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...
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.
The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase II␣ promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy.epilepsy ͉ hippocampus ͉ NpHR ͉ paroxysmal depolarizing shift (PDS) ͉ stimulation train-induced bursting (STIB)
The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.
Neurons are perpetually receiving vast amounts of information in the form of synaptic input from surrounding cells. The majority of input occurs at thousands of dendritic spines, which mediate excitatory synaptic transmission in the brain, and is integrated by the dendritic and somatic compartments of the postsynaptic neuron. The functional role of dendritic spines in shaping biochemical and electrical signals transmitted via synapses has long been intensely studied. Yet, many basic questions remain unanswered, in particular regarding the impact of their nanoscale morphology on electrical signals. Here, we review our current understanding of the structure and function relationship of dendritic spines, focusing on the controversy of electrical compartmentalization and the potential role of spine structural changes in synaptic plasticity.
Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D2 autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.
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