Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring

Accepted Manuscript Title: Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring Author: Magdalena G˛assowska Irena Baranowska-Bosiacka Joanna Moczydłowska Małgorzata Frontczak-Baniewicz Magdalena Gewartowska Lidia Stru˙zy´nska Izabela Gutowska Dariusz Chlubek Agata Adamczyk PII: DOI: Reference:

S0300-483X(16)30258-X http://dx.doi.org/doi:10.1016/j.tox.2016.10.014 TOX 51774

To appear in:

Toxicology

Received date: Revised date: Accepted date:

27-7-2016 22-10-2016 27-10-2016

Please cite this article as: G˛assowska, Magdalena, Baranowska-Bosiacka, Irena, Moczydłowska, Joanna, Frontczak-Baniewicz, Małgorzata, Gewartowska, Magdalena, Stru˙zy´nska, Lidia, Gutowska, Izabela, Chlubek, Dariusz, Adamczyk, Agata, Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring.Toxicology http://dx.doi.org/10.1016/j.tox.2016.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring

Magdalena Gąssowskaa, Irena Baranowska-Bosiackab, Joanna Moczydłowskaa, Małgorzata

*

Frontczak-Baniewiczc, Magdalena Gewartowskac, Lidia Strużyńskad, Izabela Gutowskae, Dariusz Chlubekb, *Agata Adamczyka

* Both authors contributed equally to the final version of the manuscript. a

Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy

of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland; b

Department of Biochemistry and Medical Chemistry, Pomeranian Medical University,

Powstańców Wlkp. 72, 70-111 Szczecin, Poland; c

Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of

Sciences, Pawińskiego 5, 02-106 Warsaw, Poland; d

Laboratory of Pathoneurochemistry, Department of Neurochemistry, Mossakowski Medical

Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland e

Department of Biochemistry and Human Nutrition, Pomeranian Medical University,

Broniewskiego 24, 71-460 Szczecin, Poland

Corresponding author: Magdalena Gąssowska Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland; Tel/fax: +48886192507 1

E-mail addresses: [email protected]

Irena Baranowska-Bosiacka Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Powstańców Wlkp. 72, 70-111 Szczecin, Poland; E-mail addresses: [email protected]

Agata Adamczyk Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland; Tel/fax: +48886192507 E-mail addresses: [email protected]

Graphical abstract

2

ABSTRACT Lead (Pb), environmentally abundant heavy-metal pollutant, is a strong toxicant for the developing central nervous system. Pb intoxication in children, even at low doses, is found to affect learning and memorizing, with devastating effects on cognitive function and intellectual development. However, the precise mechanism by which Pb impairs synaptic plasticity is not fully elucidated. The purpose of this study was to investigate the effect of pre- and neonatal exposure to low dose of Pb (with Pb concentrations in whole blood below 10 µg/dL) on the synaptic structure and the pre- and postsynaptic proteins expression in the developing rat brain. Furthermore, the level of brain-derived neurotrophic factor (BDNF) was analyzed. Pregnant female Wistar rats received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring, while the control animals received drinking water. During the feeding of pups, mothers from the Pb-group were continuously receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-Day old pups were sacrificed and the ultrastructural changes as well as expression of presynaptic (VAMP1/2, synaptophysin, synaptotagmin-1, SNAP25, syntaxin-1) and postsynaptic (PSD-95) proteins were analyzed in: forebrain cortex, cerebellum and hippocampus. Our data revealed that pre- and neonatal exposure to low dose of Pb promotes pathological changes in synapses, including nerve endings swelling, blurred and thickened synaptic cleft structure as well as enhanced density of synaptic vesicles in the presynaptic area. Moreover, synaptic mitochondria were elongated, swollen or shrunken in Pb-treated animals. These structural abnormalities were accompanied by decrease in the level of key synaptic proteins: synaptotagmin-1 in cerebellum, SNAP25 in hippocampus and syntaxin-1 in cerebellum and hippocampus. In turn, increased level of synaptophysin was noticed in the cerebellum, while the expression of postsynaptic PSD-95 was significantly 3

decreased in forebrain cortex and cerebellum, and raised in hippocampus. Additionally, we observed the lower level of BDNF in all brain structures in comparison to control animals. In conclusion, perinatal exposure to low doses of Pb caused pathological changes in nerve endings associated with the alterations in the level of key synaptic proteins. All these changes can lead to synaptic dysfunction, expressed by the impairment of the secretory mechanism and thereby to the abnormalities in neurotransmission as well as to the neuronal dysfunction.

Keywords: Lead (Pb) neurotoxicity; synaptic proteins; synaptic dysfunction; synaptic ultrastructure

1. INTRODUCTION Effective brain function requires chemical communication between neurons and is mediated by synapses that undergo fast and efficient release of neurotransmitters tightly coupled with Ca2+- signaling (Neal et al., 2010; Zhang et al., 2015). Neurotransmitters are packaged in synaptic vesicles (SVs), which are then anchoring/docking to the presynaptic membrane at the active zone (AZ). The docked vesicles then go through a maturation process called priming to become fusion competent. This stage of synaptic vesicle exocytosis is dependent on the formation of the fusion machinery complex (SNARE complex). The SNARE complex consists of three compartmentally defined proteins: the vesicle-associated membrane protein synaptobrevin (isoform 1 and 2) (v-SNARE/VAMP1/2), a protein with a plasma membrane anchor syntaxin-1, and the cytoplasmic synaptosomal-associated protein of 25kDa (SNAP25), which is associated with membrane via palmitoylation (Li et al., 2009; Südhof, 2013; Südhof and Rizo, 2011). SNAP25, VAMP2 and syntaxin-1 proteins exist in all 4

types of synapses, playing a crucial role in SVs exocytosis. Other protein associated with SVs release machinery, commonly used to assess presynaptic elements is synaptophysin. Before the vesicle docks to the plasma membrane, synaptophysin dissociates from VAMP1/2, allowing VAMP1/2 to bind syntaxin-1 and initiate formation of the fusion pore (Südhof and Rothman, 2009; Valtorta et al., 2004). Subsequently, the docked SVs are primed by an ATPdependent process that renders the vesicles component to respond to a Ca2+-signal. Increase in intracellular Ca2+ level, caused by depolarization of the presynaptic membrane and the calcium-channels opening, triggers exocytosis (fusion pore opening) by binding to calciumsensor protein: synaptotagmin-1. Consequently, neurotransmitters are released and bind to the receptors on the postsynaptic membrane (Fig. 1). Neurotransmitter receptors and signalling enzymes together with cytoskeletal proteins are clustered in the postsynaptic density through the scaffolding proteins (Toscano and Guilarte, 2005). One of them is a postsynaptic density protein 95 (PSD-95), which is a member of the membrane-associated guanylate kinase (MAGUK) family and is highly concentrated in glutamatergic synapses in strong colocalization with the N-methyl-D-aspartate (NMDA) receptor (NMDAR). PSD-95, by increasing the number and the size of dendritic spines and regulation NMDAR-dependent changes in the number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), contributes to synapse stabilization and plasticity (Steiner et al., 2008; Torres et al., 2015). In developing neurons, the stabilization of the sites of the presynaptic release of neurotransmitters is controlled by retrograde signals from the postsynaptic side (BaranowskaBosiacka et al., 2012a; Neal and Guilarte, 2010). One of these retrograde signals is brainderived neurotrophic factor (BDNF) (Neal and Guilarte, 2010). The production and release of BDNF depends on the postsynaptic NMDA receptor activation, as a result of the fusion of the SVs and release of glutamate. The NMDAR-dependent release of BDNF may be critical in 5

the creation of sites of presynaptic liberation of neurotransmitters (Walz et al., 2006). Moreover, BDNF signalling affects the gene expression of both pre- and postsynaptic proteins (Tartaglia et al., 2001). The synaptic exocytosis was designed to provide fast and efficient neurotransmission, and disruption of this process may impair neuronal development and brain function. Disturbances of a variety of proteins playing a role in synaptic structure, SVs trafficking and fusion represent one of the reasons for the cognitive dysfunctions observed in neurodevelopmental as well as in neurodegenerative disorders including Autism, Schizophrenia and Alzheimer’s disease (AD) (Tampellini, 2015; Van Spronsen and Hoogenraad, 2010). It is likely, that these neurological disorders are not only the result of genetic and life-style factors but also of early life exposure to environmental risk factors among which is the lead (Pb) (Fuentes-Albero et al., 2015; Lindsky and Shneider, 2005; Rahbar et al., 2014; Stanfield et al., 2012; Yassa, 2014). Synapses have been shown to be the crucial target for Pb, a potent and ubiquitous environmental neurotoxin especially for developing brain (Baranowska-Bosiacka et al., 2012a; Neal and Guilarte, 2010). One of the most prominent effects of Pb exposure in children, even at low doses (with Pb concentrations in whole blood below 10 µg/dL), is decreased capacity to learn, memorizing with devastating effect on cognitive and intellectual development (Rahman et al., 2011; Yu et al., 2015; Zhang et al., 2015). Exposure to Pb is known to affect the presynaptic neurotransmitter release in both in vivo and in vitro models (Lasley and Gilbert, 2002; Xiao et al., 2006). The effect of Pb on the alterations of synaptic proteins has been reported in a variety of models of acute and chronic toxicity (Neal et al., 2012, 2010; Yu et al., 2015). However, the precise mechanism by which Pb disrupts synaptic transmission is not fully elucidated. Moreover, there is a lack of knowledge whether pre- and neonatal exposure to Pb at blood concentrations considered “safe for humans” may affect 6

synaptic structure and process of exocytosis. The developing brain in prenatal and early postnatal periods is experiencing intensive cellular proliferation, differentiation and synaptogenesis as well as is very sensitive to environmental pollutants, especially heavy metals. The exposure to Pb during the early phase of life can be one of the possible causal factors for abnormal brain development (Kim et al. 2010; Li et al. 2009). In our previous study, we revealed that pre- and neonatal exposure to low dose of Pb cause GSK-3β and CDK5-dependent Tau pathology (a significant increase of Tau protein level and its phosphorylation), which may lead to the impairment of cytoskeleton stability and neuronal dysfunction (Gąssowska et al., 2016). Therefore, the aim of this study is to examine whether pre- and neonatal exposure to Pb in a low dose may lead to changes in synaptic endings structure as well as the disturbances in synaptic proteins in a developing rat brain. We investigated the ultrastructural changes in synapses, the expression of presynaptic VAMP1/2, synapthophysin, synaptotagmin-1, SNAP25, syntaxin-1 proteins and postsynaptic PSD-95, as well as the BDNF level. We focused our attention on the forebrain cortex (FC), cerebellum (C) and hippocampus (H) as these regions have been reported to be sensitive to the toxicity of Pb (Baranowska-Bosiacka et al., 2011; Collins et al., 1982; Strużyńska et al., 2007). Our data indicated that pre- and neonatal Pb administration caused pathological changes in synaptic structure associated with the alterations in the level of key synaptic proteins. All these changes may suggest the synaptic dysfunction, what in consequence may lead to the impairment of neuronal connectivity and synaptic transmission.

2. MATERIALS AND METHODS 2.1 Reagents 7

The following antibodies were used in the current study: anti-SNAP25, antisynaptotagmin-1 (Cell Signaling Technology, Beverly, MA, USA), anti-syntaxin-1, antiVAMP1/2, anti-synaptophysin, anti-PSD-95 (Santa Cruz Biotechnology, CA, USA), antiGAPDH, anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG (GE Health Care UK, Little Chalfont, Buckinghamshire, UK), anti-BDNF (Santa Cruz Biotechnology, CA, USA).

2.2 Animals – in vivo model Procedures involving animals were carried out in strict accordance with international standards of animal care guidelines, and effort was made to minimize suffering and the number of animals used. Experiments were approved by the Local Ethical Committee on Animal Testing at the Pomeranian Medical University in Szczecin, Poland (approval No 30/2008). Three-month old female (250±20 g) Wistar rats (n = 6) were kept for a week in a cage with sexually mature males (2:1). All animals were allowed free access to food and water and were kept in a room with a controlled temperature under a LD 12/12 regime. After a week, they were separated from the males, and each female was placed in an individual cage. Pregnant females were divided into two groups: control and Pb-treated. Females from the Pbtreated group (n = 3) received 0.1% lead acetate (PbAc) in drinking water ad libitum, starting from the first day of gestation. The solution of PbAc was prepared daily in disposable plastic bags (hydropac, Anilab, Poland) from solid reagent directly at the desired concentration, and was not acidified. Pregnant females from the control group (n = 3) received drinking water until weaning of the offspring. The volume of intaken liquids did not differ significantly between the Pb-treated and control groups. Offspring (males and females) stayed with their mothers and were fed by them. During the feeding of pups, mothers from the Pb-treated group 8

were still receiving PbAc in drinking water ad libitum. Pups were weaned at postnatal day 21 (PND 21) and placed in separate cages. From that moment, the young rats from both the Pbtreated and control groups received only drinking water ad libitum until PND 28. We chose an oral route of exposure to 0.1% lead acetate as it mimics environmental exposure and is used as a common rodent model of lead poisoning (Kang et al., 2009; Xu et al., 2005). In addition, the previous study of Baranowska-Bosiacka et al. (BaranowskaBosiacka et al., 2012b), revealed that this treatment protocol results in a concentration of Pb in whole blood (Pb-B) of rat offspring below the threshold considered as “safe for humans” (10 µg/dL) (CDC, 2007). Because the aim of the current study was to obtain Pb-B concentration below 10 µg/dL, we ceased Pb administration after the period of breast feeding. For the ultrastructure study of neuronal cells, we randomly selected 8 young animals (4 control and 4 Pb-treated animals); for the study of gene expression for BDNF as well as gene expression for synaptic proteins, we randomly selected 16 young animals (8 from each group). The immunoreactivity of synaptic proteins was estimated in group of 16 randomly selected young animals (8 from each group). The proportion of male and female pups in the Pb-treated and control groups were not significantly different (p = 0.5, Fisher exact test). The unanaesthetized pups were sacrificed by decapitation using scissors; the brains were quickly removed and dissected into three regions: cerebellum (C), hippocampus (H), and forebrain cortex (FC), and then placed in liquid nitrogen. The samples were stored at -80o C for further analysis.

2.3 Primary neuronal culture – in vitro model Primary cultures of cerebellar granule cells were prepared from dissociated cerebella of 8-day-old rats, according to Marchetti et al. (Marchetti et al., 1995). Cells were plated on poly-L-lysine coated glass coverslips (1106 per 3.5 mm dish) and maintained in basal Eagle 9

culture medium (BME), supplemented with 10% (v/v) fetal calf serum (FCS), 25 mM KCl, and 100 µg/mL gentamicin. Cells were cultured in humidified 95% air/CO2 atmosphere at 37ºC. Cytosine arabinoside (10 µM) was added to the culture medium 18 h after plating to minimize proliferation of non-neuronal cells. All used chemicals were obtained from SigmaAldrich, Poland. Experiments were carried out in cultures between 5 and 7 days in vitro (DIV).

2.4 Analysis of Pb by Atomic Absorption Spectroscopy Lead content in blood and brain was analyzed by graphite furnace atomic absorption spectrometry (GFAAS) with a Perkin Elmer 4100 ZL spectrometer, (Perkin Elmer, Warsaw, Poland) Zeeman correction. Brain samples were mineralized at 120o C for 16 h in a closed Teflon container with 1 mL of 65% HNO3. After cooling, samples were treated with 1 mL 30% H2O2 and mineralized for the next 24 h using the same conditions. The obtained solution was diluted with deionised water to 10 mL and analyzed by GFAAS together with blank and control samples. Whole blood samples were deproteinized with 65% HNO3 and further analyzed as described above. The detection limit was 0.3 μg/dL.

2.5 Transmission electron microscopy (TEM) analysis of brain samples Rats were anaesthetized with Nembutal (80 mg/kg b.w.) and perfused through the ascending aorta initially with 0.9% NaCl in 0.01M sodium-potassium phosphate buffer pH 7.4 and after with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4 at 20°C (Sigma-Aldrich, Poland). Material for ultrastructural studies was sampled from the forebrain cortex, cerebellum and the CA1 region of hippocampus of all rat groups. Specimens were fixed in the ice-cold fixative solution for 20 h and placed in a mixture of 1% OsO4 and 0.8% K4[Fe(CN)6]. After dehydration in a series of ethanol gradient, tissue 10

specimens were embedded in epoxy resin (Epon 812). Ultra-thin sections (60 nm) were examined by transmission electron microscopy (JEM-1200EX, Jeol, Japan). 2.6 Western blot analysis Immunochemical analysis of proteins level and phosphorylation status was performed by Western blotting method in standard conditions. The samples were mixed with Laemmli buffer and denatured at 95 oC for 5 min. After standard SDS-PAGE separation, the proteins were transferred onto nitrocellulose membranes at 100V and proteins were detected by immunodetection with specific antibodies. Antibodies were detected using chemiluminescent reaction and ECL reagent (Amersham Biosciences, Bath, UK) under standard conditions. After stripping, the immunolabeling of GAPDH was performed as a loading control.

2.7 Quantitative real time polymerase chain reaction (qRT-PCR) Quantitative analysis of mRNA expression of Vamp1, Vamp2, Stx1a, Stx1b, Dlg4, Syp, Snap25 and Syt1 were performed by two-step reverse transcription PCR. RNA was isolated by using TRI-reagent according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). Digestion of DNA contamination was performed by using DNase I according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). RNA quantity and quality were

controlled

by

spectrophotometric

analysis

using

a

NanoDrop

ND-1000

spectrophotometer (NanoDrop Technologies, USA) and gel electrophoresis. A reverse transcription was performed by using the High Capacity cDNA Reverse Transcription Kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed on an ABI PRISM 7500 apparatus using the commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol: Vamp1-Rn00565308_m1; Vamp2-Rn00360268_g1; 11

Stx1a-Rn00587278_m1; Rn01528256_m1;

Stx1b-Rn01510167_m1;

Snap25-Rn00578534_m1;

Dlg4-Rn00571479_m1;

Syt1-Rn00436862_m1

and

SypGapdh-

Rn01775763_g1. Gapdh was analyzed as a reference gene. Real-time conditions were as follows: 95 °C (15 sec), 40 cycles at 95 °C (15 sec), and 60 °C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. Each sample was analyzed in three technical replicates, and mean Ct values were used for further analysis. The relative quantity of a target, normalized to the endogenous control Gapdh gene and relative to a calibrator, is expressed as 2-∆∆Ct (-fold difference), where Ct is the threshold cycle, ∆Ct = (Ct of target genes) – (Ct of endogenous control gene,), and ∆∆Ct = (∆Ct of samples for target gene) – (∆Ct of calibrator for the target gene).

2.8 Fluorescent visualization of the BDNF in primary neuronal culture Cells were cultured on polylysine covered microscopic glasses. Cells were fixed in 4% buffered paraphormaldehyde solution for 10 minutes in room temperature (RT). After washing in PBS cells were permeabilized with 0.5% TRITON-X100 solution for 15 min. in RT. Then cells were incubated with goat anti-BDNF antibody (Santa Cruz Biotech, USA) in 1:100 dilution for 1 hour in RT. After that the cells were double washed in PBS (5 min each) and incubated with secondary antibody anti goat - DyLight 549 (Jackson Lab). After double wash in PBS cells ‘nuclei were counterstained with DAPI (20 min, RT). Microphotographs were collected in fluorescent Axio Observer. Z1 inverted microscope (Zeiss, Germany), combined with Axio Cam MRm (black-white camera) and Axio Vision Rel.4.8 software (Zeiss, Germany). Reflectors: 49 for DAPI and 43 HE for DyLight 549. Yellow color for BDNF was artificially used for better contrast.

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2.9 Statistical analysis The results were expressed as mean values ± S.E.M. Differences between the means of control and Pb-treated group were analyzed using a Student’s t-test. Statistical significance was accepted at p<0.05. The statistical analyses were performed using Graph Pad Prism version 6.0 (Graph Pad Software, San Diego, CA).

3. RESULTS 3.1 Lead concentration in whole blood and brain The results of the Pb concentration in brain and blood were published in detail in our previous studies (Baranowska-Bosiacka et al. 2013; Gąssowska et al., 2016). The Pb regimen used in experiment (0.1% PbAc in drinking water from the first day of gestation till weaning at postnatal day 21) caused significantly higher Pb concentrations with respect to control rats (drinking water) measured at postnatal day 28 both in whole blood (Pb-B) and in all examined parts of the brain of intoxicated rats. Briefly: Pb concentration in whole blood in Pb group was significantly higher than in control group (6.86 µg/dL and 0.93µg/dL, respectively). In examined parts of brain we observed higher Pb concentration range: 7.10-7.48µg/dL for Pb group and 0.04-0.26 µg/dL for control group. Pb concentrations in whole blood correlated strongly positively with Pb concentrations in the brain (FC: Rs=+0.72; C: Rs=+0.63; H: Rs=+0.81; p<0.005 for all). We did not observe statistically significant changes between the parts of the brain examined in the Pb-treated group (p=0.51) (Baranowska-Bosiacka et al. 2013; Gąssowska et al., 2016).

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3.2 Lead exposure induced ultrastructural changes in the synapses. The features of synaptic alterations in TEM analysis The electron microscopy analysis of neurons and other brain cells in the examined regions of control rats brain (forebrain cortex, cerebellum and the CA1 region of the hippocampus) revealed ultrastructurally unchanged neuropil as well as normal structure of synapses, synaptic vesicles (SVs) and mitochondria (M). Synapses in the control groups exhibited a proper distribution of synaptic vesicles in the cytoplasm. Multiple vesicles were in direct contact with presynaptic membrane. The synaptic cleft was narrow with prominent and clearly stained postsynaptic density. The nerve endings have not revealed the features of the swelling. The mitochondrial structure was integral and clearly visible (Fig. 2 -1.A and 1.B; 3 1.A and 1.B and Fig. 4 -1.A and 1.B). The ultrastructural images of brain tissue of rats subjected to pre- and neonatal administration of Pb revealed pathological alterations in all examined structures. Pb-treatment causes the changes in synapses and certain features are observed in all examined parts of brain including nerve endings swelling (*) (Fig. 2 -2.A; 3 -2.A and Fig. 4 -2.A), enhanced packing density of synaptic vesicles in presynaptic area (SV) (Fig. 2 -2.A and 2.B; 3 -2.A and 2.B and Fig. 4 -2.A – 2.D) as well as blurred and thickened structure of synaptic cleft (short arrows). In the vast majority of synapses the synaptic cleft was not visible and the postsynaptic density was blurred i.e. appears to be thickened and possess hardly discernible membranes (short arrows) (Fig. 2 -2.A and 2.B; 3 -2.A and 2.B and Fig. 4 -2.A – 2.D). Furthermore, in forebrain cortex and hippocampus only few synaptic vesicles (SVs) were in contact with presynaptic membrane (long arrows) (Fig. 2 -2.A and Fig. 4 -2.A). Moreover, the ultrastructure of mitochondria was altered; some of them were elongated, swollen or shrunken. Mitochondrial cristae and membrane were fused and blurred (M) (Fig. 2 -2.A and 2.B; 3 -2.A and 2.B and Fig. 4 -2.A – 2.D). 14

3.3 Lead exposure decreased the BDNF level The confocal microscope analysis of images of primary cultured cerebellar neurons with BDNF probe indicated that only some cells from Pb-intoxicated rats showed a yellow fluorescence coming from BDNF, compared to the control group (Fig. 5A). The above data indicate reduction of BDNF level in cerebellar neurons. The obtained result is complement to our previous study where, using the same model of Pb-intoxication, we revealed significant decrease in BDNF level in forebrain cortex and hippocampus (Baranowska-Bosiacka et al. 2013). Unlike decreased BDNF level, its gene expression was unchanged in all examined brain structures (Fig. 5B).

3.4 Lead exposure altered the levels of synaptic vesicles proteins The changes in synaptic ultrastructure as well as the decrease in BDNF level observed in Pb-treated group encouraged us to investigate the possible effects at the molecular level. Seeing that BDNF is involved in synaptic protein modulation, the dysfunction of BDNF signalling could lead to changes in gene expression of pre- and postsynaptic proteins. Therefore, in the next experiment we examined the expression of key presynaptic proteins involved in synaptic vesicle targeting and/or release: synaptobrevin 1 and 2 (VAMP1/2), synaptophysin (Syp) and Ca2+-sensing synaptotagmin-1 (Syt-1). The effects of Pb exposure on VAMP1/2, Syp and Syt-1 expression and protein level in the forebrain cortex, cerebellum and hippocampus were summarized in Figure 6, 7 and 8. Our data indicated significant increase in the protein level of synaptophysin in cerebellum (by about 20%) without changes in gene expression, compared to the control group (Fig. 7 B.1 and B.2). On the other hand, in the same brain structure we observed significant decrease in the level of synaptotagmin-1 with concomitant lack of changes in mRNA level (Fig. 7 C.1 and C.2). The quantitative RT-PCR 15

and Western blot analysis revealed a lack of changes in both mRNA and protein level of Syp and Syt-1 in other brain structures of Pb-treated rats (Fig. 6 B.1 - C.2 and 8 B.1 - C.2). Moreover, Pb exposure had no effect on the level of VAMP1/2 in all examined structures despite decrease in the Vamp1 and Vamp2 mRNA expression in the forebrain cortex and hippocampus and increased the levels of Vamp2 mRNA in cerebellum (Fig. 6 A.1, A.2; 7 A.1, A.2 and 8 A.1, A.2).

3.5 Lead exposure affected t-SNARE proteins The disturbances observed in the structure of synaptic cleft (blurred and not visible) after Pb intoxication led us to analyse SNAP25 and syntaxin-1 proteins, which are associated with the presynaptic plasma membrane and play essential role in the SVs docking and vesicular release machinery. We examined the effect of Pb-exposure on the protein levels and gene expression of SNAP25 and syntaxin-1. As presented in Fig. 11 A.2, Pb-intoxication significantly reduced the level of SNAP25 by about 13% in the hippocampus, compared to the control group. Decreased level of SNAP25 protein was accompanied with reduction of its gene expression by about 30% (Fig. 11 A.1). Pb exposure had no effect on SNAP25 in the forebrain cortex and cerebellum (Fig. 9 A.1, A.2 and Fig. 10 A.1, A.2). In addition, the protein level of syntaxin-1 was significantly decreased by about 24% in the cerebellum (Fig. 10 B.2) and by about 31% in the hippocampus (Fig. 11 B.2), comparing to the respective control groups, whereas mRNA levels of both Stx1a and Stx1b were significantly reduced in the hippocampus (Fig. 11 B.1) and unchanged in cerebellum (Fig. 10 B.1). Moreover, the quantitative RT-PCR and Western blot analysis revealed a lack of changes in both gene expression and protein level

16

of SNAP25 and syntaxin-1 in the forebrain cortex of Pb-treated animals (Fig. 9 A.1, A.2, B.1 and B.2).

3.6 Lead exposure evoked changes in the level of PSD-95 in all brain structures In the next step, we examined the expression of the crucial scaffolding protein of glutamatergic synapses – PSD-95 after Pb low-level intoxication. The qRT-PCR analysis indicated that the Dlg4 expression was significantly elevated in the forebrain cortex and hippocampus without changes in cerebellum (Fig. 12 A.1, B.1 and C.1). The alterations in mRNA level were accompanied with significant rise in the level of PSD-95 in the hippocampus (Fig. 12 C.2), while in the forebrain cortex and cerebellum we observed decreased immunoreactivity of this protein by about 16% and 17%, respectively, as compared to the relevant control groups (Fig. 12 A.2 and B.2).

4. DISSCUSION

Despite clinical evidence suggesting that the environmental contaminant lead (Pb) has the neurotoxic effect, the mechanism remains undefined. This is the first study to demonstrate that pre- and neonatal exposure to Pb leading to Pb-B below 10 µg/dL (concentration considered “safe for human”) induces synaptic dysfunction in rat brain. In Pb-exposed rodents we observed swollen synapses with enhanced density of synaptic vesicles, as well as the blurred and thickened structure of synaptic cleft. Moreover, TEM study showed alterations in synaptic mitochondria structure, which were shrunken, swollen or elongated. These structural abnormalities were accompanied by disorders of key synaptic proteins including: synaptotagmin-1, SNAP25 and syntaxin-1 as well as postsynaptic PSD-95. Additionally, the lower level of BDNF was observed in the brain of Pb-exposed animals comparing to control. 17

These synaptic abnormalities indicate that, under conditions of pre- and neonatal exposure to Pb even below threshold level, damage of the molecular machinery of exocytosis may occur, resulting in impairment of synaptic transmission and in consequence neuronal dysfunction. Impairment of neurotransmitters release from presynaptic nerve terminals associated with alterations in the presynaptic protein expression (mainly loss of synaptophysin and synaptobrevin) after prolonged Pb exposure was shown by Neal et al. (Neal et al., 2010). Stanfield et al. demonstrated a decrease in the expression of synaptophysin and VAMP1/2 in developing hippocampal neurons treated with Pb (Stanfield et al., 2012). Moreover, Yu et al. showed alterations of synaptic proteins, particularly decrease in synaptophysin expression in the hippocampus of mouse offspring induced by developmental Pb exposure (Yu et al., 2015). In turn, the study of Li et al. revealed that the expression of syntaxin-1A and VAMP2 raised but the expression of SNAP25 was decreased in hippocampus of mouse pups as a result of early-life Pb administration (Li et al., 2009). These observations partially agree with our results. We revealed significant decrease in the level of SNAP25 in hippocampus, syntaxin-1 in cerebellum and hippocampus and synaptotagmin-1 in cerebellum. The divergences in the results may be due to differences in the experimental model of Pb toxicity (period of exposure, species, dose) or differences in the level of Pb in brain tissues. It is noteworthy that the final concentrations of Pb interacting directly with neurons in our study remained in the range "safe for human" and were lower than those observed in the above-cited experiments. Probably for the same reasons, we observed the lack of alterations in the expression of VAMP1/2 in all examined brain parts and synaptophysin in forebrain cortex and hippocampus.

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Synaptic transmission underlying learning and memory formation requires the combined action between presynaptic proteins as well as interaction between presynaptic proteins and proteins forming the postsynaptic densities. This process is initiated when an action potential triggers neurotransmitters release from a presynaptic nerve terminal as a result of SVs exocytosis. The process of exocytosis requires the formation of membrane fusion machinery (SNARE complex) consisting of the SV proteins such as: VAMP1/2, syntaxin-1 as well as SNAP25 protein. Into the process of vesicular releases other proteins such as synaptotagmin-1 or synaptophysin are also involved. The cross-interaction between these proteins seems to be essential to the processes of neurotransmitters release and neurotransmission, and thus to learning and memory formation (Südhof, 2004; Li et al., 2009). Therefore, it is possible that changes in the level of both integral proteins of synaptic vesicles, as well as presynaptic membrane proteins may lead to damage of the synaptic vesicle exocytosis. Our data indicated that while presynaptic vesicles retain normal level of the VAMP1/2, synaptophysin and the synaptotagmin-1 (except the cerebellum), the levels of plasma membrane proteins necessary to the SNARE complex formation were significantly reduced. It is possible that the pathological alterations in the structure of synaptic cleft (blurred and not visible), observed in Pb-treated animals and described in literature (Chen et al., 2015; Ding et al., 2013) may be associated with lower level of syntaxin-1 and SNAP25. Deficiency in these proteins could contribute to the presynaptic membrane structure dysfunction and could lead to the disturbances in the exocytosis mechanism. In addition, we observed enhancement of SVs density in the presynaptic area. Probably the reduced levels of syntaxin-1 in cerebellum and hippocampus, SNAP25 in hippocampus as well as Ca2+-sensing protein: synaptotagmin-1 in cerebellum lead to sequestration of SVs in the reserve pool with decreased releasable pool directly docking to the presynaptic membrane (only few SVs are visible in physically contact with the presynaptic membrane in presynaptic active zone (PAZ) 19

in Pb-treated groups). Similarly to our results, the study of Zhang et al. provide evidence that chronic exposure to Pb resulted in an accumulation of synaptic vesicles in the presynaptic part of synapses (in the resting vesicle pool) with simultaneous reduction in number of ready releasable pool/docked SVs in hippocampal CA1 synapses in young adult rats (Zhang et al., 2015). There is also the possibility that decrease in BDNF level, observed in the current and our previous study (Baranowska-Bosiacka et al., 2013), could be responsible for accumulation of SVs in the presynaptic part of synapses. BDNF plays a role as retrograde signal and has been implicated in synaptic connectivity and ultrastructure (Cohen-Cory et al., 2010; Ventriglia et al., 2013). In the glutamatergic synapses, the activation of the presynaptic TrkB receptor by BDNF increases the number of docked SVs and enhances vesicular release (BaranowskaBosiacka et al., 2012a; Neal and Guilarte, 2010). Altered functionality of BDNF has been observed in different neurodegenerative diseases, including Huntington disease, Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Zuccato and Cattaneo, 2009). The lack of BDNF has been selectively observed in neurons containing neurofibrillary tangles in AD and the loss of BDNF production has been associated with mutations of alpha-synuclein in early-onset familial PD (Kohno et al., 2004; Lee et al., 2005; Murer et al., 1999). The versatility of BDNF is emphasized by its contribution to a range of adaptive neuronal responses including a longterm potentiation (LTP), long-term depression (LTD), as well as homeostatic regulation of intrinsic neuronal excitability (Cunha et al., 2010). It is documented that failure of positive reverse signal can impair incorporation of synaptic vesicle proteins synaptobrevin and synaptophysin as well as vesicular release (Neal and Guilarte, 2010; Pozzo-Miller et al., 1999). Neal et al. demonstrated that the concentration of BDNF in cultured hippocampal neurons incubated with Pb was decreased (Neal et al., 2010). Similarly the study of Stanfield 20

et al. showed that the levels of both proBDNF as well as the extracellular mature BDNF were reduced by Pb exposure in hippocampal neurons (Stanfield et al., 2012). Our electron microscopic study revealed also that in Pb-treated rats, neurons had ultrastructurally altered mitochondria in all examined brain structures; some of them were elongated, swollen or shrunken with not visible structure of cristae. Disturbances in the structure of mitochondria in Pb-exposed rats were observed previously (Deveci, 2006; Jabłońska et al., 1994). Degenerated or larger than normal mitochondria were present in the brain cortex of rats receiving PbAc in drinking water for 60 days (Deveci, 2006). Also the study of Zhang et al. demonstrated a significant increase in the number of mitochondria with diameter greater than 300 nm (Zhang et al., 2015). Because the synaptic vesicle cycle involves numerous ATP-consuming steps and is tightly controlled by the cytosolic calcium concentration, synaptic mitochondria appear to be essential for synaptic transmission, organization and movement of SVs to the readily releasable pool (Vos et al., 2010). Therefore, it is likely that the enhanced density of SVs in presynaptic area together with explicit reduction of vesicles which are in contact with presynaptic membrane may be caused, at least in part, by dysfunction of mitochondria and reduced ATP availability. Moreover, damage to the cytoskeleton appears to be able to impair axonal transport of organelles causing synapse starvation, depletion of ATP, and ultimately neuronal damage (Eckert et al., 2014; Gąssowska et al., 2016; Reddy, 2011). Process of synaptic transmission requires the combined action between presynaptic proteins and proteins of postsynaptic densities. PSD-95 is one of the most abundant proteins found in the postsynaptic density of excitatory synapses (Béïque and Andrade, 2003). This scaffold protein is involved in regulation of long-term neuronal synaptic plasticity, associated with NMDA and AMPA receptor signalling; promotes the maturation and strengthening of 21

excitatory synapses (El-Husseini, 2000; Kim et al., 2007). Reduction of PSD-95 has been observed in many pathological states of brain, including neurodegenerative diseases such as AD (Shao et al., 2011; Sultana et al., 2010; Tu et al., 2014) and PD (Nash et al., 2005). Shao et al. showed reduction of PSD-95 as well as synaptic destruction and dysfunction in transgenic mouse model of AD and suggested the role of Aβ and Tau protein in PSD-95 alteration (Shao et al., 2011). In our previous study we indicated that pre- and neonatal exposure to Pb result in accumulation of Tau protein with parallel excessive its hyperphosphorylation at Ser396 and Ser199/202 in forebrain cortex and cerebellum (Gąssowska et al., 2016). Therefore, it is likely that Pb-induced Tau protein pathology in our experimental conditions may contribute to observed decrease in the level of PSD-95 in these brain structures. The result of research may suggest the dysfunction of the excitatory synapses. On the other hand, increase in PSD-95 level in the hippocampus could result from up regulation of protein expression as a compensatory mechanism against Pb-induced changes in developmental processes. Enhanced level of PSD-95 in hippocampus may reflect an adaptive response to changes in synaptic proteins levels and synaptic destruction. Although we observe elevated PSD-95, this increase (possibly an abortive compensatory reaction) does not seem to prevent the evident alterations of synaptic morphology and-likely loss of their function.

5. CONCLUSIONS Summarizing, the results of this study provide evidence that exposure to Pb during pre- and neonatal period of life causes pathological changes in ultrastructure of synapses in rat brain. These structural abnormalities were accompanied by decrease in the level of key synaptic proteins: synaptotagmin-1 in cerebellum, SNAP25 in hippocampus and syntaxin-1 in 22

cerebellum and hippocampus. Moreover, the expression of PSD-95 was decreased in forebrain cortex and cerebellum, while raised significantly in hippocampus. Additionally, we observed the lower BDNF level in all brain structures. These Pb-induced changes may cause to the disturbances in the SVs movement and availability, presumably leading to the significant reduction in functional release and synaptic transmission efficiency as well as synaptic dysfunction that are implicated in many neurodegenerative and neurodevelopmental disorders.

CONFLICT OF INTEREST The authors declare that there are no conflicts of interest in the present work.

FUNDING This study was supported by the statutory budget of the Department of Biochemistry and Medical Chemistry Pomeranian Medical University in Szczecin, Poland and by the statutory budget (theme number 8) of the Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences in Warsaw, Poland.

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FIGURE CAPTIONS Figure 1. Schematic diagram showing the role of synaptic proteins in exocytosis. Synaptic vesicles (SVs) are filled with neurotransmitters (NT) by active transport fueled by an electrochemical gradient established by a proton pump (H+) that acidifies the vesicle interior. In preparation to synaptic exocytosis, SVs are docked at the active zone (AZ), where they are primed by an ATP-dependent process to convert into a state of competence for Ca2+-triggered fusion-pore opening. Different protein-protein interactions at the active zone mediate attachment of the SVs to the target membrane. Synaptobrevins (VAMP1/2) in the vesicle binds to SNAP25 and syntaxin-1, presynaptic plasma membrane proteins (t-SNARE), forming the fusion machinery complex (SNARE complex). In addition, synaptophysin (Syp) dissociates from VAMP1/2, allowing VAMP1/2 to bind syntaxin-1. When an action potential depolarizes the presynaptic membrane and opens Ca2+ channels, local increase in intracellular calcium level triggers fusion reaction by binding to Ca2+ sensor protein: synaptotagmin-1 (Syt-1). Released neurotransmitters into the synaptic cleft then bind to receptors (R) associated with postsynaptic density (PSD). After fusion, SVs are re-endocytosed, recycled and refilled with neurotransmitters.

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Figure 2. Ultrastructure of neuronal cells in forebrain cortex of control and Pb-treated rats. 1) Control group. Ultrastructurally unchanged neuronal cells are observed. A, B - unaltered structure of neuropil with normal appearance of synaptic cleft (short arrows); well-defined structure of synapses with unchanged distribution of synaptic vesicles, multiple vesicles are in direct contact with presynaptic membrane (long arrow). Ultrastructurally unchanged mitochondria (M). 2) Pb-treated group. A, B - enhanced density of synaptic vesicles in the center of presynaptic part of synapse (SV). Only few vesicles are in contact with presynaptic membrane (long arrows); synaptic clefts are blurred and thickened, without clearly marked pre- and postsynaptic membranes (short arrows). Ultrastructurally changed mitochondria (M), with blurred cristae structure (A, B). Neural cells and neuropil with features of swelling (*) are observed. Pictures are representative for each of 4 control and Pb-treated animals.

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Figure 3. Ultrastructure of neuronal cells in cerebellum of control and Pb-treated rats. 1) Control group. Ultrastructurally unchanged neuronal cells are visible. A, B - unchanged structure of neuropil with typical appearance of synaptic cleft (short arrows); well-defined structure of synapses with unaltered distribution of synaptic vesicles (SV), multiple vesicles are in direct contact with presynaptic membrane (long arrows). Ultrastructurally unchanged mitochondria (M). 2) Pb-treated group. Enhanced density of synaptic vesicles arranged in the center of presynaptic part of synapse (SV); focal swelling of nerve endings is visible (*); blurred and thickened structure of synaptic clefts without clearly marked pre- and postsynaptic membranes (short arrows). Ultrastructurally changed mitochondria (M) with blurred structure (A) or partially lacking cristae (B). Pictures are representative for each of 4 control and Pb-treated animals.

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Figure 4. Ultrastructure of neuronal cells in CA1 region of the hippocampus in brain of control and Pb-treated rats. 1) Control group. Ultrastructurally unchanged neuronal cells are observed. A, B - welldefined structure of synapses, normal appearance of synaptic cleft with clearly marked preand postsynaptic membranes (short arrows) with unaltered distribution of synaptic vesicles (long arrow). Ultrastructurally unchanged mitochondria (M). V – capillary blood vessel. 2) Pb-treated group. Enhanced density of synaptic vesicles in presynaptic part of synapse (SV) and only few vesicles are in contact with presynaptic membrane (A, long arrows). Blurred and thickened structure of synaptic clefts without clearly marked pre- and postsynaptic membranes (short arrows); focal swelling of nerve endings visible as white places in the cytoplasm (*). Ultrastructurally changed mitochondria (M) with blurred structure (A, B, D) or partially lacking cristae (C). Pictures are representative for each of 4 control and Pb-treated animals.

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Figure 5. The effect of perinatal exposure to Pb on the BDNF level and gene expression in the rat brain. A) The changes in BDNF level in cultured cerebellar neurons were analysed using confocal microscope. Images of cultured cerebellar neurons incubated with BDNF probe showed yellow fluorescence coming from BDNF from control (CTRL) and Pb-intoxicated (Pb) rats. B) The gene expression for BDNF in forebrain cortex, cerebellum and hippocampus of control and Pb-exposed rats were measured with qRT-PCR. Data represent the means ± S.E.M. for 8 independent experiments.

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Figure 6. The effect of perinatal exposure to Pb on the expression of synaptic vesicles proteins: VAMP1/2, Syp and Syt-1 in the forebrain cortex. The gene expression for VAMP1 and VAMP2 (A.1), Syp (B.1) and Syt-1 (C.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of SVs proteins were monitored using Western blot analysis. Representative pictures and densitometric analysis of VAMP1/2 (A.2), Syp (B.2) and Syt-1 (C.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5, ** p<0.01 versus control using a Student’s t-test.

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Figure 7. The effect of perinatal exposure to Pb on the expression of synaptic vesicles proteins: VAMP1/2, Syp and Syt-1 in the cerebellum. The gene expression for VAMP1 and VAMP2 (A.1), Syp (B.1) and Syt-1 (C.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of SVs proteins were monitored using Western blot analysis. Representative pictures and densitometric analysis of VAMP1/2 (A.2), Syp (B.2) and Syt-1 (C.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5, ** p<0.01 versus control using a Student’s t-test.

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Figure 8. The effect of perinatal exposure to Pb on the expression of synaptic vesicles proteins: VAMP1/2, Syp and Syt-1 in the hippocampus. The gene expression for VAMP1 and VAMP2 (A.1), Syp (B.1) and Syt-1 (C.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of SVs proteins were monitored using Western blot analysis. Representative pictures and densitometric analysis of VAMP1/2 (A.2), Syp (B.2) and Syt-1 (C.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5, ** p<0.01 versus control using a Student’s t-test.

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Figure 9. The effect of perinatal exposure to Pb on the expression of presynaptic plasma membrane proteins (t-SNAREs): SNAP25 and syntaxin-1 in the forebrain cortex. The gene expression for SNAP25 (A.1), syntaxin-1 (B.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of t-SNARE proteins was monitored using Western blot analysis. Representative pictures and densitometric analysis of SNAP25 (A.2) and syntaxin-1 (B.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments.

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Figure 10. The effect of perinatal exposure to Pb on the expression of presynaptic plasma membrane proteins (t-SNAREs): SNAP25 and syntaxin-1 in the cerebellum. The gene expression for SNAP25 (A.1), syntaxin-1 (B.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of t-SNARE proteins was monitored using Western blot analysis. Representative pictures and densitometric analysis of SNAP25 (A.2) and syntaxin-1 (B.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5 versus control using a Student’s t-test.

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Figure 11. The effect of perinatal exposure to Pb on the expression of presynaptic plasma membrane proteins (t-SNAREs): SNAP25 and syntaxin-1 in the hippocampus. The gene expression for SNAP25 (A.1), syntaxin-1 (B.1) of control and Pb-exposed brains was measured with quantitative RT-PCR. Immunoreactivity of t-SNARE proteins was monitored using Western blot analysis. Representative pictures and densitometric analysis of SNAP25 (A.2) and syntaxin-1 (B.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5, ** p<0.01 versus control using a Student’s t-test.

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Figure 12. The effect of perinatal exposure to Pb on the expression of PSD-95 in the rat brain. The gene expression for PSD-95 in the forebrain cortex (A.1), cerebellum (B.1) and hippocampus (C.1) of control and Pb-exposed brains was measured with quantitative RTPCR. The levels of PSD-95 proteins were determined using Western blot analysis. Representative pictures and densitometric analysis of PSD-95 in forebrain cortex (A.2), cerebellum (B.2) and hippocampus (C.2) are shown. Results were normalized to GAPDH levels. Data represent the means ± S.E.M. for 8 independent experiments. * p<0.5, *** p<0.001 versus control using a Student’s t-test.

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