RESUMEN
Sequential neural dynamics encoded by "time cells" play a crucial role in hippocampal function. However, the role of hippocampal sequential neural dynamics in associative learning is an open question. In this manuscript, we used two-photon Ca2+ imaging of dorsal CA1 pyramidal neurons in head-fixed mice performing a go-no-go associative learning task. We found that pyramidal cells responded differentially to the rewarded or unrewarded stimuli. The stimuli were decoded accurately from the activity of the neuronal ensemble, and accuracy increased substantially as the animal learned to differentiate the stimuli. Decoding the stimulus from individual pyramidal cells that responded differentially revealed that decision-making took place at discrete times after stimulus presentation. Lick prediction decoded from the ensemble activity of cells in dCA1 correlated linearly with lick behavior indicating that sequential activity of pyramidal cells in dCA1 constitutes a temporal memory map used for decision-making in associative learning.
RESUMEN
The properties of H(+)-ATPase from rat liver lysosomes were analyzed by reconstituting proton pump activity from solubilized enzyme and Escherichia coli phospholipids in proteoliposomes devoid of anion-channels. The reconstitution procedure involved solubilization of the ATPase with n-octyl-beta-D-thioglucoside in the presence of asolectin, and incorporation of the solubilized enzyme into E. coli phospholipid liposomes by dilution, freeze-thawing, and sonication. Proton pump activity of reconstituted H(+)-ATPase as detected by the ATP-dependent quenching of acridine orange fluorescence indicated that ATP can be replaced with dATP and to a lesser extent with GTP, but not with any other nucleotide, that Mg2+ can be replaced with Mn2+, but not with Ca2+, Sr2+, or Ba2+, that Zn2+, Pd2+, Cd2+, and Hg2+ were inhibitory, and that the enzyme was sensitive to inhibitors of v-type H(+)-ATPase, including bafilomycin A1, N-ethylmaleimide, DCCD, DIDS, and tri-n-butyltin. The enzyme showed unique sensitivity to anions and was activated by chloride, fluoride, and bromide from inside, but not from outside the vesicles. It was inhibited by sulfate, sulfite, and thiocyanate from outside the vesicles, and by nitrate from both inside and outside the vesicles.
Asunto(s)
Lisosomas/enzimología , Proteolípidos/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , Cationes Bivalentes/farmacología , Detergentes , Escherichia coli , Cinética , Liposomas , Fosfatidilcolinas , Fosfolípidos , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/aislamiento & purificación , Ratas , Solubilidad , Especificidad por Sustrato , Tioglucósidos/farmacologíaRESUMEN
Lysosomal H(+)-ATPase was purified to homogeneity from rat liver lysosomes. It is a bafilomycin A1-sensitive Mg(2+)-ATPase, which reacts with antibodies against the 16- and 70-kDa subunits of vacuolar H(+)-ATPase (Nezu, J., Motojima, K., Tamura, H., and Ohkuma, S. (1992) J. Biochem. (Tokyo) 112, 212-219), and has been separated from both the N-ethylmaleimide (NEM)-sensitive/bafilomycin A1-insensitive Mg(2+)-ATPase (ATPase I) and the NEM-insensitive Mg2+/Ca(2+)-ATPase (ATPase II) (Hayashi, H., Arai, K., Sato, O., Shimaya, A., Sai, Y., and Ohkuma, S. (1992) Chem. Pharm. Bull. 40, 2783-2786). The purified enzyme had the subunit structure of vacuolar H(+)-ATPase, consisting of 110-, 70-, 56-, 42-, 39-, 34-, (32-,) and 16-kDa proteins. It had optimal activity at a pH of 7.0-8.0, with an apparently single Km value for ATP of 95 microM. It hydrolyzed ATP > or = dATP >> GTP, ITP >> UTP, but not CTP, and was inhibited by ADP. It demonstrated divalent cation specificity in the order of Mg2+, Mn2+ > Fe2+, Co2+ > Ca2+. Among various anions, Cl-, Br-, and F- activated ATPase activity, whereas NO3- inhibited activity. It was inhibited not only by bafilomycin A1 but also by NEM, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and dicyclohexylcarbodi-imide. The purified enzyme was incorporated into proteoliposomes where its proton pump activity was reconstituted. This suggested that the isolated enzyme maintains its H+ translocation activity. These findings suggest that the isolated enzyme is an anion-sensitive vacuolar type H(+)-ATPase that functions as a lysosomal proton pump.