Article Figures & Data
Figures
- Figure 1.
Deleting EAAC1 does not alter the glial uptake capacity, but speeds the time course of synaptically released glutamate clearance. A, Examples of raw and peak normalized STCs in WT (black) and KO mice (red). Each trace represents the averages of 20 trials. B, The bar chart shows the summary of the kinetic properties of STCs in WT (white) and KO mice (red). STCs decay faster in the absence of EAAC1 (rise time, p = 0.17; t50 **p = 0.002; <t
***p = 0.0002; WT n = 8, KO n = 13). C, Left: schematic of the experimental design. Electrical stimuli were delivered through a bipolar electrode in stratum radiatum to evoke glutamate release from Schaffer collateral fibers. Right: Raw WT (black) and KO (red) STCs. D, Deconvolution-derived clearance of synaptically released glutamate is faster in KO mice (*p = 0.02). Mean ± SEM. E, The STC clearance derived from centroid analysis is also faster in KO mice (*p = 0.02). F, Left: Schematic of the experimental design. MNI-l-glutamate (100 μm) was uncaged over the entire field of view. Right: Raw WT and KO FTCs. G, Deconvolution-derived clearance of caged glutamate is similar in WT and KO mice (p = 0.61). H, The FTC clearance derived from centroid analysis is similar in WT and KO mice (p = 0.29). Data in C–H have been obtained from the same population of astrocytes (WT n = 7, KO n = 6). I, Relationship between the STC charge transfer and the amplitude of the fiber volley preceding the STC. No difference in the extent of glutamate transport per afferent fiber recruitment is observed between WT and KO mice (WT n = 5, KO n = 6; raw STC charge p = 0.22; raw STC FV amplitude p = 0.42). J, STCs have a similar sensitivity to the GLT-1 antagonist DHK (300 μm) in WT (n = 5) and KO mice (n = 4, amplitude p = 0.71, t50 p = 0.47, <t p = 0.37). The inset shows representative examples of STCs recorded in control conditions and in the presence of DHK, in WT and KO mice. - Figure 2.
The structure of the neuropil does not change between WT and EAAC1 KO mice. A, Biocytin-filled CA1 pyramidal cell in WT (left) and KO mice (middle). Bottom panels are close-up views of dendritic regions in stratum radiatum. Right bar charts: Summary of spine density and size analysis in WT (n = 9) and KO mice (n = 6, density WT-KO p = 0.07, Power = 0.61; spine head diameter WT-KO p = 0.18, Power = 0.41). B, Electron micrographs showing the structure of the hippocampal neuropil in stratum radiatum 1 and 3 h after acute slice preparation and in perfused animals (right panel) in a WT and KO mouse. C, The total density of synapses does not change in vitro and is similar to that of perfused tissue in WT (n = 3) and KO mice (n = 3; 1 h WT-KO p = 0.39, Power = 0.20; 3 h WT-KO p = 0.14, Power = 0.44; perfused WT-KO p = 0.44, Power = 0.17). D, Examples of the analytical approach used to calculate the ECS fraction from EM data. An EM picture is shown together with its corresponding binary image. The ECS is drawn in black. E, Estimates of the ECS. No change is detected between WT and KO mice (1 h WT-KO p = 0.80, Power = 0.08; 3 h WT-KO p = 0.53, Power = 0.14; perfused WT-KO p = 0.41, Power = 0.19), but the ECS tends to be smaller after perfusion (WT 1 h-perfused *p = 0.04, 3 h-perfused p = 0.14; KO 1 h-perfused 0.08, 3 h-perfused *p = 0.03).
- Figure 3.
Diffusion in the hippocampal neuropil is similar in WT and KO mice. A, Representative images of TRdx3 diffusion in stratum radiatum of WT (top) and KO mice (bottom). Middle: Fluorescence intensity profiles and theoretical fits for these images. Right: A linear regression of γ2/4 over time yields an estimate of D* for TRdx3. B, Summary of the apparent diffusion coefficient (D*), tortuosity (λ), and hydrodynamic diameter (dH) for various fluorescent probes measured at 34°C in WT and KO neuropil. (D* TRdx3 WT n = 11, KO n = 9, p = 0.36, Power = 0.11; TRdx10 WT n = 8, KO n = 8, p = 0.18, Power = 0.38; TRdx40 WT n = 10, KO n = 8, p = 0.89, Power = 0.06; TRdx70 WT n = 14, KO n = 12, p = 0.82, Power = 0.10; QD655 WT n = 5, KO n = 3 p = 0.57, Power = 0.12) (λ TRdx3 p = 0.94, Power = 0.15; TRdx10 p = 0.20, Power = 0.36; TRdx40 p = 0.84, Power = 0.09; TRdx70 p = 0.46, Power = 0.40) (dH TRdx3 p = 0.60, Power = 0.13; TRdx10 p = 0.21, Power = 0.35; TRdx40 p = 0.81, Power = 0.08; TRdx70 p = 0.12, Power = 0.49). The numerical values are presented in Table 1. C, By using a model of parallel plane geometry for the hippocampal neuropil (Thorne and Nicholson, 2006), we show a similar diffusion behavior of the Texas red probes in WT and KO slices. The predicted values of λθ = 0 (the estimated tortuosity for a vanishingly small molecule) are λ = 2.18 ± 0.13 (WT) and λ = 2.06 ± 0.13 (KO). The estimated ECS width is 155.1 ± 9.5 nm (WT) and 120.4 ± 8.4 nm (KO). D, Fluorescence intensity profiles for Alexa Fluor 350 pressure applied in free medium (left), WT (middle) or KO slices (right). Each image represents the average of 10 consecutive trials, and the intensity profiles are measured 200 ms and 1 s after the puff. E, Summary of the diffusion coefficient and tortuosity measured in each condition at room temperature (Top: Dfree 0.64 ± 0.16 μm2/ms, n = 6; D*WT 0.32 ± 0.08 μm2/ms, n = 8; D*KO 0.30 ± 0.07 μm2/ms, n = 9, Dfree-D*WT ***p = 0.0002, Dfree-D*KO ***p = 6.1E-5, D*WT-D*KO p = 0.68, Power = 0.13). Bottom: λWT 1.45 ± 0.17; λKO 1.48 ± 0.17, p = 0.71, Power = 0.09). The estimated values of the diffusion coefficients at 34°C, obtained by multiplying the previous values by Q10 = 1.3 are: Dfree 0.84 ± 0.21 μm2/ms; D*WT 0.41 ± 0.10 μm2/ms; D*KO 0.39 ± 0.09 μm2/ms.
- Figure 4.
A larger population of NMDARs is activated when postsynaptic glutamate transport is impaired. A, NMDAR EPSCs in WT mice in control conditions (black) and in D-AA (100 μm; green) or CPP (1 μm; blue), at ±50 mV. Peak-scaled EPSCs are shown in gray. B, Summarized effects of D-AA (green; n = 11), l-AP5 (250 μm; brown; n = 9) and CPP (blue; n = 19) on the decay of NMDAR EPSCs at ±50 mV in WT mice (CPP+50 mV-D-AA+50 mV *p = 0.014; CPP+50 mV-l-AP5+50 mV *p = 0.036; CPP−50 mV-D-AA−50 mV p = 0.21; CPP−50 mV-l-AP5−50 mV p = 0.92). C, As in A, in KO mice. D, As in B, with summarized effects of D-AA (n = 8), l-AP5 (n = 10), and CPP (n = 10) in KO mice (CPP+50 mV-D-AA+50 mV *p = 0.021; CPP+50 mV-l-AP5+50 mV p = 0.107, CPP−50 mV-D-AA−50 mV *p = 0.023, CPP−50 mV-l-AP5−50 mV *p = 0.040; n = 6). Drug application in A–D was performed in separate groups of cells. EPSC analysis was performed 20 min after drug application, after confirming that the effects on the EPSC profile were stable. E, Left: Raw traces of composite peak-normalized EPSCs recorded at −70 mV, in 0 Mg2+. Right: the average peak-normalized decay is plotted on a semilog scale (WT n = 13, KO n = 27). Mean ± SEM F, Summary of the NMDAR and AMPAR contribution to composite EPSCs. Initial slope and Q30-80 were normalized by the corresponding mean values in WT mice. The NMDAR component is larger in KO than in WT mice (*p = 0.016). Mean ± SEM. G, Left: Raw traces of peak-normalized composite EPSCs recorded at +50 mV. Right: The average peak-normalized decay is plotted on a semilog scale (WT n = 11, KO n = 11). Mean ± SEM. H, As in F, at +50 mV (p = 0.34). Mean ± SEM.
- Figure 5.
EAAC1 limits activation of NR2B-containing NMDARs. A, NMDAR EPSCs in WT and KO mice in control conditions (black, WT; red, KO) and after application of ifenprodil (5 μm), at ±50 mV. Gray, peak-scaled EPSCs in ifenprodil. B, NMDAR EPSCs at −50 with internal NMDG+, in WT mice. C, Summarized effects of NR2B antagonists on EPSC amplitude at ±50 mV [ifenprodil WT+50 mV **p = 0.002, WT−50 mV p = 0.43, n = 9 (white circle), KO+50 mV **p = 0.002, KO−50 mV **p = 0.007, n = 10 (red circle), WT-KO+50 mV p = 0.38, WT-KO−50 mV *p = 0.040)] The hollow white square represents the mean reduction of EPSC amplitude in ifenprodil at −50 mV in WT mice, with internal NMDG+ (ifenprodil+NMDG+ WT−50 mV **p = 0.003, n = 7). D, NMDAR EPSCs in WT and KO mice in ifenprodil, in control conditions (black, WT; red, KO) and after D-AA (100 μm, green). Gray: peak-scaled EPSCs. E, Summary of D-AA effects on EPSC t50 in ifenprodil at ±50 mV, in WT (white circle) and KO mice (red circle) (WT D-AA+50 mV p = 0.24, WT D-AA−50 mV **p = 0.006, n = 8; KO D-AA+50 mV p = 0.37, KO D-AA−50 mV *p = 0.043, n = 5, WT-KO+50 mV p = 0.13, WT-KO−50 mV p = 0.13).
- Figure 6.
TBS-induced LTP is impaired in KO mice, but is rescued by ifenprodil. A, Time course of baseline-normalized fEPSC slope in WT slices (n = 9). Traces at the top are average responses in test and control pathways 5–0 min before, and 5–10 min and 25–30 min after TBS. The induction protocol consisted of 5 bursts at 5 Hz (100 Hz and 0.06 s each). B, Summarized effect of TBS on fEPSC slope in control and test pathways (WT Ctrl-Test **p = 0.001). C, Same as in A, in KO mice (n = 7). D, As in B, in KO mice (KO Ctrl-Test p = 0.45; WT Test-KO Test *p = 0.013). E, As in A, in KO mice and in the presence of ifenprodil (5 μm; n = 6). F, As in B, in KO mice and in the presence of ifenprodil (KO Ctrl-Test **p = 0.003; KO Test-KO Test ifenprodil *p = 0.029; WT Test-KO Test ifenprodil p = 0.50). All data in this figure represent mean ± SEM. G, Time course of baseline-normalized fEPSC slope in WT slices (n = 13) in the presence of DHK (15 μm). H, Summarized effect of TBS on fEPSC slope in control and test pathways (WT DHK Ctrl-Test *p = 0.016).
- Figure 7.
LTD, maximal LTP, and depotentiation are not affected by EAAC1. A, Left: Time course of baseline-normalized fEPSC slope before and after a LFS protocol (1 Hz, 15 min) in WT mice (n = 6). Right: summarized effects (WT Ctrl-Test **p = 0.002). B, Summary of the effect of ifenprodil (5 μm) on LFS-induced LTD, in WT slices (n = 8, WT Ctrl ifenprodil-Test ifenprodil *p = 0.048; WT Test-WT Test ifenprodil p = 0.75). C, As in A in KO mice (n = 7, KO Ctrl-Test **p = 0.006; WT Test-KO Test p = 0.77). D, As in B, in KO mice (n = 8, KO Ctrl ifenprodil-Test ifenprodil *p = 0.02, KO Test-KO Test ifenprodil p = 0.60; WT Test ifenprodil-KO Test ifenprodil p = 0.53). E, Left: Time course of baseline-normalized fEPSC slope following three tetani (100 Hz, 1 s) applied every 15 min and one LFS depotentiating protocol (1 Hz, 10 min) in WT mice (n = 8). Center: Summarized effect of tetanic stimulations and subsequent LFS on fEPSC slope, in WT mice (WT T3-Depot **p = 0.003). Right: fEPSC slope expressed relative to a 5 min baseline beginning 10 min after each tetanic stimulation (WT Depot/T3 ***p = 0.0008). F, As in E, in KO mice (KO T3-Depot **p = 0.007; KO Depot/T3 **p = 0.002). All data are displayed as mean ± SEM.
- Figure 8.
EAAC1 tips the balance between TBS-induced potentiation and depression. A, Average time course of baseline-normalized fEPSC slope following three tetanic stimulations (100 Hz, 1 s) and one TBS (5 bursts at 5 Hz, 100 Hz and 0.06 s each) in WT mice (n = 13; left). The bar chart summarizes the average magnitude of fEPSC slope, normalized to the initial baseline value, 10–15 min after each tetanus and 25–30 min after TBS (center: WT T1–T2 *p = 0.02, WT T3-TBS p = 0.23). Data in the right panel represent the fEPSC slope measured 10–15 min after T2, T3, and TBS relative to a baseline fEPSC slope measured 10–15 min after T1, T2, and T3, respectively (WT T2/T1 *p = 0.02; WT TBS/T3 p = 0.50). B, As in A, in KO mice (n = 11; KO T3-TBS *p = 0.038; KO TBS/T3 *p = 0.017). C, As in B, but ifenprodil was applied 8.3 min after T3 (n = 17; KO T3-TBS ifenprodil p = 0.058; KO TBS/T3 ifenprodil p = 0.079). In KO mice, maximal LTP was not significantly different in the absence (B) and presence (C) of ifenprodil (KO T1–T1 ifenprodil p = 0.051; KO T2–T2 ifenprodil p = 0.057; KO T3–T3 ifenprodil p = 0.062). D, Average time course of baseline-normalized fEPSC slope following two LFS stimulations (1 Hz, 15 min) and one TBS (5 bursts at 5 Hz, 100 Hz and 0.06 s each), in WT mice (n = 14; left). The bar chart summarizes the extent of change in fEPSC slope at the end of each test period (center: WT LFS1-LFS2 *p = 0.02, WT LFS2-TBS **p = 0.003). The plot on the right shows changes in fEPSC slope in respect to baseline measured before each stimulation protocol (WT LFS2/LFS1 **p = 0.002, WT TBS/LFS2 ***p = 0.0006). E, As in D, in KO mice (n = 12; center: KO LFS1-LFS2 p = 0.59, KO LFS2-TBS **p = 0.003; right: KO LFS2/LFS1 p = 0.80, KO TBS/LFS2 ***p = 0.0008). All data are displayed as mean ± SEM.
- Figure 9.
Three-dimensional Monte Carlo model of the synaptic and extrasynaptic environment. A, The first diagram illustrates the arrangement of NMDARs (blue symbols) and EAAC1 molecules (green symbols) in the postsynaptic area directly facing the presynaptic terminal (r = 250 nm). The PSD is the inner white circle (r = 110 nm) and the perisynaptic area the 70-nm-wide ring at the edge of the apposition zone. NMDARs are randomly scattered in the PSD at d = 300 μm−2, and at d = 60 μm−2 beyond the PSD. In this example, for EAAC1, d = 1080 μm−2. The next images show the arrangement of the presynaptic and postsynaptic elements and of neighboring astrocytic processes (wedges). The ECS width between the synapse and the wedges is 20 nm, 50 nm, 100 nm, 200 nm, respectively. The white dots represent glial glutamate transporters, present at d = 10,800 μm−2. At t = 0, 2000 glutamate molecules were released from a point source 20 nm above the center of the PSD (see Materials and Methods for details). B, Top: The four panels show the simulated astrocytic STCs obtained at the different values of ECS width described in A. The red traces represent simulated STCs obtained in the absence of EAAC1. The green traces represent simulated STCs obtained in the presence of perisynaptic EAAC1 (d = 10,800 μm−2), for different levels of efficiency of glutamate transport (dark green, EAAC1 efficiency = 65%, like that of glial transporters; green, EAAC1 efficiency = 15%; light green, EAAC1 efficiency = 0%). The insets display STC profiles, normalized by that observed in 20 nm ECS width, with no glial or neuronal transporters. Middle: Summary results of the influence on <t
STC (filled circles and continuous lines) and ampSTC (filled squares and dotted lines) of varying the density of expression of EAAC1, its transport efficiency, and the width of ECS around an active synapse. Bottom: Effect of varying EAAC1's transport efficiency on perisynaptic NMDAR activation. C, Effect of glial transporters and EAAC1 (efficiency = 15%) on the glutamate concentration profile monitored in the extracellular volume above the PSD (0 nm), the perisynaptic ring (215 nm), and a 100-nm-wide extrasynaptic region centered 425 nm away from the center of the PSD (425 nm). The concentration of glial transporters ([T]) is set to 10,800 μm−2. The concentration of EAAC1 ([EAAC1]) is set to 0 μm−2 in the black and red traces, whereas it matches the one of glial transporters (10,800 μm−2) in the green traces (both glial and neuronal transporters are present in these simulations). Glial transporters curtail the decay of the glutamate transient within the synapse, and also decrease the peak glutamate concentration extrasynaptically. EAAC1 decreases the peak but, due to its low transport efficiency, prolongs the decay of the glutamate transient in perisynaptic and extrasynaptic regions. D, Summary of the effects of glial transporters (red) and EAAC1 (green) on the peak (solid lines) and the integral (dashed lines) of the glutamate concentration at different distances from the release site. The gray shaded area represents the region in which EAAC1 is expressed. E, Example traces showing the effect of glial and glutamate transporters on the activation of perisynaptic NMDARs. F, Summary of the effects of glial transporters (red) and EAAC1 (green) on the peak open probability of NMDARs at increasing distances from the release site, for different values of ECS width. The dashed line represents the relative effect of EAAC1 compared with what measured in the presence of glial transporters only. EAAC1 limits NMDAR activation particularly in the perisynaptic region. The level to which this effect extends in the extrasynaptic compartment depends on the ECS width.
Tables
- Table 1.
Summary of the diffusion parameters estimated through the integrative optical imaging approach in free solution, in WT and KO slices
Probe Dfree (μm2/ms) D* (μm2/ms) λ dH (nm) WT KO WT KO WT KO TRdx3 0.33 ± 0.11 (12) 0.074 ± 0.025 (11) 0.080 ± 0.039 (9) 2.19 ± 0.36 2.07 ± 0.47 9.13 ± 3.02 8.32 ± 3.80 TRdx10 0.37 ± 0.13 (8) 0.080 ± 0.006 (8) 0.085 ± 0.010 (8) 2.16 ± 0.08 2.09 ± 0.12 7.71 ± 0.56 7.23 ± 0.86 TRdx40 0.18 ± 0.13 (9) 0.021 ± 0.004 (10) 0.020 ± 0.004 (8) 2.99 ± 0.30 3.02 ± 0.37 30.58 ± 6.42 31.39 ± 8.10 TRdx70 0.19 ± 0.09 (7) 0.008 ± 0.005 (14) 0.009 ± 0.002 (12) 5.48 ± 1.51 4.84 ± 0.59 103.47 ± 55.47 76.27 ± 18.28 QD655 0.075 ± 0.048 (7) 0.0004 ± 0.0004 (5) 0.0008 ± 0.0015 (3) N.A. N.A. N.A. N.A. -
N.A., not applicable. The numbers in parentheses represent the number of experiments done.
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Supplemental Data
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