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Using animals selectively bred for high and low trait anxiety to investigate the role of Neuropeptide S in anxiety disorders
ABSTRACT NOT AVAILABLE AT THE TIME OF GOING TO PRINT
S9/3 USING ANIMALS SELECTIVELY BRED FOR HIGH AND LOW TRAIT ANXIETY TO INVESTIGATE THE ROLE OF NEUROPEPTIDE S IN ANXIETY DISORDERS David Slattery; University of Regensburg, Regensburg, Germany
doi:10.1016/j.npep.2017.02.034
S9/5
GENOME EDITING TO EXPLORE THE EFFECTS OF ALLELIC VARIATION AND EPIGENETICS ON GENE REGULATION IN HEALTH AND DISEASE Alasdair MacKenzie, Elizabeth Hay, Evgeni Ivanov; University of Aberdeen, Aberdeen, UK We have used comparative genomics to identify many of the gene regulatory regions that control the tissue specific expression of a number of different neuropeptide genes. Characterisation of these sequences, and their disease associated allelic variants, were subsequently achieved using a combination of primary cell reporter assays and transgenic mouse analysis. However, our studies have been limited by a lack of a cheap and efficient method of deleting these regulatory sequences from the mouse genome that would allow functional analysis in-vivo. CAS9/CRISPr technology permits the targeted introduction of double stranded cuts into specific regions of the mouse genome. These cuts are then repaired by the cells natural repair mechanisms that can be exploited to delete or introduce specific sequences. In order to determine if CAS9/CRISPr could be used to remove enhancers from the mouse genome we identified single guide RNA (srRNA) target sequences flanking the ECR1 sequence (CNR1 locus) and the GAL5.1 sequence (Galanin locus). sgRNA molecules were transcribed from a cloning free T7 template and injected into the cytoplasm of single-cell mouse embryos with CAS9 mRNA to give a 90% survival. These embryos were transferred into host mothers and gave a 25% implantation rate. Subsequent PCR analysis of resulting mice showed that over 60% were homozygous for the targeted knockout. Although these mice are still to be screened for off-target effects it is very clear that CRISPr technologies represent the most important development in our understanding of neuropeptide gene regulatory mechanisms since the discovery of recombinant DNA technology.
EC
TE
DP
Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behaviour (HAB) versus low anxiety-related behaviour, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behaviour. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity-an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioural consequence of these differences revealed that intracerebroventricular NPS reversed the hyper-anxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date.
the i.c.v. infusion of the vehicle, artificial cerebrospinal fluid (aCSF), were prevented in GAL-treated animals. GAL was also able to modulate the changes in M1 and M2 MR densities observed in aCSF-treated rats, and to increase the activity of MR in the hippocampus. The administration of aCSF was also accompanied by an increased number of positive fibers and cells for GAL along the cortical tract of the injection. Furthermore, GAL avoided the decrease of GAL receptor density in hippocampus observed in vehicle-treated rats. The acetylcholinesterase-positive neurons in BF were reduced in both groups of treatment. The ex vivo models of organotypic culture showed a neuroprotective effect of GAL under diverse conditions (oxidative stress, IgG-saporin and beta-amyloid oligomers). In summary, GAL improves memory-related abilities and preserves the cell viability.
OF
S9/2
Abstracts
RO
138
S9/4 0078
RR
doi:10.1016/j.npep.2017.02.033
CO
EFFECTS OF GALANIN TREATMENT ON MEMORY AND THE CHOLINERGIC SYSTEM
UN
Status: Accepted Presentation type: Oral Author's preference: Oral
doi:10.1016/j.npep.2017.02.035
Gabriel Barreda, Laura Lombardero, Maria Teresa Giralt, Iván Manuel, Rafael Rodríguez; University of the Basque Country (EHU/UPV), Bilbao/ Basque Country, Spain The cholinergic pathways, which originate in the basal forebrain (BF) and are responsible for the control of cognitive processes including learning and memory, are regulated by some neuropeptides, such as galanin (GAL) that is also involved in both neurotrophic and neuroprotective actions. The present study has evaluated the effect of GAL, both in vivo and ex vivo. The effect on cognition was evaluated after a subchronic treatment with GAL, by analyzing the passive avoidance response. The modulation of muscarinic receptor (MR) densities and activities was studied in the same treated animals. Some cognitive deficits induced by
S10/1 A ROLE FOR GALANIN N-TERMINAL FRAGMENT (1-15) IN ANXIETY AND DEPRESSION IN RATS Carmelo Millóna, Antonio Flores-Burguessa, Manuel Narváeza, Dasiel Borroto-Escuelab, Luis Santína, Jose Angel Narváeza, Kjell Fuxeb, Zaida Díaz-Cabialea; aUniversidad de Málaga, Málaga, Spain; bKarolinska Institute, Stockholm, Sweden
S9/3 USING ANIMALS SELECTIVELY BRED FOR HIGH AND LOW TRAIT ANXIETY TO INVESTIGATE THE ROLE OF NEUROPEPTIDE S IN ANXIETY DISORDERS David Slattery; University of Regensburg, Regensburg, Germany
doi:10.1016/j.npep.2017.02.034
S9/5
GENOME EDITING TO EXPLORE THE EFFECTS OF ALLELIC VARIATION AND EPIGENETICS ON GENE REGULATION IN HEALTH AND DISEASE Alasdair MacKenzie, Elizabeth Hay, Evgeni Ivanov; University of Aberdeen, Aberdeen, UK We have used comparative genomics to identify many of the gene regulatory regions that control the tissue specific expression of a number of different neuropeptide genes. Characterisation of these sequences, and their disease associated allelic variants, were subsequently achieved using a combination of primary cell reporter assays and transgenic mouse analysis. However, our studies have been limited by a lack of a cheap and efficient method of deleting these regulatory sequences from the mouse genome that would allow functional analysis in-vivo. CAS9/CRISPr technology permits the targeted introduction of double stranded cuts into specific regions of the mouse genome. These cuts are then repaired by the cells natural repair mechanisms that can be exploited to delete or introduce specific sequences. In order to determine if CAS9/CRISPr could be used to remove enhancers from the mouse genome we identified single guide RNA (srRNA) target sequences flanking the ECR1 sequence (CNR1 locus) and the GAL5.1 sequence (Galanin locus). sgRNA molecules were transcribed from a cloning free T7 template and injected into the cytoplasm of single-cell mouse embryos with CAS9 mRNA to give a 90% survival. These embryos were transferred into host mothers and gave a 25% implantation rate. Subsequent PCR analysis of resulting mice showed that over 60% were homozygous for the targeted knockout. Although these mice are still to be screened for off-target effects it is very clear that CRISPr technologies represent the most important development in our understanding of neuropeptide gene regulatory mechanisms since the discovery of recombinant DNA technology.
EC
TE
DP
Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behaviour (HAB) versus low anxiety-related behaviour, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behaviour. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity-an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioural consequence of these differences revealed that intracerebroventricular NPS reversed the hyper-anxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date.
the i.c.v. infusion of the vehicle, artificial cerebrospinal fluid (aCSF), were prevented in GAL-treated animals. GAL was also able to modulate the changes in M1 and M2 MR densities observed in aCSF-treated rats, and to increase the activity of MR in the hippocampus. The administration of aCSF was also accompanied by an increased number of positive fibers and cells for GAL along the cortical tract of the injection. Furthermore, GAL avoided the decrease of GAL receptor density in hippocampus observed in vehicle-treated rats. The acetylcholinesterase-positive neurons in BF were reduced in both groups of treatment. The ex vivo models of organotypic culture showed a neuroprotective effect of GAL under diverse conditions (oxidative stress, IgG-saporin and beta-amyloid oligomers). In summary, GAL improves memory-related abilities and preserves the cell viability.
OF
S9/2
Abstracts
RO
138
S9/4 0078
RR
doi:10.1016/j.npep.2017.02.033
CO
EFFECTS OF GALANIN TREATMENT ON MEMORY AND THE CHOLINERGIC SYSTEM
UN
Status: Accepted Presentation type: Oral Author's preference: Oral
doi:10.1016/j.npep.2017.02.035
Gabriel Barreda, Laura Lombardero, Maria Teresa Giralt, Iván Manuel, Rafael Rodríguez; University of the Basque Country (EHU/UPV), Bilbao/ Basque Country, Spain The cholinergic pathways, which originate in the basal forebrain (BF) and are responsible for the control of cognitive processes including learning and memory, are regulated by some neuropeptides, such as galanin (GAL) that is also involved in both neurotrophic and neuroprotective actions. The present study has evaluated the effect of GAL, both in vivo and ex vivo. The effect on cognition was evaluated after a subchronic treatment with GAL, by analyzing the passive avoidance response. The modulation of muscarinic receptor (MR) densities and activities was studied in the same treated animals. Some cognitive deficits induced by
S10/1 A ROLE FOR GALANIN N-TERMINAL FRAGMENT (1-15) IN ANXIETY AND DEPRESSION IN RATS Carmelo Millóna, Antonio Flores-Burguessa, Manuel Narváeza, Dasiel Borroto-Escuelab, Luis Santína, Jose Angel Narváeza, Kjell Fuxeb, Zaida Díaz-Cabialea; aUniversidad de Málaga, Málaga, Spain; bKarolinska Institute, Stockholm, Sweden