Distribution Of Lead In Rat Hippocampus Results In Impaired Dendritic Spine Plasticity

Lead is a widespread environmental heavy metal toxicant, which can cause children’s learning and memory decline and cognitive dysfunction. Recent studies have found that blood lead concentrations can not fully reflect the neurotoxic effects of lead, while the specific distribution and accumulation of lead in the brain may be the base of lead-induced neurotoxicity.Synaptic plasticity is the neurobiological basis of learning and memory function, including transfer efficiency associated functional plasticity and information storage related structural plasticity. Previous studies mainly focused on the functional plasticity of synapse, but changes on functional plasticity can not completely explain the mechanism of neurotoxicity of lead. As the basis of functional plasticity, structural plasticity of synspse plays an important role in lead-induced learning and memory dysfunction. Paying more attention to structural plasticity might be a new way to clarify the mechanism of neurotoxicity of lead.Plasticity of dendritic spines is an important component of structural plasticity of synapse, including changes in the density and shape of dendritic spines, which plays a significant role in the excitatory synaptic transmission and closely associated with learning and memory function. Neuroligin1(NLGN1), a postsynaptic adhesion protein, which is located on excitatory synapse, regulats excitatory synapse formation and morphology. However, whether or not NLGN1 is involved in lead-induced learning and memory dysfunction deserves further study.Aims:In this study, by establishing the low-level gestational lead exposure rat model and primary cultured rat hippocampal neurons model, we observed the distribution of lead in the brain, investigated the effects of lead on the density and morphology changes of dendritic spines, and the role of NLGN1 played in this process. The results will help to further clarify the molecular mechanisms of lead-induced learning and memory impairment, and provide new theoretical bases for exploring potential targets for drug therapy.Methods:1. Establishment of lead exposure model.1). Female SD rats were exposed to water containing 0, 0.005%, 0.01%, or 0.02% of lead acetate(Pb AC2) beginning 2 weeks before mating, throughout gestation, and until postnatal day 10.2). Primary cultured rat hippocampal neurons were treated with Neurobasal mediem containing a dose of 0, 0.1, 1μmol / L Pb AC2 on day in vitro(DIV) 7, and tested on DIV 12.2. Atomic absorption spectrophotometry was employed to detect the blood and brain Pb levels.3. AMG and XFM were performed to detect the distribution of lead in rat brain.4. To observe the effects of lead exposure on LTP induction in rat hippocampus, whole-cell patch-clamp recordings were performed.5. Nissl staining was perfoemed to detect the number of hippocampal CA1 neurons, Golgi staining to observe hippocampal CA1 neuronal morphology and dendritic spines.6. The cell viability of primary cultured rat hippocampal neurons was assessed by MTT assay.7. Western blotting was performed to observe NLGN1, PSD-95 and NR-2A protein expression in hippocampal tissue and primary cultured rat hippocampal neurons.8. Immunofluorescence staining was performed to detect primary cultured rat hippocampal neuronal dendritic spines.Results:1. The distribution of lead in rat brain1) After lead exposure, compared with the control group, lead exposure groups at P0, 3, 7, 10, and 21 rats blood lead levels significantly increased, whereas the P30 lead-exposed rats blood lead levels had no significant difference.2) AMG, atomic absorption spectrophotometry and XFM results suggested that lead exposure significantly increased lead accumulation in the brain, especially in the hippocampus of rats.2. Effects of lead exposure on synaptic plasticity of rat hippocampal neurons1) Compared with the control group, the excitatory postsynaptic current(EPSC) amplitudes of treatments were significantly reduced.2) After lead exposure, compared with the control group, there were no significant changes in the lead-exposed rat hippocampal CA1 neuronal densities and dendritic branchings, while the number of dendritic spines was significantly reduced, among which thin, filopodium and mushroom significantly decreased.3) The number of dendritic spines of lead exposure group in primary cultured hippocampal neurons was significantly reduced when compared with the control group.3. Effects of lead exposure on NLGN1, PSD-95 and NR-2A protein levels1) After lead exposure, compared with the control group, the NLGN1, PSD-95 and NR-2A protein levels of lead-exposed groups in rat hippocampus were significantly reduced.2) Compared with the control group, the NLGN1, PSD-95 and NR-2A protein levels of lead-exposed groups in primary cultured hippocampal neurons were significantly reduced.Conclusion:1. During gestational lead exposure, lead accumulated in the brain and unevenly distributed. Hippocampus was the main region of lead accumulation.2. Lead exposure caused hippocampal LTP inhibition, suggesting the damage to the functional plasticity of hippocampal synapse.3. Lead exposure significantly reduced the density of dendritic spines, including of the shapes of thin, filopodium and mushroom, suggesting the damage to the structural plasticity of hippocampal synapse.4. Lead can down-regulate hippocampal neurons NLGN1, PSD-95 and NR-2A protein levels, thus affecting the formation, development and maturation of dendritic spines.