Journal: The Journal of Neuroscience
Article Title: Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development
Figure Lengend Snippet: Downregulation of the α2δ3 subunit impairs spontaneous GABA release and leads to suppression of neuronal firing in developing hippocampal neurons. A , B , The shRNA-mediated knockdown of the α2δ3 subunit results in a significant decrease of the α2δ3 surface expression in HEK293T was examined via fluorescence labeling of HA-tagged α2δ3 subunits in HEK293T cells ( A , B ), compared with the effect of scrambled (scr) shRNA. Scale bar, 20 µm. C , D , Downregulation of the α2δ3 subunit in rat hippocampal neurons. Scale bar, 20 µm. E , F , Western blots of HEK293T cells expressing the HA-tagged α2δ3 subunit together with the scrambled shRNA control or the α2δ3 shRNA. G-I , Western blots of hippocampal cultures infected with the HA-tagged α2δ3 construct ( G ) or with the scrambled shRNA control, as well as the α2δ3 shRNA (blue) ( H , I ). J , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in K , N . K , Downregulation of the α2δ3 subunit, but not the GFP expression, significantly decreases the mean mIPSC frequency in rat hippocampal neurons. L . Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. M , The mean mEPSC amplitude is not affected by either α2δ3 knockdown or by lentiviral expression of the GFP. N , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. O , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in P , Q . P , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ3 overexpression (blue) or α2δ3 knockdown (petrol). Thirty of 60 channels from each array are shown. Scale bar, 20 s. Q , The α2δ3 overexpression in young neurons strongly enhances neuronal activity, whereas the shRNA-mediated α2δ3 knockdown leads to dramatic suppression of the mean firing rate. * p
Article Snippet: For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428).
Techniques: shRNA, Expressing, Fluorescence, Labeling, Western Blot, Infection, Construct, Over Expression, Activity Assay