Alzheimer's disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimer's disease has not been achieved. We extracted soluble amyloid-beta protein (Abeta) oligomers directly from the cerebral cortex of subjects with Alzheimer's disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Abeta from Alzheimer's disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Abeta dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Abeta N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimer's disease cortex did not impair LTP unless they were first solubilized to release Abeta dimers, suggesting that plaque cores are largely inactive but sequester Abeta dimers that are synaptotoxic. We conclude that soluble Abeta oligomers extracted from Alzheimer's disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

Alzheimer's disease (AD) is characterized by decreased synapse density in hippocampus and neocortex, and synapse loss is the strongest anatomical correlate of the degree of clinical impairment. Although considerable evidence supports a causal role for the amyloid-␤ protein (A␤) in AD, a direct link between a specific form of A␤ and synapse loss has not been established. We demonstrate that physiological concentrations of naturally secreted A␤ dimers and trimers, but not monomers, induce progressive loss of hippocampal synapses. Pyramidal neurons in rat organotypic slices had markedly decreased density of dendritic spines and numbers of electrophysiologically active synapses after exposure to picomolar levels of soluble oligomers. Spine loss was reversible and was prevented by A␤-specific antibodies or a small-molecule modulator of A␤ aggregation. Mechanistically, A␤-mediated spine loss required activity of NMDA-type glutamate receptors (NMDARs) and occurred through a pathway involving cofilin and calcineurin. Furthermore, NMDARmediated calcium influx into active spines was reduced by A␤ oligomers. Partial blockade of NMDARs by pharmacological antagonists was sufficient to trigger spine loss. We conclude that soluble, low-n oligomers of human A␤ trigger synapse loss that can be reversed by therapeutic agents. Our approach provides a quantitative cellular model for elucidating the molecular basis of A␤-induced neuronal dysfunction.

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.

Spines are neuronal protrusions, each of which receives input typically from one excitatory synapse. They contain neurotransmitter receptors, organelles, and signaling systems essential for synaptic function and plasticity. Numerous brain disorders are associated with abnormal dendritic spines. Spine formation, plasticity, and maintenance depend on synaptic activity and can be modulated by sensory experience. Studies of compartmentalization have shown that spines serve primarily as biochemical, rather than electrical, compartments. In particular, recent work has highlighted that spines are highly specialized compartments for rapid large-amplitude Ca(2+) signals underlying the induction of synaptic plasticity.

Spine Ca(2+) is critical for the induction of synaptic plasticity, but the factors that control Ca(2+) handling in dendritic spines under physiological conditions are largely unknown. We studied [Ca(2+)] signaling in dendritic spines of CA1 pyramidal neurons and find that spines are specialized structures with low endogenous Ca(2+) buffer capacity that allows large and extremely rapid [Ca(2+)] changes. Under physiological conditions, Ca(2+) diffusion across the spine neck is negligible, and the spine head functions as a separate compartment on long time scales, allowing localized Ca(2+) buildup during trains of synaptic stimuli. Furthermore, the kinetics of Ca(2+) sources governs the time course of [Ca(2+)] signals and may explain the selective activation of long-term synaptic potentiation (LTP) and long-term depression (LTD) by NMDA-R-mediated synaptic Ca(2+).

BackgroundLaser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope.ResultsWe describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality.ConclusionsWe present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and v-ATPase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag GTPases and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Km and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

In vivo calcium imaging through microendoscopic lenses enables imaging of previously inaccessible neuronal populations deep within the brains of freely moving animals. However, it is computationally challenging to extract single-neuronal activity from microendoscopic data, because of the very large background fluctuations and high spatial overlaps intrinsic to this recording modality. Here, we describe a new constrained matrix factorization approach to accurately separate the background and then demix and denoise the neuronal signals of interest. We compared the proposed method against previous independent components analysis and constrained nonnegative matrix factorization approaches. On both simulated and experimental data recorded from mice, our method substantially improved the quality of extracted cellular signals and detected more well-isolated neural signals, especially in noisy data regimes. These advances can in turn significantly enhance the statistical power of downstream analyses, and ultimately improve scientific conclusions derived from microendoscopic data.