Quantitative ultrastructural analysis of hippocampal excitatory synapses

Abstract

From three-dimensional reconstructions of CA1 excitatory synapses in the rodent hippocampus and in culture, we have estimated statistical distributions of active zone and postsynaptic density (PSD) sizes (average area approximately 0.04 micron2), the number of active zones per bouton (usually one), the number of docked vesicles per active zone (approximately 10), and the total number of vesicles per bouton (approximately 200), and we have determined relationships between these quantities, all of which vary from synapse to synapse but are highly correlated. These measurements have been related to synaptic physiology. In particular, we propose that the distribution of active zone areas can account for the distribution of synaptic release probabilities and that each active zone constitutes a release site as identified in the standard quantal theory attributable to Katz (1969).

Fig. 5.

Depiction of 26 active zones, as viewed from within the bouton, reconstructed from serial sections. The black bars correspond to the active zone of a single section (thus the thickness of the bar equals 60 nm), and theshaded circles indicate the position of docked vesicles. Note that because the section is approximately twice as thick as the vesicle diameter, the location of the vesicle within the section is unknown; all vesicles have been placed in the center of the section for illustration, but the actual vesicles would not be arranged in the neat rows as shown here. The point of representing the vesicles here is to indicate the extent to which areas of the active zone seem to be unoccupied. Many active zones could be described as round or oval, although distortions of these idealized forms are common. Two active zones were perforated. The occupancy of active zones by docked vesicles varies greatly.

Fig. 1.

Sample electron micrographs used in our study.A, Electron micrograph depicting several synapses within the stratum radiatum in CA1 in the mouse hippocampus. Thearrows indicate the borders of the active zones on the presynaptic side and of the postsynaptic densities on the spine. Examples of vesicles that are defined as docked in our study are marked by arrowheads. B, C, Electron micrograph showing the ultrastructure of two synapses from a hippocampal neuron grown in culture. Scale bars: A, 0.5 μm; B, C, 0.25 μm.

Fig. 2.

Three-dimensional reconstructions of synapses from serial sections. A, Two views of a brain synapse that is included in our study. The spine head (spine) is surrounded by two varicosities (axon). One of these varicosities forms a contact on the spine; the other is located beneath the spine. The right image presents a view from the right side of the structure in the left image. The bouton is transparent for the visualization of the active zone (red) and the nine docked vesicles (gray). Nondocked vesicles are, for clarity, excluded. B, The reconstruction of two shaft synapses from cultured hippocampal neurons. Both synapses originate from the same varicosity (axon). The two different shades ofyellow represent the two different dendritic shafts (dend. I, dend. II). Dend. II branches at the site of the synapse. The leftand right images depict views of the same structure rotated by ∼30°. The red active zones and their docked vesicles are visible through the transparentaxon; again nondocked vesicles are excluded for clarity.

Fig. 3.

Correlation of active zone size with PSD size. A, Scatter plot comparing the sizes of 79 active zones from mouse brain with their corresponding PSD. The line indicates the linear regression with a correlation coefficient of 0.97.B, Same comparison of 21 active zones with PSDs from hippocampal neurons in culture. The correlation coefficient is 0.97.

Fig. 4.

Comparison of active zone sizes with the number of docked vesicles for the brain synapses. A, Frequency distribution of active zone sizes for 79 active zones. Thesolid line is a γ density function (y = a ×e^−bx, witha = 3437 and b = 53). Mean = 0.039 μm²; SD = 0.022 μm². B, Distribution of the number of docked vesicles for 79 active zones. The solid lineis a γ density function (y =a ×e^−bx, witha = 4.98 and b = 0.165). Mean = 10.3 vesicles per active zone; SD = 5.6.C, Scatter plot of active zone size and corresponding number of docked vesicles. The fitted linear regression is indicated as a solid line, and the correlation coefficient is 0.927.

Fig. 6.

Comparison of the distribution of the active zone areas with the release probability. The solid linerepresents the release probability of hippocampal CA1 synapses as measured by Murthy et al. (1997). The dashed line gives the distribution of release probabilities predicted from the distribution of active zone areas (Fig. 4A) as described in . The theoretical and observed distributions are not significantly different (Kolmogorov–Smirnov test,P > 0.2).