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Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats

BACKGROUND: Lead (Pb(2+)) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca(2+) signaling. How perinatal Pb(2+) exposure affects Ca(2+)-related gene regulation remains unclear. However, Ca(2+) activates the L-Type voltage se...

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Autores principales: Neuwirth, Lorenz S., Phillips, Greg R., El Idrissi, Abdeslem
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967126/
https://www.ncbi.nlm.nih.gov/pubmed/29793500
http://dx.doi.org/10.1186/s12929-018-0450-4
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author Neuwirth, Lorenz S.
Phillips, Greg R.
El Idrissi, Abdeslem
author_facet Neuwirth, Lorenz S.
Phillips, Greg R.
El Idrissi, Abdeslem
author_sort Neuwirth, Lorenz S.
collection PubMed
description BACKGROUND: Lead (Pb(2+)) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca(2+) signaling. How perinatal Pb(2+) exposure affects Ca(2+)-related gene regulation remains unclear. However, Ca(2+) activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission. METHOD: A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam’s litters each ranged from N = 6–10 pups per litter (M = 8, SD = 2), irrespective of Pb(2+) treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb(2+)− and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter’s obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb(2+) N = 12) were used in the present study.  Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Post-centrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 μL of DEPC treated H(2)O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb(2+) exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABA(AR)-β3, NKCC(1), KCC(2), and GAD 80, 86, 65, and 67 isoforms. RESULTS: Perinatal Pb(2+) exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb(2+) exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb(2+) competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb(2+) at PND 2 and 7, and both PFC and HP were negatively impacted by Pb(2+) at PND 22. Additionally, Pb(2+) altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb(2+) at PND 22. CONCLUSIONS: Perinatal Pb(2+) exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb(2+) has on the GABAergic system preceding what is known regarding Pb(2+) insults unto the glutamatergic system.
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spelling pubmed-59671262018-05-30 Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats Neuwirth, Lorenz S. Phillips, Greg R. El Idrissi, Abdeslem J Biomed Sci Research BACKGROUND: Lead (Pb(2+)) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca(2+) signaling. How perinatal Pb(2+) exposure affects Ca(2+)-related gene regulation remains unclear. However, Ca(2+) activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission. METHOD: A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam’s litters each ranged from N = 6–10 pups per litter (M = 8, SD = 2), irrespective of Pb(2+) treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb(2+)− and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter’s obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb(2+) N = 12) were used in the present study.  Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Post-centrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 μL of DEPC treated H(2)O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb(2+) exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABA(AR)-β3, NKCC(1), KCC(2), and GAD 80, 86, 65, and 67 isoforms. RESULTS: Perinatal Pb(2+) exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb(2+) exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb(2+) competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb(2+) at PND 2 and 7, and both PFC and HP were negatively impacted by Pb(2+) at PND 22. Additionally, Pb(2+) altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb(2+) at PND 22. CONCLUSIONS: Perinatal Pb(2+) exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb(2+) has on the GABAergic system preceding what is known regarding Pb(2+) insults unto the glutamatergic system. BioMed Central 2018-05-24 /pmc/articles/PMC5967126/ /pubmed/29793500 http://dx.doi.org/10.1186/s12929-018-0450-4 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Neuwirth, Lorenz S.
Phillips, Greg R.
El Idrissi, Abdeslem
Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title_full Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title_fullStr Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title_full_unstemmed Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title_short Perinatal Pb(2+) exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats
title_sort perinatal pb(2+) exposure alters the expression of genes related to the neurodevelopmental gaba-shift in postnatal rats
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967126/
https://www.ncbi.nlm.nih.gov/pubmed/29793500
http://dx.doi.org/10.1186/s12929-018-0450-4
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