The Journal of Neuroscience, December 8, 2004, ():
Retina-Driven Dephosphorylation of the NR2A Subunit Correlates with Faster NMDA Receptor Kinetics at Developing Retinocollicular Synapses
J. Neurosci. Townsend et al.
24: 11098
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplement A – Surgical methods for implanting drug-infused Elvax plastic in the P06 mouse retina.
A) Under halothane anesthesia, the eyelid of P6 mouse pups was carefully opened. A scalpel blade was used to make small incision at the ciliary margin, and a sliver of Elvax plastic was inserted into the vitreous humor (see methods). The eyelid was sutured closed and secured with Vetbond.
B) The placement of drug-infused Elvax in one eye allowed for the selective reduction in visual activity to the contralateral sSC. The slow diffusion properties of Elvax permit the sustained release of glutamate receptor antagonists within the eye. The un-operated eye and its corresponding sSC served as a negative control.
C) On P11, the eyelids were opened, and each eye assessed for pupillary constriction in response to light. The animals were then anesthetized and sacrificed. The operated eye was dissected to verify the proper placement of the Elvax and appropriate healing of the surgical incision. Both the ipsilateral and contralateral superior colliculus was sectioned, and used for electrophysiological or biochemical experiments.
- supplemental material
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Supplement B – Synaptosome preparation from sSC neurons
Schematic representation of the synaptosome protocol adapted from Dunah & Standaert 2001. Superficial layers of the superior colliculus were homogenized in TEVP buffer (see methods) containing 320mM sucrose. Samples were centrifuged at 1000xg to pellet nuclei and large tissue fragments. The soluble fraction (S1) was then centrifuged at 10,000xg. The S2 fraction contained soluble proteins, while the P2 fraction was enriched for plasma membranes and organelles. The P2 fraction was then treated with hypo-osmotic TEVP buffer plus 0.2% TritonX-100. Synaptic fractions were largely resistant to mild detergent, and pelletted at 25,000xg in the P3 fraction.
The enrichment procedure was tracked using Western blotting. Fractions were run on 8% gels and probed with antibodies for various proteins unique to subcellular compartments. Histone 2B, a marker for nuclei, was highly enriched in the nuclear fraction (P1). Soluble proteins such as LDH were predominant in the S2 fraction. The synaptosomal fraction (P3) contained the synaptic marker PSD-95 with only trace amounts of the dendritic protein MAP2. This technique was used in Figures 5 & 6 to enrich for NR2A.
- supplemental material
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Supplement C – Curve fits for average spontaneous NMDAR currents at P08 and P11
A) Synaptosoft MiniAnalysis software was used to analyze spontaneous NMDAR currents in the sSC. Individual events over a 2-8 min period were averaged and fit with a single exponential curve. Estimates of rise-time, peak amplitude and decay-time (90-37%) were made for the average response. This technique was used to estimate the decay-time in Figures 1-4.
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