Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling

We have estimated the densities of synapses per unit volume in the whole mouse brain. To do this, we combined Spinning Disc confocal Microscopy (SDM) that acquires images of synaptic proteins labeled with fluorophores in large brain areas, and FIB-SEM, a three-dimensional electron microscopy technique that provides a resolution beyond that of the confocal microscope, but in relatively small areas. The postsynaptic scaffold proteins PSD95 and SAP102 were genetically labeled to visualize excitatory synapses with SDM. We calculated the densities of synaptic puncta (with SDM) and the actual number of synapses (with FIB-SEM) in the CA1 region of the hippocampus. We then calculated the quantitative relationship between the densities of fluorescent puncta and the number of synapses. Finally, we applied this conversion factor to other regions of the brain where densities of puncta were available. We observed three different groups of brain regions, one with the highest densities of synapses (isocortex, olfactory areas, hippocampal formation and cortical subplate), a second group with low densities of synapses (pallidum, hypothalamus, brainstem and cerebellum), and a third group comprising structures which showed a wide range of synapse densities (striatum and thalamus).