The Journal of Neuroscience, February 15, 2006, ():
Glutamate Receptor Exocytosis and Spine Enlargement during Chemically Induced Long-Term Potentiation
J. Neurosci. Kopec et al.
26: 2000
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Table 1
Spines potentiate as autonomous units. Autocorrelation and Fourier analysis were conducted on spines and dendrites of cells expressing either SEP-GluR1, SEP-GluR2, SEP-NR2A, or SEP-NR2B. For each subunit and analysis type the fold and absolute change of receptor and volume in spines and dendrites was analyzed for either positive or negative neighbor or regional correlations. Non-significant (N.S.) denotes p > 0.05.
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Supplemental Figure 1
Individual spine and edge analysis method. (a) Example of how pairs of spine and dendrite regions of interest (ROIs) are defined. (b) Integrated fluorescence within the ROI is plotted verses depth (Z-stack) in the tissue. Z boundaries are defined by the full width at half max after background subtraction. (c) Example of how edge ROIs are positioned on the edge of dendrites between spines. ROIs are drawn here on a ratio image for clarity. Analysis of edge ROIs is performed on raw fluorescence data.
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Supplemental Figure 2
Total spine and total dendrite analysis method. Left panel: Example images of dendritic segment expressing SEP-GluR1. ROI for analyzing spines and dendrite combined drawn in blue. ROI for analyzing entire dendrite drawn in red. Individual spine ROIs drawn as in Supp. Fig 1 but are not shown here for clarity. Right panel: Integrated fluorescence from ROIs. Spine data is the sum of all spine ROIs. Calculation for dendrite surface GluR1 vs. spine surface GluR1 shown at bottom. This data originates from the dendritic segment shown at the left. Data in the manuscript is the combined analysis of all dendritic segments.
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Supplemental Figure 3
Enrichment is independent of receptor expression level. Average enrichment of SEP-GluR1 from 8 cells is plotted against each cell’s mean SEP-GluR1 dendritic fluorescence.
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Supplemental Figure 4
Synaptic incorporation of NR2A and NR2B. (a) Whole-cell patch clamp recordings from CA1 pyramidal cells expressing either an NR2A or NR2B mutant that permits inward current at holding potential of –60 mV. The ratio of NMDA receptor mediated current (measured as the average current between 110 to 160 ms) to AMPA mediated current at –60 mV holding potential was plotted for non-transfected cells (n = 6), cells transfected with NR2A (n = 7) or NR2B mutant (n = 9), and cells transfected with the receptor mutants in the presence of APV (NR2A(N/Q) +APV n = 5; NR2B(N/Q) +APV n = 4). * denotes p < 0.01. Error bars SEM. (b) Representative paired recording of one non-transfected cell and an adjacent cell transfected with NR2A mutant. (c) Representative recording of a cell transfected with NR2A mutant before and after application of APV. (d) Representative paired recording of one non-transfected cell and an adjacent cell transfected with NR2B mutant. (e) Representative recording of a cell transfected with NR2B mutant before and after application of APV. For comparison, currents are scaled to the same amplitude for b-e.
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Supplemental Figure 5
Expression of recombinant NR2A does not affect LTP. CA1 neurons were infected at 16-18 days in vitro with Sindbis virus expressing GFP-NR2A and allowed to express for 36 to 72 hrs. Infected cells show similar LTP compared with non-infected cells.
