Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging ...are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.
Extant fold-switching proteins are widespread Porter, Lauren L.; Looger, Loren L.
Proceedings of the National Academy of Sciences - PNAS,
06/2018, Letnik:
115, Številka:
23
Journal Article
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A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch ...folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ∼100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ∼100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5–4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health.
Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also ...be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.
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•AAV can be endowed with robust retrograde functionality through directed evolution•Up to two orders of magnitude increase in retrograde transport over existing variants•Efficiency comparable to synthetic tracers•Sufficient payload expression for circuit interrogation and gene manipulation
Projection neurons are a critical component of large-scale networks distributing the results of local circuit computations between distant brain regions, but their specific contribution is often hard to pinpoint because of the difficulty of gaining selective genetic access. Tervo et al. introduce a designer variant of adeno-associated virus, rAAV2-retro, that allows for efficient mapping, monitoring, and manipulation of projection neurons.
Understanding how activity patterns in specific neural circuits coordinate an animal's behavior remains a key area of neuroscience research. Genetic tools and a brain of tractable complexity make
a ...premier model organism for these studies. Here, we review the wealth of reagents available to map and manipulate neuronal activity with light.
The spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and ...electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.
•Astrocyte branches/territories were studied using optical and pharmacogenetic tools•Spontaneous astrocyte Ca2+ signals were not due to action potentials or glutamate•Evoked astrocyte Ca2+ signals were slow, territorial and triggered by spike trains•Astrocyte Ca2+ signaling was tightly gated by neurotransmitter clearance
Haustein et al. use optical and genetic methods to explore the rules under which astrocytes are engaged in a model neuronal circuit. The findings show that astrocytes listen to synapses during bursts of action potentials in mossy fiber axons.
Astrocytes tile the entire CNS, but their functions within neural circuits in health and disease remain incompletely understood. We used genetically encoded Ca(2+)and glutamate indicators to explore ...the rules for astrocyte engagement in the corticostriatal circuit of adult wild-type (WT) and Huntington's disease (HD) model mice at ages not accompanied by overt astrogliosis (at approximately postnatal days 70-80). WT striatal astrocytes displayed extensive spontaneous Ca(2+)signals, but did not respond to cortical stimulation, implying that astrocytes were largely disengaged from cortical input in healthy tissue. In contrast, in HD model mice, spontaneous Ca(2+)signals were significantly reduced in frequency, duration, and amplitude, but astrocytes responded robustly to cortical stimulation with evoked Ca(2+)signals. These action-potential-dependent astrocyte Ca(2+)signals were mediated by neuronal glutamate release during cortical stimulation, accompanied by prolonged extracellular glutamate levels near astrocytes and tightly gated by Glt1 glutamate transporters. Moreover, dysfunctional Ca(2+)and glutamate signaling that was observed in HD model mice was largely, but not completely, rescued by astrocyte specific restoration of Kir4.1, emphasizing the important contributions of K(+)homeostatic mechanisms that are known to be reduced in HD model mice. Overall, our data show that astrocyte engagement in the corticostriatal circuit is markedly altered in HD. Such prodromal astrocyte dysfunctions may represent novel therapeutic targets in HD and other brain disorders.
We report how early-onset astrocyte dysfunction without detectable astrogliosis drives disease-related processes in a mouse model of Huntington's disease (HD). The cellular mechanisms involve astrocyte homeostasis and signaling mediated by Kir4.1, Glt1, and Ca(2+) The data show that the rules for astrocyte engagement in a neuronal circuit are fundamentally altered in a brain disease caused by a known molecular defect and that fixing early homeostasis dysfunction remedies additional cellular deficits. Overall, our data suggest that key aspects of altered striatal function associated with HD may be triggered, at least in part, by dysfunctional astrocytes, thereby providing details of an emerging striatal microcircuit mechanism in HD. Such prodromal changes in astrocytes may represent novel therapeutic targets.
Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing ...fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.
Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to ...track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
We review the principles of development and deployment of genetically encoded calcium indicators (GECIs) for the detection of neural activity. Our focus is on the popular GCaMP family of green GECIs, ...culminating in the recent release of the jGCaMP8 sensors, with dramatically improved kinetics relative to previous generations. We summarize the properties of GECIs in multiple colour channels (blue, cyan, green, yellow, red, far‐red) and highlight areas for further improvement. With their low‐millisecond rise‐times, the jGCaMP8 indicators allow new classes of experiments following neural activity in time frames approaching the underlying computations.
figure legend GCaMP calcium sensors are widely used to report neuronal activity via fluorescence readout.
The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent ...protein-based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as chronically in awake, behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain and will greatly facilitate dissecting complex structural and functional relationships of neural networks.
► Thy1-GCaMP2.2c and Thy1-GCaMP3 transgenic lines were generated ► Spontaneous and evoked calcium transients can be detected in acute brain slices ► Calcium transients can be detected in dendrites and spines in vivo ► Calcium transients can be detected in neuronal populations in vivo
Chen et al. generate transgenic mice expressing genetically encoded calcium sensors and demonstrate the utility of these mice in optically monitoring neuronal activity in vivo.
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