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...

Dendritic spines on pyramidal neurons receive the vast majority of excitatory input and are considered electrobiochemical processing units, integrating and compartmentalizing synaptic input. Following synaptic plasticity, spines can undergo morphological plasticity, which possibly forms the structural basis for long-term changes in neuronal circuitry. Here, we demonstrate that spines on CA1 pyramidal neurons from organotypic slice cultures show bidirectional activity-dependent morphological plasticity. Using two-photon time-lapse microscopy, we observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, whereas theta burst stimulation led to the formation of new spines. Moreover, without stimulation the number of spine retractions was on the same order of magnitude as the stimulus-induced spine gain or loss. Finally, we found that the ability of neurons to eliminate spines in an activity-dependent manner decreased with developmental age. Taken together, our data show that hippocampal neurons can undergo bidirectional morphological plasticity; spines are formed and eliminated in an activity-dependent way.

Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.LTP ͉ nanoscopy ͉ spine neck diameter ͉ superresolution ͉ synaptic plasticity

It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60-80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.

Long-lasting changes in synaptic strength are thought to play a pivotal role in activity-dependent plasticity and memory. There is ample evidence indicating that in hippocampal long-term potentiation (LTP) the synthesis of new proteins is crucial for enduring changes. However, whether protein degradation also plays a role in this process has only recently begun to receive attention. Here, we examine the effects of blocking protein degradation on LTP. We show that pharmacological inhibition of proteasome-dependent protein degradation, just like inhibition of protein synthesis, disrupts expression of late (L-)LTP. However, when protein degradation and protein synthesis are inhibited at the same time, LTP is restored to control levels, calling into question the commonly held hypothesis that synthesis of new proteins is indispensable for L-LTP. Instead, these findings point to a more facetted model, in which L-LTP is determined by the combined action of synthesis and degradation of plasticity proteins.

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

We tracked pathogenic myelin basic protein-specific CD4+ effector T cells in early central nervous system (CNS) lesions of experimental autoimmune encephalomyelitis (EAE) by combining two-photon imaging and fluorescence video microscopy. We made two key observations: (a) the majority of the cells (65%) moved fast (maximal speed 25 μm/min) and apparently nondirected through the compact tissue; and (b) a second group of effector T cells (35%) appeared tethered to a fixed point. Polarization of T cell receptor and adhesion molecules (lymphocyte function-associated antigen 1) towards this fixed point suggests the formation of immune synapses. Nonpathogenic, ovalbumin-specific T cells were not tethered in the CNS and did not form synapse-like contacts, but moved through the tissue. After intrathecal injection of antigen, 40% of ovalbumin-specific T cells became tethered. Conversely, injection of anti–major histocompatibility complex class II antibodies profoundly reduced the number of stationary pathogenic T cells within the CNS (to 15%). We propose that rapid penetration of the CNS parenchyma by numerous autoimmune effector T cells along with multiple autoantigen-presentation events are responsible for the fulminate development of clinical EAE.

Highlights d Induction of synaptic LTP prompts withdrawal of perisynaptic astroglia d The underlying mechanisms involve NKCC1 transporter and cofilin d Reduced synaptic astroglial coverage boosts extrasynaptic glutamate escape d LTP induction thus enhances NMDAR-dependent intersynaptic cross-talk