title: Establishment and characterization of immortalized neuronal cell lines derived from the spinal cord of normal and trisomy 16 fetal mice, an animal model of Down syndrome.
abstract: We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein-2 [MAP-2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo-1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACDP) (100 microM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N-methyl-D-aspartate (NMDA) and kainate, but not alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), produced [Ca2+)]i rises in both cell types. MTh cells exhibited faster time-dependent decay phase kinetics in glutamate-induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]-choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Down's syndrome.
authors.affiliation: Laboratory of Pharmacology, School of Medicine and Valparaíso Center for Cellular and Molecular Neuroscience, University of Valparaíso, Valparaíso, Chile.
authors.forename: Ana María
authors.initials: AM
authors.lastname: Cárdenas
authors.forename: David D
authors.initials: DD
authors.lastname: Allen
authors.forename: Christian
authors.initials: C
authors.lastname: Arriagada
authors.forename: Alexis
authors.initials: A
authors.lastname: Olivares
authors.forename: Lori B
authors.initials: LB
authors.lastname: Bennett
authors.forename: Raúl
authors.initials: R
authors.lastname: Caviedes
authors.forename: Alexies
authors.initials: A
authors.lastname: Dagnino-Subiabre
authors.forename: Isabel E
authors.initials: IE
authors.lastname: Mendoza
authors.forename: Juan
authors.initials: J
authors.lastname: Segura-Aguilar
authors.forename: Stanley I
authors.initials: SI
authors.lastname: Rapoport
authors.forename: Pablo
authors.initials: P
authors.lastname: Caviedes
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed / Q000378:metabolism / Q000473:pathology; D002794:Choline / Q000378:metabolism; D004195:Disease Models, Animal; D004314:Down Syndrome / Q000503:physiopathology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009474:Neurons / Q000737:chemistry* / Q000473:pathology; D009538:Nicotine / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology; D017470:Receptors, Glutamate / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000166:cytology* / Q000196:embryology / Q000473:pathology; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics; D000072105:Superoxide Dismutase-1; D014314:Trisomy
chemical_list: D018690:Excitatory Amino Acid Agonists; D017470:Receptors, Glutamate; D009538:Nicotine; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D002794:Choline; D000109:Acetylcholine; D002118:Calcium; D009638:Norepinephrine