1. Extracellular Glutamate in the Nucleus Accumbens Is Nanomolar in Both Synaptic and Non-synaptic Compartments 
Delia N. Chiu, Craig E. Jahr 
Cell Reports 
Volume 18, Issue 11, Pages (March 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 2576-2583DOI: (10.1016/j.celrep.2017.02.047)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Standing NMDAR-Mediated Current in MSNs Reflects Nanomolar Glutamate
(A) Current traces from an MSN voltage-clamped at +40 mV aligned to baseline (dashed line) show inhibition of a standing current by 100 μM D-AP5 and outward current evoked by 5 μM NMDA.
(B) The current blocked by 100 μM D-AP5 was 0.87% ± 0.13% of maximal NMDA current (n = 19).
(C) The concentration of glutamate that corresponds to the D-AP5-sensitive current is 25.6 nM ± 3.2 nM in juvenile rats (P15–P22, n = 19) and 31.7 nM ± 8.7 nM in adult rats (8–12 weeks, n = 8). n.s., not significant.
In summary graphs, mean values (±SEM) are indicated in black; data from individual cells are indicated in gray.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Variance Analysis of Calcium Signals Reveals NMDAR Detection of glue at MSN Spines
(A) Top: 2PLSM image of MSN filled with Alexa Fluor 594 and Fluo-5F. Scale bar, 20 μm. Middle: MSN dendritic spine and shaft showing line scan position (dashed line). Scale bar, 2 μm. Bottom: line scan showing fluorescence in the green channel increasing sharply during depolarization in the spine but not the dendrite. Timing of depolarization and shutter opening indicated above.
(B) Fluo-5F fluorescence (upper traces, average in black) and variance (lower traces) from 320 trials from the dendrite shaft and spine shown in (A). Symbols indicate the times used to calculate baseline (open) and step (closed) fluorescence and variance. Predicted variance (dashed line) reflects the sum of dark noise and shot noise of the mean fluorescence signal. Timing of depolarization and shutter opening are indicated as in (A).
(C) Baseline (open symbols) and step (closed symbols) variance versus mean fluorescence for the spine (triangles) and dendrite shaft (circles) shown in (A). Measurements of pipette fluorescence (squares) reflect shot noise; the slope of the line fit (green) was used to calculate predicted variance. Variance due to dark noise is indicated by a dashed line.
(D) Summary graphs show measured variance normalized to predicted variance in spines and dendrite shafts. D-AP5 reduced step variance in spines, but not dendrite shafts, and did not affect baseline variance. Data are represented as mean ± SEM; individual pairs are in gray; ∗∗p =
(E) Removal of three of 320 trials shown in (B) greatly reduced step variance for the spine. The variance of the remaining trials is predicted by shot noise, as indicated also in (C) with a closed diamond.
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5. Figure 3 glue at Spines Remains Nanomolar with Uptake Blocked
(A) Current traces from an MSN voltage-clamped at +40 mV during application of 100 μM DL-TBOA and 5 μM NMDA.
(B) Steady-state currents evoked by 100 μM TBOA corresponded to activation by 123.2 nM ± 10.5 nM glutamate in juvenile rats (n = 16) and 142.5 nM ± 21.2 nM in adult rats (n = 4). n.s., not significant.
(C) The average frequency of NMDAR-mediated calcium transients increased in 100 μM DL-TBOA. In each condition, the sum of trials that showed NMDAR-mediated calcium transients divided by the sum of all trials is denoted by the gray lines (control: 78/10,513 = 0.74%; DL-TBOA: 55/1,709 = 3.22%). ∗∗∗p =
(D) Normalized baseline fluorescence in 20 μM NMDA and 100 μM DL-TBOA in spines (triangles) and dendrites (circles).
(E) Frame scans from the green channel during application of 20 μM NMDA (top) and 100 μM DL-TBOA (bottom). Drug application duration indicated by the bars above the images. Vhold = −70 mV throughout. Scale bar, 2 μm.
(F) Current traces from an MSN voltage-clamped at +40 mV during application of 100 μM DL-TBOA and 100 μM DL-TBOA plus 1 μM glutamate.
In summary graphs, mean values (±SEM) are indicated in black; data from individual cells are indicated in gray.
Cell Reports  , DOI: ( /j.celrep )
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6. Figure 4 MSNs Depolarize in the Equivalent of 0.5 μM Glutamate
Voltage traces from MSNs recorded in the absence of TTX, picrotoxin, NBQX, and d-serine. Application of 20 μM NMDA, equipotent to 0.5 μM glutamate, led to firing and sustained depolarization. In the same cell, application of 100 μM DL-TBOA resulted in a peak depolarization of 7 mV.
Cell Reports  , DOI: ( /j.celrep )
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