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Comparative Study
. 2001 Sep 1;21(17):6666-72.
doi: 10.1523/JNEUROSCI.21-17-06666.2001.

Three-dimensional comparison of ultrastructural characteristics at depressing and facilitating synapses onto cerebellar Purkinje cells

M A Xu-Friedman  1 , K M Harris, W G Regehr
Affiliations
Comparative Study

Three-dimensional comparison of ultrastructural characteristics at depressing and facilitating synapses onto cerebellar Purkinje cells

M A Xu-Friedman et al. J Neurosci. .

Abstract

Cerebellar Purkinje cells receive two distinctive types of excitatory inputs. Climbing fiber (CF) synapses have a high probability of release and show paired-pulse depression (PPD), whereas parallel fiber (PF) synapses facilitate and have a low probability of release. We examined both types of synapses using serial electron microscopic reconstructions in 15-d-old rats to look for anatomical correlates of these differences. PF and CF synapses were distinguishable by their overall ultrastructural organization. There were differences between PF and CF synapses in how many release sites were within 1 microm of a mitochondrion (67 vs 84%) and in the degree of astrocytic ensheathment (67 vs 94%). However, the postsynaptic density sizes for both types of synapses were similar (0.13-0.14 microm(2)). For both types of synapses, we counted the number of docked vesicles per release site to test whether this number determines the probability of release and synaptic plasticity. PF and CF synapses had the same number of anatomically docked vesicles (7-8). The number of docked vesicles at the CF does not support a simple model of PPD in which release of a single vesicle during the first pulse depletes the anatomically docked vesicle pool at a synapse. Alternatively, only a fraction of anatomically docked vesicles may be release ready, or PPD could result from multivesicular release at each site. Similarities in the number of docked vesicles for PF and CF synapses indicate that differences in probability of release are unrelated to the number of anatomically docked vesicles at these synapses.

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Figures

Fig. 1.
Fig. 1.
Overview of anatomical and physiological differences between cerebellar CFs and PFs. A,Top, Image of an individual CF labeled by injections of DiI in the inferior olive. The fiber is shown in a sagittal slice taken from cerebellar vermis, and a single optical section was imaged using a confocal microscope. The CF enters from the bottom and makes a complex arborization, with multiple processes running parallel to each other along the Purkinje cell dendrite, but the synapses themselves are not discernable. A, Bottom, Paired-pulse depression at the CF. A CF was stimulated twice at an interval of 20 msec, and the EPSCs were recorded in the Purkinje cell. The second EPSC is 60% of the size of the first. B,Top, Image of a band of PFs labeled with Texas Red dextran in a transverse slice (imaged using a confocal microscope). Each PF runs perpendicular to the Purkinje cell dendritic arbor. Varicosities, which correspond to synaptic contacts, are scattered along the length of the PF. B, Bottom, Paired-pulse facilitation at the PF. A band of PFs was stimulated twice at an interval of 20 msec, and the EPSCs were recorded in the Purkinje cell. The second EPSC is 2.5 times larger than the first. Physiology traces are adapted from Dittman et al. (2000).
Fig. 2.
Fig. 2.
Serial EM sections of CFs. A,B, Two sample release sites from the same CF. CF axons are shaded blue, Purkinje cell dendrites and spines are shaded pink, and astrocytes are shadedyellow. The release site in A is larger than average, whereas the release site in B is smaller than average. In A, the synaptic cleft and PSD are clearly identifiable in the top three panels. InB, two spines are visible emerging from the parent dendrite at the top of each panel. The PSD for one spine is visible in the bottom three panels, and the second makes a PSD in later sections. C, Close-ups of the active zone in A. The presynaptic terminal is on the left, and the postsynaptic spine, with PSD clearly visible, is on the right. A vesicle was classified as docked (green) if it was located opposite a PSD and directly touched the presynaptic membrane. Nondocked vesicles close to the membrane are indicated byarrows.
Fig. 3.
Fig. 3.
3-D reconstruction of a CF terminating on a Purkinje cell from serial EM. A, Purkinje cell proximal dendrite. The soma is off the bottom, and distal into the molecular layer is toward the top. Spines that make contact with a CF are included, and their PSDs are labeled inred. The dendrite is viewed at an angle, so as an indication of scale, the dendrite is 3.8 μm in diameter at the position indicated by the arrow. B, CF axon, colored in blue. PSDs on the Purkinje cell are labeled in red. Labels A–D inB correspond to the segments of CF reconstructed in more detail in Figure 4. C, CF and Purkinje cell superimposed.
Fig. 4.
Fig. 4.
3-D reconstructions of sample CF release sites. Reconstructions of each PSD with associated docked vesicles are shown unsmoothed next to the axon. Axons are light blue, mitochondria are tan, vesicles are dark blue (uniformly represented as 40 nm spheres), and PSDs on the opposing spine are transparent red. A, Two release sites. The first is located on the main branch of the CF axon, with a locally high density of vesicles and many mitochondria. The second is located in a bouton with no mitochondria.B, Two release sites, made en passant, with a high density of vesicles and three mitochondria nearby. The rightmost part (with a large density of vesicles and mitochondria) branches toward additional release sites (data not shown). C, Three release sites made en passant, with a high density of vesicles and two mitochondria (partially cut off). D, Two release sites, both in boutons, only one of which has a mitochondrion.
Fig. 5.
Fig. 5.
Serial EM sections of PFs. A,B, Two example series. PFs are shadedblue, Purkinje cell spines are shadedpink, and astrocytes are shaded yellow. Every panel contains a clearly defined synaptic cleft and PSD. The release site in A is larger than average, whereas the release site in B is smaller than average. Although neither PF has a mitochondrion in the sections shown here, the release site in A has a mitochondrion in a section that is 1.5 μm away, and the site in B has one in a section that is 0.2 μm away. C, Close-up of the active zone inA. The presynaptic terminal is on theleft, and the postsynaptic spine, with PSD clearly visible, is on the right. Docked vesicles are labeled ingreen, and nondocked vesicles close to the membrane are indicated by arrows.
Fig. 6.
Fig. 6.
3-D reconstructions of sample PF release sites. Reconstructions of each PSD with associated docked vesicles are shown unsmoothed under the axon. The color scheme is as described in the legend to Figure 4. A–C, PFs with one to two mitochondria near the release site. D, E, PFs without mitochondria. F, PF with two release sites onto different spines in the same varicosity.
Fig. 7.
Fig. 7.
Comparison of ultrastructural characteristics at PF and CF release sites. Data are presented as cumulative histograms. PF distributions are shown as thin lines, and CF distributions are shown as thick lines.A, PSD area. B, Docked vesicle number.C, Distance from active zone to nearest mitochondrion.D, Degree of ensheathment.
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