To image synaptic activity within neural circuits, we have tethered the genetically-encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detects the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: the locations of synapses are identified and the reporter displays a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicate that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrates that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum as well as graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of β-actin mRNA and fewer dendritic β-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Substantial progress has been made toward understanding the neuropathology, genetic origins, and epidemiology of neurodegenerative diseases, including Alzheimer's disease; tauopathies, such as frontotemporal dementia; α-synucleinopathies, such as Parkinson's disease or dementia with Lewy bodies; Huntington's disease; and amyotrophic lateral sclerosis with dementia, as well as prion diseases. Recent evidence has implicated dendritic spine dysfunction as an important substrate of the pathogenesis of dementia in these disorders. Dendritic spines are specialized structures, extending from the neuronal processes, on which excitatory synaptic contacts are formed, and the loss of dendritic spines correlates with the loss of synaptic function. We review the literature that has implicated direct or indirect structural alterations at dendritic spines in the pathogenesis of major neurodegenerative diseases, focusing on those that lead to dementias such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as frontotemporal dementia and prion diseases. We stress the importance of in vivo studies in animal models.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pulldown assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the nonneuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the proteinprotein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Synaptic failure is an immediate cause of cognitive decline and memory dysfunction in Alzheimer’s disease. Dendritic spines are specialized structures on neuronal processes, on which excitatory synaptic contacts take place and the loss of dendritic spines directly correlates with the loss of synaptic function. Dendritic spines are readily accessible for both in vitro and in vivo experiments and have, therefore, been studied in great detail in Alzheimer’s disease mouse models. To date, a large number of different mechanisms have been proposed to cause dendritic spine dysfunction and loss in Alzheimer’s disease. For instance, amyloid beta fibrils, diffusible oligomers or the intracellular accumulation of amyloid beta have been found to alter the function and structure of dendritic spines by distinct mechanisms. Furthermore, tau hyperphosphorylation and microglia activation, which are thought to be consequences of amyloidosis in Alzheimer’s disease, may also contribute to spine loss. Lastly, genetic and therapeutic interventions employed to model the disease and elucidate its pathogenetic mechanisms in experimental animals may cause alterations of dendritic spines on their own. However, to date none of these mechanisms have been translated into successful therapeutic approaches for the human disease. Here, we critically review the most intensely studied mechanisms of spine loss in Alzheimer’s disease as well as the possible pitfalls inherent in the animal models of such a complex neurodegenerative disorder.

Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A 2a adenosine receptor, a G s -coupled receptor. Usp4 binds to the carboxyl terminus of the A 2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A 2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.

Imaging of optical reporters of neural activity across large populations of neurones is a widely used approach for investigating the function of neural circuits in slices and in vivo. Major challenges in analysing such experiments include the automatic identification of neurones and synapses, extraction of dynamic signals, and assessing the temporal and spatial relationships between active units in relation to the gross structure of the circuit. We have developed an integrated set of software tools, named SARFIA, by which these aspects of dynamic imaging experiments can be analysed semi-automatically. Key features are image-based detection of structures of interest using the Laplace operator, determining the positions of units in a layered network, clustering algorithms to classify units with similar functional responses, and a database to store, exchange and analyse results across experiments. We demonstrate the use of these tools to analyse synaptic activity in the retina of live zebrafish by multi-photon imaging of SyGCaMP2, a genetically encoded synaptically localised calcium reporter. By simultaneously recording activity across tens of bipolar cell terminals distributed throughout the IPL we made a functional map of the ON and OFF signalling channels and found that these were only partially separated. The automated detection of signals across many neurones in the retina allowed the reliable detection of small populations of neurones generating “ectopic” signals in the “ON” and “OFF” sublaminae. This software should be generally applicable for the analysis of dynamic imaging experiments across hundreds of responding units.

The kinetics of amyloid plaque formation and growth as one of the characteristic hallmarks of Alzheimer’s disease (AD) are fundamental issues in AD research. Especially the question how fast amyloid plaques grow to their final size after they are born remains controversial. By long-term two-photon in vivo imaging we monitored individual methoxy-X04-stained amyloid plaques over 6 weeks in 12 and 18 months old Tg2576 mice. We found that in 12 months old mice, newly appearing amyloid plaques were initially small in volume and subsequently grew over time. The growth rate of plaques was inversely proportional to their volume; thus amyloid plaques that were already present at the first imaging time point grew over time but slower compared to new plaques. Additionally, we analyzed 18 months old Tg2576 mice in which we neither found newly appearing plaques nor a significant growth of pre-existing plaques over 6 weeks of imaging. In conclusion, newly appearing amyloid plaques are initially small in size but grow over time until plaque growth can not be detected anymore in aged mice. These results suggest that drugs that target plaque formation should be most effective early in the disease, when plaques are growing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-010-0787-6) contains supplementary material, which is available to authorized users.