Rat hippocampal neurons were transfected using 0.5 to 0.8 µg DNA per well in a 24-well plate and NeuroMag at a 3:1 ratio.
This paper shows the high efficiency of NeuroMag from OZ Biosciences to transfect long term cultured neurons (DIV>12).article reference: PLoS One. 2014 Apr 4;9(4):e94037.
PSD95 Suppresses Dendritic Arbor Development in Mature Hippocampal Neurons by Occluding the Clustering of NR2B-NMDA Receptors.
Bustos FJ, Varela-Nallar L, Campos M, Henriquez B, Phillips M, Opazo C, Aguayo LG, Montecino M, Constantine-Paton M, Inestrosa NC, van Zundert B.
Abstract
Considerable
evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B
are critical mediators of synaptic plasticity and dendritogenesis;
however, how they differentially regulate these processes is unclear.
Here we investigate the roles of the NR2A and NR2B subunits, and of
their scaffolding proteins PSD-95 and SAP102, in remodeling the
dendritic architecture of developing hippocampal neurons (2-25 DIV).
Analysis of the dendritic architecture and the temporal and spatial
expression patterns of the NMDARs and anchoring proteins in immature
cultures revealed a strong positive correlation between synaptic
expression of the NR2B subunit and dendritogenesis. With maturation, the
pruning of dendritic branches was paralleled by a strong reduction in
overall and synaptic expression of NR2B, and a significant elevation in
synaptic expression of NR2A and PSD95. Using constructs that alter the
synaptic composition, we found that either over-expression of NR2B or
knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature
neurons. Reactivation of dendritogenesis could also be achieved in
mature cultured neurons, but required both manipulations simultaneously,
and was accompanied by increased dendritic clustering of NR2B. Our
results indicate that the developmental increase in synaptic expression
of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby
restricts reactivation of dendritic branching. Experiments with
shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that
C-terminus of the NR2B subunit (tail) was sufficient to induce robust
dendritic branching in mature hippocampal neurons, and suggest that the
NR2B tail is important in recruiting calcium-dependent signaling
proteins and scaffolding proteins necessary for dendritogenesis.
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