Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses

Abstract

To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers ofAMPARs.Two-dimensionalviewsofreplicasrevealedthatAMPARsformmicroclustersinbothsynapsestoasimilarextent,resultinginlarger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

Mechanisms Underlying Signal Filtering at a Multisynapse Contact

“…The diameter of each E-face IMP apparently associated with panAMPAR immunolabeling was ϳ14 nm, and the synaptic E-face IMP clusters, defined as densely packed IMPs at a distance of Ͻ14 nm from each other, were demarcated freehand. IMP cluster size demarcated by this method was comparable to that of the postsynaptic density (PSD) visualized in conventional ultrathin sections, and, thus, the demarcation likely well represents the extent of postsynaptic specialization (Tarusawa et al, 2009). The center of gravity and the areas of individual IMP clusters were measured using iTEM software (SIS).…”

“…Data were obtained only from complete synapses where the whole IMP cluster is visible within a continuous fractured plane. Immunoparticles within a demarcated IMP cluster and those located outside but within 30 nm from the edge of the IMP cluster were regarded as synaptic labeling, considering possible deviation of the immunoparticles from antigens (Tarusawa et al, 2009). Labeling of glutamate transporters was searched for on (1) the P-faces of the RG fiber using vGluT2 labeling as a marker, (2) the corresponding postsynaptic E-faces adjacent to the membrane identified in step 1, (3) the corresponding P-faces of the replica complementary to the postsynaptic E-faces identified in step 2, and (4) the glial P-faces surrounding the presynaptic and postsynaptic membrane identified in steps 1-3, which were also identified by the morphological signature of glial membrane such as the frequent presence of reticular and/or aggregated arrangements of IMPs.…”

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…SDS-FRL was performed using some modifications (37,(63)(64)(65) of the original technique developed by Fujimoto (66). Sagittal slices (thickness: 200 μm) were prepared from the cerebellar vermis of Sprague-Dawley rats (P14, P15, and P29) in the same way as for electrophysiological experiments, followed by immersion fixation with 2% paraformaldehyde in phosphate buffer (PB; 0.1 M; pH 7.4) for 2 h at room temperature (22-24°C).…”

Distinct cerebellar engrams in short-term and long-term motor learning

“…The outline of synaptic sites was demarcated free hand along the edge of densely packed intramembrane particles at a distance of <15 nm from each other (Fig. 2 A and B, blue) (34), and their areas were measured with Scion Image software (Scion). The density of receptors was then defined by the particles per size of the postsynaptic area.…”