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.

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.

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.

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.

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.

The peripheral benzodiazepine receptor is a sensitive indicator of domoic acid neurotoxicity.

To evaluate the utility of the peripheral benzodiazepine receptor (PBR) as a biomarker of neurotoxicity, we measured receptor levels after sub-seizure doses of domoic acid (0-3.0 mg/kg) in rats using [3H]PK-11195 autoradiography. PBR expression in limbic structures was significantly increased 5 days, but not 24 or 48 h after injection of 3.0 mg/kg domoic acid. The largest increase in [3H]PK-11195 binding (> 500% above control) was found in the CA3 subfield of the hippocampus. Other limbic structures including the CA1 hippocampal subfield, subiculum, dentate gyrus and amygdala also showed significant increases in PBR expression, as did the striatum and substantia nigra pars reticulata. Smaller but significant increases were also observed 5 days after injection of 1.5 mg/kg, but not in animals treated with 0.75 mg/kg domoic acid. No pathology was observed after routine histological staining of brain tissue. Spatial learning and memory, a process thought to be associated with the hippocampus, was assessed in the Morris water maze. Groups treated with 1.5 and 3.0 mg/kg, but not 0.75 mg/kg domoic acid were significantly impaired in water maze performance. These findings suggest that the PBR could provide a sensitive and specific biomarker of neurotoxicity.

Enhanced expression of peripheral benzodiazepine receptors in trimethyltin-exposed rat brain: a biomarker of neurotoxicity.

We have used the binding of the selective, high affinity, isoquinoline carboxamide, [3H]-PK11195, to measure the levels of Peripheral Benzodiazpine Receptors (PBR) in the brain of rats exposed to the well characterized neurotoxicant trimethyltin (TMT). The results demonstrate that autoradiograms of saggital sections of rats injected with a 8 mg/kg TMT dose, express a high level of [3H]-PK11195 binding in brain regions known to be damaged by TMT. The highest level of [3H]-PK11195 binding in the TMT-exposed rats occurred in the CA3/CA4 subfield of the hippocampus, followed by the primary olfactory cortex, the posteriomedial cortical amygdaloid nucleus, subiculum, and entorhinal cortex. These findings are consistent with the neuropathology of TMT in rats. The increase in [3H]-PK11195 binding in the brain of TMT-exposed rats was significant at 7 days after injection and remained elevated up to 42 days after exposure, the last time point measured in the study. This pattern is very similar to that observed for levels of the astrocyte intermediate filament protein, GFAP. The enhanced binding of [3H]-PK11195 in TMT-exposed rats was the result of a significant increase (p < 0.005) in the number of PBR with no change in affinity. The Bmax for [3H]-PK11195 binding in hippocampi from TMT-treated rats at 4 weeks post-injection was 606 +/- 25 (n = 4) fmoles/mg protein and 329 +/- 41 (n = 4) fmoles/mg protein for control. These findings suggest that the quantitative assessment of [3H]-PK11195 binding to PBR in the brain could represent a potential biomarker for assessing chemical-induced neurotoxicity. Since this ligand has been labeled with single photon (123I) or positron emitting (11C, 18F) radioisotopes, it can potentially be used with non-invasive imaging techniques such as Single Photon Emission Tomography (SPECT) or Positron Emission Tomography (PET) for human studies.