS task began to appear by the fourth month of Mn exposure but only became consistently significantly impaired beginning at the ninth month of Mn exposure. Performance on the 5-CSRT became significantly affected by the third month of Mn exposure. These data suggest that in addition to the paired associate learning task, cognitive processing speed (as measured by the 5-CSRT) may be a sensitive measure of Mn toxicity and that brain circuits involved in performance of the SOSS task may be somewhat less sensitive to disruption by chronic Mn exposure.

Early-life lead exposure recapitulates the selective loss of parvalbumin-positive GABAergic interneurons and subcortical dopamine system hyperactivity present in schizophrenia.

Environmental factors have been associated with psychiatric disorders and recent epidemiological studies suggest an association between prenatal lead (Pb(2+)) exposure and schizophrenia (SZ). Pb(2+) is a potent antagonist of the N-methyl-D-aspartate receptor (NMDAR) and converging evidence indicates that NMDAR hypofunction has a key role in the pathophysiology of SZ. The glutamatergic hypothesis of SZ posits that NMDAR hypofunction results in the loss of parvalbumin (PV)-positive GABAergic interneurons (PVGI) in the brain. Loss of PVGI inhibitory control to pyramidal cells alters the excitatory drive to midbrain dopamine neurons increasing subcortical dopaminergic activity. We hypothesized that if Pb(2+) exposure in early life is an environmental risk factor for SZ, it should recapitulate the loss of PVGI and reproduce subcortical dopaminergic hyperactivity. We report that on postnatal day 50 (PN50), adolescence rats chronically exposed to Pb(2+) from gestation through adolescence exhibit loss of PVGI in SZ-relevant brain regions. PV and glutamic acid decarboxylase 67 kDa (GAD67) protein were significantly decreased in Pb(2+) exposed rats with no apparent change in calretinin or calbindin protein levels suggesting a selective effect on the PV phenotype of GABAergic interneurons. We also show that Pb(2+) animals exhibit a heightened locomotor response to cocaine and express significantly higher levels of dopamine metabolites and D2-dopamine receptors relative to controls indicative of subcortical dopaminergic hyperactivity. Our results show that developmental Pb(2+) exposure reproduces specific neuropathology and functional dopamine system changes present in SZ. We propose that exposure to environmental toxins that produce NMDAR hypofunction during critical periods of brain development may contribute significantly to the etiology of mental disorders.

Chronic manganese exposure impairs visuospatial associative learning in non-human primates.

Manganese (Mn) is an essential trace metal nutrient, however, excess Mn can be neurotoxic. The degree to which chronic environmental or occupational exposures to Mn in adults cause neuropsychological dysfunction is of considerable interest. Descriptions of neuropsychological dysfunction following chronic Mn exposure have been somewhat inconsistent though, likely owing to different measures of exposure in different populations, complicated by factors of mixed exposures and differences in neuropsychological tests administered. We previously described up-regulation of the mRNA expression for amyloid-beta (A-beta) precursor-like protein 1 (APLP1) and the presence of A-beta diffuse plaques in frontal cortex of Mn-exposed monkeys. The present study examined Mn-induced changes in performance on a paired associate learning (PAL) task that has been suggested as a marker for preclinical Alzheimer's disease. Aspects of performance of this task were affected early following initiation of Mn exposure. Thus, PAL performance may be a sensitive and valuable tool for the early, preclinical detection of incipient dementia and it may also be a sensitive tool for detecting cognitive dysfunction from Mn exposure. The current cognitive data, combined with our previous findings, suggest that frontal cortex may be a particularly sensitive target for the effects of Mn on cognition and that chronic Mn exposure may initiate or accelerate a process that could lead to or predispose to Alzheimer's like pathology and cognitive dysfunction.

Astrocytes release D-serine by a large vesicle.

Long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus depends on the activation of N-methyl-D-aspartate receptors (NMDARs), which can be regulated by Ca²âº-dependent release of D-serine from astrocytes. The detailed mechanism underlying astrocytic d-serine release is still unknown. In hippocampal slices prepared from Sprague-Dawley rats, we found that clamping astrocytic [Ca²âº] at 100-150 nM or puffing artificial cerebrospinal fluid (ACSF) into the extracellular space (weak mechanical stimulation) enhanced the synaptic activation of NMDARs. The enhancement was blocked by the NMDAR glycine site antagonist 5,7-dichlorokynurenic acid, glycine saturation, and infusion of astrocytes with D-amino acid oxidase and the serine racemase inhibitor L-erythro-3-hydroxyaspartate, suggesting the involvement of astrocytic D-serine release. Intracellular 100-150 nM [Ca²âº] or puffing ACSF stimulated astrocytes to generate D-serine-containing large vesicles (1-3 µm), exocytotic fusion of which released D-serine. The formation of astrocytic large vesicles involved the intracellular fusion of small vesicles and/or other organelles. Spontaneous fusion of large vesicles occurred occasionally in astrocytes at rest, contributing to baseline D-serine levels, which increased the rising slope of NMDAR post-burst potentiation (PBP) without altering the PBP peak amplitude. Thus, under physiological conditions, astrocytic D-serine release by large vesicles facilitated weak theta-burst (TBS consisting of five bursts), but not strong TBS (TBS consisting of 10 bursts) stimulation-induced LTP.

Effects of chronic manganese exposure on working memory in non-human primates.

Human exposure to manganese (Mn) has been associated with a variety of cognitive deficits including learning and memory deficits. However, results from epidemiological studies have been inconsistent in describing the nature of such cognitive deficits. The present study was conducted to evaluate the effects of chronic Mn exposure on memory functioning in non-human primates and to correlate behavioral outcome with brain Mn levels in an attempt to explain outcome variability seen in prior studies. Cynomolgus macaque monkeys were trained to perform memory-related tasks (spatial working memory, non-spatial working memory, reference memory) and exposed to manganese sulfate (15-20 mg/kg/week) over an exposure period lasting 227.5+/-17.3 days. Blood manganese levels were in the upper range of levels reported for human environmental, medical or occupational exposures. By the end of the manganese exposure period, animals developed mild deficits in spatial working memory, more significant deficits in non-spatial working memory and no deficits in reference memory. Linear regression analyses showed that for most brain regions sampled, there was a significant inverse relationship between working memory task performance and brain Mn concentration. These results suggest that chronic exposure to levels of manganese achieved in this study may have detrimental effects on working memory and that Mn levels achieved in several brain regions are inversely related to working memory performance.

Environmental lead exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats.

Exposure to environmentally relevant levels of lead (Pb(2+)) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats [Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC, Guilarte TR (2000) N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience 99:233-242; Guilarte TR, Toscano CD, McGlothan JL, Weaver SA (2003) Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Ann Neurol 53:50-56]. Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) [Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P-V, Abrous DN (2003) Spatial memory performance of aged rats in the water maze predicts level of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385-14390]. In this study, we examined whether continuous exposure to environmentally relevant levels of Pb(2+) during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb(2+)-exposed rats received bromodeoxyuridine (BrdU) injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were killed either 1 day or 4 weeks after the last injection. The total number of newborn cells in the DG of Pb(2+)-exposed rats was significantly decreased (13%; P<0.001) 1 day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb(2+)-exposed rats was significantly decreased by 22.7% (P<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb(2+) exposure on cellular fate. In Pb(2+)-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology. That is, the overall length-density of the DCX-positive apical dendrites in the outer portion of the DG molecular layer was significantly reduced up to 36% in the suprapyramidal blade only. We also found that the area of Timm's-positive staining representative of the mossy fibers terminal fields in the CA3 stratum oriens (SO) was reduced by 26% in Pb(2+)-exposed rats. These findings demonstrate that exposure to environmentally relevant levels of Pb(2+) during early life alters granule cell neurogenesis and morphology in the rat hippocampus. They provide a cellular and morphological basis for the deficits in synaptic plasticity and spatial learning documented in Pb(2+)-exposed animals.

Methamphetamine-induced deficits of brain monoaminergic neuronal markers: distal axotomy or neuronal plasticity.

We examined the effects of methamphetamine (METH) on monoaminergic (i.e. dopamine and serotonin) axonal markers and glial cell activation in the rat brain. Our findings indicate that the loss of dopamine transporters (DAT), serotonin transporters (5-HTT), vesicular monoamine transporter type-2 (VMAT-2) and glial cell activation induced by METH in the striatum and in the central gray are consistent with a degenerative process. Our novel finding of METH effects on monoaminergic neurons in the central gray may have important implications on METH-induced hyperthermia. In other brain regions examined, DAT and 5-HTT deficits after METH administration were present in the absence of lasting changes in VMAT-2 levels or glial cell activation. Brain regions exhibiting protracted deficits in DAT and/or 5-HTT and VMAT-2 levels also expressed increased levels of [(3)H]-R-PK11195 binding to peripheral benzodiazepine receptors, a quantitative marker of glial cell activation. Immunohistochemical assessment of microglia and astrocytes confirmed the PBR results. Microglia activation was more pronounced than astrocytosis in affected regions in most METH-exposed brains with the exception of a small number of rats that were most severely affected by METH based on loss of body weight. In these rats, both microglia and astrocytes were highly activated and expressed a distinct regional pattern suggestive of widespread brain injury. The reason for the pattern of glial cell activation in this group of rats is not currently known but it may be associated with METH-induced hyperthermia. In summary, our findings suggest two neurotoxic endpoints in the brain of METH-exposed animals. Brain regions exhibiting DAT and 5-HTT deficits that co-localize with decreased VMAT-2 levels and glial cell activation may represent monoaminergic terminal degeneration. However, the DAT and 5-HTT deficits in brain regions lacking a deficit in VMAT-2 and glial cell activation may reflect drug-induced modulation of these plasma membrane proteins.

Low level Pb(2+) exposure affects hippocampal protein kinase C gamma gene and protein expression in rats.

We examined the effect of chronic exposure to lead (Pb(2+)) on protein kinase C (PKC) in 50-day-old rat hippocampus. Cytosolic and membrane fractions of hippocampus from Pb(2+)-exposed rats showed reduced expression of PKC gamma protein. In contrast, a significant elevation of PKC gamma mRNA was observed in pyramidal and dentate granule cell layers. Protein expression of alpha, beta I, beta II and epsilon isoenzymes were unchanged in Pb(2+)-exposed rats, as was [(3)H]phorbol 12,13 dibutyrate (PDBu) binding in tissue slices. Differences were not observed in Ca(2+)-dependent or -independent PKC activity, or in PKC-specific back-phosphorylation of hippocampal homogenates from Pb(2+)-exposed rats. Reduced subcellular levels of PKC gamma in Pb(2+)-exposed rats suggest that signal transduction in the hippocampus may be selectively altered and may be important in manifesting Pb(2+)-induced impairments of synaptic plasticity, learning and memory.

Is methamphetamine abuse a risk factor in parkinsonism?

Parkinsons disease (PD) is a neurodegenerative disorder with increased incidence in individuals beyond 50 years of age. The etiology of PD is currently not known, but it appears that environmental factors may play an important role. The molecular basis of PD is the nearly complete loss of the neurotransmitter dopamine (DA) in the basal ganglia (caudate/putamen). The decrease in dopamine levels is the result of degeneration of dopamine-containing neurons in the substantia nigra. This biochemical deficit in the nigrostriatal pathway leads to the emergence of motor impairments typical of PD. Methamphetamine (METH) is a psychostimulant drug with increasing use in certain segments of the population in the United States and worldwide. In experimental animal models and human studies, METH administration has been shown to decrease markers of dopaminergic neuron terminal integrity in the basal ganglia. A long-standing question has been whether the reductions in dopaminergic markers induced by METH constitute degenerative changes or reflect drug-induced modulation. Resolving this question is important because the irreversible loss of dopaminergic function may increase the likelihood of Parkinsonism with advancing age.

Molecular changes in glutamatergic synapses induced by Pb2+: association with deficits of LTP and spatial learning.

What are the molecular bases for the neurotoxicity that occurs after developmental exposure to low levels of Pb2+, and are these effects persistent and detrimental in adults? Our inability to understand specific mechanisms behind Pb2+ neurotoxicity has long been one of many problem areas of this preventable childhood disease. The sensitivity of the developing brain to Pb2+-induced neurotoxicity is an outcome of the many unique characteristics that comprise the developing central nervous system. The developing brain can be exposed to significant concentrations of Pb2+ during vulnerable periods of development such as synapse formation, gene and protein expression, and other diverse molecular changes associated with these processes. Recently, changes in NMDA receptor subunits were identified in animals that showed cognitive deficits induced by exposure to Pb2+. This molecular association is important because it provides new evidence in the characterization of developmental Pb2+ neurotoxicity that supports physiological findings of impairments in synaptic plasticity and behavior. This review updates information from molecular studies that can be directly associated with impairments of behavior and synaptic plasticity, and outlines the functional consequences of molecular differences in Pb2+-exposed animals that illuminate potential mechanisms of Pb2+-induced neurotoxicity.

N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning.

The present study demonstrates that impairments of spatial learning and hippocampal long-term potentiation in rats chronically exposed to lead are associated with changes in gene and protein expression of N-methyl-D-aspartate receptor subunits. Rats exposed to 750 and 1500 ppm lead acetate were found to exhibit deficits in acquisition of a water maze spatial learning task. Furthermore, lead-exposed rats show dose-dependent reductions in the maintenance of in vivo hippocampal long-term potentiation induced in entorhinal cortex-dentate gyrus synapses. We found an unexpected, but significant (P<0.05), correlation between spatial learning and long-term potentiation when control and lead-exposed rats were analysed as a single, combined population. Dentate gyrus NR1 subunit messenger RNA was reduced 18% and 28% by exposure to 750 and 1500 ppm lead acetate, respectively. NR2A subunit messenger RNA was reduced 18% but only in the dentate gyrus of rats exposed to 1500 ppm lead acetate. No significant changes in dentate NR2B messenger RNA expression were measured in either of the lead-exposed groups. NR1 subunit protein was reduced 24% and 58% in hippocampal homogenates from rats exposed to 750 and 1500 ppm lead acetate. In contrast, no changes in NR2A or NR2B subunit protein were observed in the same hippocampal homogenates. These data show that reductions of specific N-methyl-D-aspartate receptor subunits are associated with deficits of both hippocampal long-term potentiation and spatial learning, induced in rats by chronic exposure to environmentally relevant levels of lead. These findings strongly suggest that the effects of lead on N-methyl-D-aspartate receptors may be the mechanistic basis for lead-induced deficits in cognitive function.

Hippocampal expression of N-methyl-D-aspartate receptor (NMDAR1) subunit splice variant mRNA is altered by developmental exposure to Pb(2+).

N-methyl-D-aspartate receptors (NMDAR) play an important role in synaptic plasticity and brain development. We have previously shown that NR1-pan mRNA is significantly increased in the hippocampus of rats chronically exposed to low levels of lead (Pb(2+)) during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res., 790 (1998) 98-107]. It is not known whether this Pb(2+)-induced increase in NR1-pan mRNA is associated with changes in specific splice isoforms. To study this effect, we used in situ hybridization of oligonucleotides to probe for the NR1-a, NR1-b, NR1-1, NR1-2, and NR1-4 isoforms which are most abundantly expressed in the rat hippocampus. Developmental exposure to increasing levels of Pb(2+) resulted in significant increases in NR1-a mRNA throughout the pyramidal and granule cell layers of the rat hippocampus at postnatal day 14 (PN14). NR1-b mRNA was increased in the pyramidal cell layer of Pb(2+)-exposed rats at PN21. Splicing of the C-terminus cassettes was also regulated by developmental exposure to Pb(2+). NR1-2 mRNA was increased in CA4 pyramidal cells and in dentate granule cells of PN21 Pb(2+)-exposed rats. Notably, expression of NR1-4 mRNA in CA3 pyramidal cells was increased in Pb(2+)-exposed rats at PN14 and decreased at PN21. No significant Pb(2+) effect was measured for NR1-1 mRNA expression. These data indicate that alternative splicing of the NR1 gene shows selective anatomical and temporal regulation by Pb(2+) in the developing rat hippocampus. This study provides further support to the hypothesis that NMDARs are important targets for Pb(2+)-induced neurotoxicity.

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity.

The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.

Divalent cations modulate N-methyl-D-aspartate receptor function at the glycine site.

The modulation of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) by divalent cations was examined using (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten 5,10-imine maleate ([(3)H]MK-801) binding as a functional indicator of NMDAR function. Ca(2+) and Mg(2+) produce a biphasic effect on the binding of [(3)H]MK-801 to the NMDAR channel in extensively washed adult rat brain membranes. Concentrations of Ca(2+) and Mg(2+) between 1 and 600 microM potentiate binding, but higher concentrations inhibit binding. The potentiating effect of Ca(2+) and Mg(2+) on [(3)H]MK-801 binding is due to an increase in the maximal number of binding sites (B(max)) with no effect on binding affinity (K(d)). Ca(2+)- and Mg(2+)induced potentiation is the result of an apparent increase in the affinity of the NMDAR for glycine. The ontogeny of NMDAR potentiation by Ca(2+) and Mg(2+) was also investigated. The number of [(3)H]MK-801 binding sites associated with divalent cation potentiation are present at low levels shortly after birth, and increase to peak level at 17 days of age before declining to adult levels. The potency of Ca(2+) and Mg(2+) to stimulate [(3)H]MK-801 binding did not change as a function of age. Lead (Pb(2+)) and zinc (Zn(2+)), potent inhibitors of the NMDAR, antagonize NMDAR potentiation by Ca(2+) and Mg(2+). These findings indicate that divalent cations differentially regulate NMDAR function by modulation of the glycine site. The NMDAR glycine site may be important in the regulation of glutamatergic neurotransmission by physiologically and toxicologically relevant cations.

Regional and temporal expression of the peripheral benzodiazepine receptor in MPTP neurotoxicity.

We used the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to evaluate the sensitivity and specificity of the peripheral benzodiazepine receptor (PBR) as a biomarker of chemical-induced neurotoxicity. Receptor autoradiography of [3H]-PK11195, a PBR selective ligand, indicated dose-dependent increases throughout the nigrostriatal dopaminergic system as early as 24 h after MPTP administration (10-80 mg/kg), which persisted for at least 21 days. The binding of [3H]-PK11195 was increased as much as 98% in the corpus striatum and 114% in the substantia nigra, following MPTP exposure. The integrity of nigrostriatal dopaminergic terminals in the corpus striatum was assessed by measuring high affinity dopamine transporter (DAT) levels and dopamine content. DAT levels were measured by [3H]-WIN 35,428 autoradiography, and dopamine content decreased with increasing MPTP dose. Reductions of both indices of dopaminergic terminal integrity correlated with increased levels of [3H]-PK11195-binding in the striatum (r2 = 0.84 for DAT and 0.93 for dopamine content). Tyrosine hydroxylase (TH) immunohistochemistry demonstrated dose-dependent reductions of dopaminergic neurons in the substantia nigra pars compacta, with a 67% loss measured 7 days after treatment with 80 mg/kg MPTP. The loss of TH-positive neurons was correlated (r2 = 0.95) with increased levels of [3H]-PK11195 binding in the substantia nigra. These findings demonstrate that the PBR is both sensitive and specific for identifying brain regions involved in MPTP neurotoxicity.

NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development.

Chronic exposure to lead (Pb2+) produces deficits of learning and memory in children and spatial learning deficits in developing rats. The N-methyl-D-aspartate receptor (NMDAR) has been identified as a principal target for Pb2+-induced neurotoxicity. Age-dependent changes in NMDAR subunit gene expression were observed in hippocampi of rats chronically exposed to Pb2+ during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res. 790 (1998) 98-107]. These changes were present at blood Pb2+ levels ranging from 20-60 microg/dl. Littermates were used in the present study to determine whether the changes in gene expression were reflected in protein levels. NR1, NR2A, and NR2B subunit protein levels were measured in rat hippocampus and cortex at post-natal days (PND) 7, 14, 21, and 28 by Western blot and densitometric analysis. A treatment effect was apparent for NR2A subunit protein expression in the hippocampus (F1,28=10.224, p<0.01). NR2A subunit protein was reduced by 40%, 19%, and 27% from control levels in PND14, 21, and 28 Pb2+-exposed rats, respectively. Mean comparisons indicated that rats at PND14 exhibited the most significant reduction of NR2A (p<0.001). These data concur with our previous finding of reduced NR2A mRNA found in hippocampal pyramidal and granule cells of Pb2+-exposed rats. Pb2+ exposure during development had no effect on NR1 or NR2B subunit protein expression in the hippocampus at any age. No effect was observed on any subunit in the cortex at any age. The developmental profile of the NMDAR-2A subunit protein in the hippocampus is specifically changed by chronic exposure to Pb2+. These data suggest that composition of subunits comprising NMDAR may be altered in Pb2+-exposed rats.

Understanding the NIH review process: a brief guide to writing grant proposals in neurotoxicology.

During the past two years, the National Institutes of Health have made significant changes in the review process for investigator-initiated research grant applications in neurotoxicology. First, study sections that formerly dealt with toxicology and alcohol, respectively, have been merged. Neurotoxicology grant applications are now reviewed by ALTX-3, a study section in which the majority of members have expertise in the neuronal, biochemical or behavioral effects of alcohol, but usually not other neurotoxicants. Second, the NIH has instituted new review criteria, in which significance, approach, innovation, investigator expertise, and research environment must all be explicitly addressed by the reviews. In this article, past and present members of the ALTX-3 study section describe the NIH review process, with emphasis on how neurotoxicology applications are handled, and provide guidelines for preparing competitive applications.

Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure.

The N-methyl-D-aspartate (NMDA) receptor has shown to play an important role in the cognitive deficits associated with developmental lead (Pb) exposure. In this study, we examined the effects of low-level Pb exposure on NMDA receptor subunit gene expression in the developing rat brain. The pattern of NR1, NR2A, NR2B, and NR2C subunit mRNA in situ hybridization was consistent with previous studies. Brain levels of NR1 and NR2A mRNAs were lowest shortly after birth, increasing to reach peak levels by 14 or 21 days of age and subsequently decreasing at 28 days of age. NR2B mRNA levels were highest during early development and decreased as the animals aged. NR2C subunit mRNA was restricted to the cerebellum and a signal was not detectable until the second week of life. Lead exposure resulted in significant and opposite effects in NR1 and NR2A subunit mRNA expression with no changes in NR2B or NR2C subunit expression. The Pb-induced changes in NR1 and NR2A subunit mRNA were mainly present in the hippocampus. Hippocampal NR1 mRNA levels were significantly increased in the CA1 (15.3%) and CA4 (26.8%) pyramidal cells from 14-day-old Pb-exposed rats. At 21 days of age, only the NR1 mRNA at the CA4 subfield remained significantly elevated (10.3%). Lead exposure caused reductions of NR2A mRNA levels (11.9-19.3%) in the pyramidal and granule cell layers of the hippocampus at 14 and 21 days of age. NR1 mRNA levels were also significantly increased (14.0%) in the cerebellum of 28-day-old rats with no change in NR2A mRNA at any age. No significant changes in subunit mRNA levels were present in cortical or subcortical regions at any age. The Pb-induced changes in hippocampal NMDA receptor subunit mRNA expression measured in the present study may lead to modifications in receptor levels or subtypes and alter the development of defined neuronal connections which require NMDA receptor activation.

Developmental lead exposure causes spatial learning deficits in adult rats.

Groups of male rats exposed to lead (Pb) during different developmental periods were tested as adults in a water maze. A highly significant (P < 0.01) impairment in water maze performance was measured in rats exposed to Pb only during gestation and lactation (maternal exposure). At the time of testing (100-106 days old), blood and brain Pb concentrations were at control levels. Significant impairments (P < 0.05) were also present in rats continuously exposed to Pb from conception through adulthood. Post-weaning Pb exposure alone did not result in impaired performance despite significantly elevated blood and brain Pb levels at the time of testing. This study supports the hypothesis that a window of vulnerability to Pb neurotoxicity exists in the developing brain and that Pb exposure can result in long-term cognitive deficits.