Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons

Abstract

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.

Figure 1. Two-photon excitation of MNI-glutamate in cultured hippocampal neurons

(a) Structure of MNI-glutamate. (b) Cumulative amplitude distributions of mEPSCs in the absence (black) or presence (green) of MNI-glutamate (5 mM). Traces of mEPSCs in the absence (black) and presence (green) of MNI-glutamate are overlaid. Each trace is an average of 20 mEPSCs. A representative of three experiments. (c) Average of 28 current traces obtained from a cell in response to photolysis of MNI-glutamate for 50 μs with 7 mW of 720-nm-wavelength light. (d) Average of 10 mEPSCs recorded from a cell that exhibited rapid mEPSCs. The smooth lines in (c) and (d) are drawn as described in Methods. (e) Superimposition of the 2pEPSC (green) and mEPSC (black) shown in (c) and (d), respectively. (f, g) Amplitudes of 2pEPSCs (green dots) along horizontal (f) and vertical (g) lines crossing the center of an AMPA receptor cluster, and fluorescence profiles of a 0.1-μm diameter bead (black dots) along the horizontal (f) and vertical axis (g). The smooth black lines represent Gaussian fitting of the data. The smooth green lines are predicted from a theory described in Methods. (h) Stability of 2pEPSCs. Insets, superimposed current traces. (i) Double-logarithmic plot of the dependence of 2pEPSC amplitudes on the incident power of the mode-locked laser. The duration of the irradiation was 0.6 ms. The slope of solid and dashed straight lines are 2.3 and 2, respectively.

Figure 2. Functional mapping of glutamate sensitivities in hippocampal neurons

(a) Fluorescence image of a neuron in culture. The red box indicates a region of interest for functional mapping. (b) Pseudocolor coding of the amplitude of a 2pEPSC. (c) Region of interest of the neuron shown in (a) for functional mapping. (d) A pseudocolor map of peak amplitudes of 2pEPSCs (glutamate-sensitivity map). (e) A smoothed glutamate-sensitivity map by linear interpolation. (f) Overlay of (c) and (e). (g) A FM1-43 fluorescence image. (h) An overlay of (e) and (g).

Figure 3. Functional mapping of glutamate sensitivities in hippocampal neurons

(a) Fluorescence image of a CA1 pyramidal neuron in slice. The arrow indicates the region of interest. (b) 2pEPSCs induced at four different sites of the dendrite indicated as A–D in (f). The traces were averages of data from four neighboring pixels. E shows an averaged trace of 10 mEPSCs recorded from the same neuron. (c–e) Fluorescence images at four z-axis sections as indicated (c), two-dimensional pseudocolor map of 2pEPSC amplitude (d), and overlays (e). (f) Three-dimensional reconstruction of the fluorescent images. (g) Maximal intensity plot of the glutamate sensitivity for the four sections shown in (d). The map has been smoothed by linear interpolation. White lines indicate contours of the dendritic structures. (h) Glutamate-sensitivity map within the dendritic contours.

Fgiure 4. Spine geometry and expression of functional AMPA receptors

Fluorescence images (top) and glutamate-sensitivity maps (bottom) are shown for various dendritic spines of CA1 pyramidal neurons in hippocampal slices. The fluorescence images were obtained from stacked images containing the respective spine. The glutamate-sensitivity map was obtained from one x–y section in which the maximal glutamate sensitivity for the spine was detected. The maps were smoothed by linear interpolation. White lines indicate the contours of the dendritic structures. Representative data from thin spines (a–e), mushroom spines (f–j), filopodia (k) and a mushroom spine in a 9-day-old animal (l) are shown.

Figure 5. Correlation between spine geometry and glutamate sensitivities

A fluorescence image of the dendrite shown in Fig. 3f. The image was generated from a stack of 40 images. (b) Correlations between the spine-head volume and glutamate sensitivities for the dendrites shown in (a). Numbered points correspond to the numbered spines in (a). (c) Correlations between the spine-head volume and glutamate sensitivities for nine different dendrites. (e) Correlation between the spine-neck length and spine-head volume for the dendrites. (f) Spatial autocorrelations of glutamate sensitivity (blue line) and of spine-head volume (red line). The mean distances between the spines obtained from the nine dendrites are also indicated.

Figure 6. Non-stationary fluctuation analysis of AMPA receptors in a mushroom spine

(a) Five examples of 2pEPSCs obtained during repetitive photolysis (4.5 mW, 2 ms) of the spine shown in the inset in (b). (b) Peak amplitudes of 2pEPSCs evoked 50 times in the presence of cyclothiazide. Inset, fluorescence picture of a mushroom spine. White dot indicates the site at which optical release of MNI-glutamate was made. (c) Five current traces shown in (a) after subtraction of the ensemble average of 2pEPSCs. The vertical dashed line in (a) and (c) represents the onset of uncaging. The white bar indicates the time window used for the fluctuation analysis. (d) Variance (σ²) of 2pEPSCs versus the mean amplitudes (I). Each plot represents an average of five sample points. N, calculated number of AMPA receptors.