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Devellis-5U24NS043562-02

ABSTRACT: Nicotinic receptors (nAChR) are widely distributed in the adult brain where they mediate excitatory postsynaptic transmission and presynaptic release of other neurotransmitters. Functional nicotinic receptors are also present in the embryonic brain. Several studies have suggested a role for nAChRs in cognitive functions (learning and memory) and cellular events such as degeneration and survival. In vitro, activation of nAChR can differentially modulate neuritic outgrowth, and enhance early gene expression prior to neuronal differentiation. Furthermore, several reports have shown that nicotine can prevent cell death induced by activation of glutamatergic receptors, NMDA-subtype. Little is known of these events in the cerebral cortex. Knowledge of the genes activated by exposure of cortical neurons to nicotine or to an excitotoxic agent such as the glutamatergic agonist NMDA could potentially lead to new strategies for adult brain protection and repair. The effect of nicotine and the cembranoid 4R, a novel nicotinic receptor non competitive antagonist, will be analyzed in primary cultures of cortical neurons, in the presence or the absence of NMDA, to identify which genes affected by NMDA receptor activation are modulated nicotine or 4R. We hypothesize that the neuroprotective effect of nAChRs is mediated by modulation of the changes in gene expressions elicited by NMDA. In order to detect early changes in gene expression, primary culture of cortical neurons prepared from 16 day old Sprague Dawley rat embryos at 11 days in culture will be treated for different times (10, 30, 60, 180, 360, min) with 1 mM NMDA and then harvested. In a second set of experiments, neuronal cultures will be treated with NMDA for 10, 30 and 60 min then the drug will be washed out and the cells will be harvested 24 h later. It was reported that 8-24 h pretreatment of neurons with 10 micromolar nicotine is neuroprotective, therefore we will treat separate cultures with either 10 micromolar nicotine or 2 micromolar 4R for 18 h and then with NMDA for 30 min or 1 h. The cells will be either harvested soon after NMDA treatment or the drugs will be washed out and the cells harvested 24 h later. We will also examine sample treated only with either 4R or nicotine for 15 min or 18 h in order to determine which genes are differentially regulated by these drugs. These experiments will allow us to detect any change in gene expression elicited soon by each treatment or with some delay. The samples in Trizol will be kept at -70 until all the 3 or 5 replicates are harvested and then the RNA will be purified. RNA from 3-5 independent experiments for each different protocol or treatment together with controls will be sent to the facility. Keywords: time-course

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE4242 | GEO | 2006/02/14

SECONDARY ACCESSION(S): PRJNA94901

REPOSITORIES: GEO

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Project description:Nicotinic receptors (nAChR) are widely distributed in the adult brain where they mediate excitatory postsynaptic transmission and presynaptic release of other neurotransmitters. Functional nicotinic receptors are also present in the embryonic brain. Several studies have suggested a role for nAChRs in cognitive functions (learning and memory) and cellular events such as degeneration and survival. In vitro, activation of nAChR can differentially modulate neuritic outgrowth, and enhance early gene expression prior to neuronal differentiation. Furthermore, several reports have shown that nicotine can prevent cell death induced by activation of glutamatergic receptors, NMDA-subtype. Little is known of these events in the cerebral cortex. Knowledge of the genes activated by exposure of cortical neurons to nicotine or to an excitotoxic agent such as the glutamatergic agonist NMDA could potentially lead to new strategies for adult brain protection and repair. The effect of nicotine and the cembranoid 4R, a novel nicotinic receptor non competitive antagonist, will be analyzed in primary cultures of cortical neurons, in the presence or the absence of NMDA, to identify which genes affected by NMDA receptor activation are modulated nicotine or 4R. We hypothesize that the neuroprotective effect of nAChRs is mediated by modulation of the changes in gene expressions elicited by NMDA. In order to detect early changes in gene expression, primary culture of cortical neurons prepared from 16 day old Sprague Dawley rat embryos at 11 days in culture will be treated for different times (10, 30, 60, 180, 360, min) with 1 mM NMDA and then harvested. In a second set of experiments, neuronal cultures will be treated with NMDA for 10, 30 and 60 min then the drug will be washed out and the cells will be harvested 24 h later. It was reported that 8-24 h pretreatment of neurons with 10 micromolar nicotine is neuroprotective, therefore we will treat separate cultures with either 10 micromolar nicotine or 2 micromolar 4R for 18 h and then with NMDA for 30 min or 1 h. The cells will be either harvested soon after NMDA treatment or the drugs will be washed out and the cells harvested 24 h later. We will also examine sample treated only with either 4R or nicotine for 15 min or 18 h in order to determine which genes are differentially regulated by these drugs. These experiments will allow us to detect any change in gene expression elicited soon by each treatment or with some delay. The samples in Trizol will be kept at -70 until all the 3 or 5 replicates are harvested and then the RNA will be purified. RNA from 3-5 independent experiments for each different protocol or treatment together with controls will be sent to the facility.

Project description:Many addictive drugs such as nicotine mediate reward and reinforcing mechanisms within the mesolimbic pathway involving midbrain dopaminergic (mDA) neurons via nicotinic acetylcholine receptors (nAChRs). Previously, genome-wide association analyses (GWAs) identified several nucleotide polymorphism (SNP) associated with increased risk of addictive phenotypes including the nonsynonymous rs16969968 SNP encoding D398->N398 variation in the CHRNA5 gene (Bierut et al., 2008; Saccone et al., 2007) . However, the etiology and the pathophysiology associated with the N398 allele is unknown. Previous animal studies using a knock-in of human CHRNA5 N398 revealed increased nicotine consumption. However, given the evolutionary distance between mice and humans, identifying intrinsic factors that affect addiction may correlate poorly, limiting the conclusiveness of these studies. In this study, induced pluripotent stem cell (iPSC) lines were derived from cryopreserved lymphocytes drawn from patients with homozygous minor alleles (N398) of rs16969968 and demonstrated nicotine addiction as well as age- and gender-matched unaffected controls carrying the major allele (D398). Cultures of primarily mDA neurons were prepared from these iPSC lines. Gene expression analysis using RT-PCR and RNAseq revealed similar expression of mRNAs encoding subunits of nAChR in mDA neurons derived from both D398 and N398 iPSC lines, however, gene ontology (GO) analysis revealed an increased enrichment of genes associated with neuroactive ligand-receptor interactions and Ca2+ signaling pathways in N398 neurons. Moreover, the N398 neuronal population responded more actively to application of nicotine in both electrophysiological analysis and Ca2+-imaging analysis. Together, these results suggest that the N398 variation affects Ca2+-signaling in neurons, which may explain the predisposition of subjects carrying this mutation for addictive behavior in subjects carrying the nonsynonymous rs16969968 SNP (Sherva et al., 2008).

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Devellis-5U24NS043562-02

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