Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood.We used time-lapse superresolution STED imaging in combination with FRAP measurements, 2-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices.We report a diversity of spine morphologies that argues against common categorization schemes, and establish a close link between compartmentalization and spine morphology, where spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after LTP. These morphological changes are predicted to lead to a substantial drop in spine head EPSP, while leaving overall biochemical compartmentalization preserved. 3 Dendritic spines form the postsynaptic component of most excitatory synapses, whose plasticity is essential for brain development and higher brain functions 1,2 . In addition to the molecular composition of the synapse, the morphology of spines is thought to be critical for synaptic function, as spine head size correlates with synaptic strength 3,4 and undergoes changes during synaptic plasticity [5][6][7][8] . Even so, our understanding of how spine structure shapes synapse function remains fragmented.It is well established that spines compartmentalize biochemical signals 9 . By contrast, the quantitative contribution of spine morphology to compartmentalization is still unknown, and only moderate correlations between spine neck length or head volume and chemical diffusion have been reported [9][10][11] . It is an open question to what extent biochemical compartmentalization is determined primarily by spine geometry or intracellular factors such as organelles or protein assemblies.Concerning electrical compartmentalization, it is not clear how electrical signals are transformed by the spine neck 9,[12][13][14] . This is an important question because synaptic strength may be adjusted through structural changes in spine necks, which has been a long-standing hypothesis 15,16 . An early electron microscopy study reported that the average spine head becomes larger and the neck wider and shorter after the induction of long-term plasticity (LTP) 17 , which was corroborated more recently by work based on 2-photon microscopy 6,18,19 . However, it is not known how these structural changes might affect biochemical and electrical compartmentalization, because 2-photon microscopy does not have sufficient spatial resolution to properly resolve spines and electron microscopy cannot be combined with functional assays. 4 Here, we combined stimulated emission depletion (STED) microscopy, fluorescence recovery after photo-bleaching (FRAP) experiments, 2-photon glutamate uncaging, and patch-clam...

Cortical synapses display remarkable structural, molecular and functional heterogeneity. Our knowledge regarding the relationship between the ultrastructural and functional parameters is still fragmented. Here we asked how the release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active zone area. The peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated Ca2+ channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active zone area. Gold particles for Cav2.1 showed a nonrandom distribution within the active zones. Our results demonstrate that the number of several active zone proteins, including presynaptic Ca2+ channels, docked vesicles and the release probability scales linearly with the active zone area.

Microglia are the main immune cells of the brain and contribute to common brain diseases. However, it is unclear how microglia influence neuronal activity and survival in the injured brain in vivo. Here we develop a precisely controlled model of brain injury induced by cerebral ischaemia combined with fast in vivo two-photon calcium imaging and selective microglial manipulation. We show that selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases.