Synaptic contacts display large diversity in their morphological and functional properties throughout the CNS. Yet relationship between ultrastructural and functional parameters has remained largely elusive. Here we addressed how the neurotransmitter release probability (Pr) and presynaptic action potential (AP)-evoked [Ca ²⁺ ] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals with different postsynaptic targets.

Two-photon [Ca ²⁺] imaging–derived optical quantal analysis combined with correlated electron microscopic 3D reconstructions revealed that Pr, (Ca²⁺( transient and the number of the docked vesicles scales linearly with the active zone (AZ) area of CA3 pyramidal cell (PC) boutons that contact other CA3 PCs. Freeze-fracture immunogold labeling revealed an AZ confined distribution of the voltage-gated Ca²⁺ channel subunit Cav2.1 and the presynaptic AZ protein Rim1/2, and their number correlates with the AZ area.

Our results demonstrate that functional, molecular and ultrastructural features scale linearly with the AZ area at CA3 PC-PC connections. The question arises whether this principle holds for those CA3 PC axon terminals that establish functionally different synapses on different types of GABAergic interneurons.

Using two-photon [Ca²⁺] imaging, and post hoc triple immunofluorescent labeling we found that the peak amplitude of [Ca²⁺] transients in boutons that contact parvalbumin expressing cells (PV-B) and assumed to have high Pr, were 33% higher than in their low Pr counterparts that synapse on mGluR1α immunopositive interneurons (mG-B). Calculating the total amount of fluxed Ca²⁺ per unit AZ area using correlated electron microscopic 3D reconstruction of the imaged boutons resulted a 2.4 times higher value in PV-Bs due to differences in the AZ area / bouton volume ratio between the two axon terminal populations. Assuming the same set of calcium channels with similar functional properties and AZ-confined distributions at both types of terminals, our data suggest that the functional Ca²⁺ channel density within the AZ of PV-Bs is ~2.4 times higher than that in mG-Bs. Our results indicate that unlike to CA3 PC–PC connections, differences in the AZ Ca²⁺ channel density may underlie the differences in the Pr and short-term plasticity of these two axon terminal populations.