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Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein ( App ) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, whereas spatial representation is significantly impaired in the App knock-in mice. This is because of the overall reduction of active place cells, but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus., Competing Interests: Y.H. was supported in part by Takeda Pharmaceutical Company, Fujitsu Laboratories, and DWANGO., (Copyright © 2021 the authors.)

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca 2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.

Wearable technologies for functional whole brain imaging in freely moving animals would advance our understanding of cognitive processing and adaptive behavior. Fluorescence imaging can visualize the activity of individual neurons in real time, but conventional microscopes have limited sample coverage in both the width and depth of view. Here we developed a novel head-mounted laser camera (HLC) with macro and deep-focus lenses that enable fluorescence imaging at cellular resolution for comprehensive imaging in mice expressing a layer- and cell type-specific calcium probe. We visualized orientation selectivity in individual excitatory neurons across the whole visual cortex of one hemisphere, and cell assembly expressing the premotor activity that precedes voluntary movement across the motor cortex of both hemispheres. Including options for multiplex and wireless interfaces, our wearable, wide- and deep-imaging HLC technology could enable simple and economical mapping of neuronal populations underlying cognition and behavior.

Wide-field imaging of neural activity at a cellular resolution is a current challenge in neuroscience. To address this issue, wide-field two-photon microscopy has been developed; however, the field size is limited by the objective size. Here, we develop a micro-opto-mechanical device that rotates within the post-objective space between the objective and brain tissue. Two-photon microscopy with this device enables sub-second sequential calcium imaging of left and right mouse sensory forelimb areas 6 mm apart. When imaging the rostral and caudal motor forelimb areas (RFA and CFA) 2 mm apart, we found high pairwise correlations in spontaneous activity between RFA and CFA neurons and between an RFA neuron and its putative axons in CFA. While mice performed a sound-triggered forelimb-movement task, the population activity between RFA and CFA covaried across trials, although the field-averaged activity was similar across trials. The micro-opto-mechanical device in the post-objective space provides a novel and flexible design to clarify the correlation structure between distant brain areas at subcellular and population levels.

In vivo two-photon calcium imaging currently allows us to observe the activity of multiple neurons up to ~900 µm below the cortical surface without cortical invasion. However, many important brain areas are located deeper than this. Here, we used an 1100 nm laser that underfilled the back aperture of the objective together with red genetically encoded calcium indicators to establish two-photon calcium imaging of the intact mouse brain and detect neural activity up to 1200 μm from the cortical surface. This imaging was obtained from the medial prefrontal cortex (the prelimbic area) and the hippocampal CA1 region. We found that neural activity before water delivery repeated at a constant interval was higher in the prelimbic area than in layer 2/3 of the secondary motor area. Reducing the invasiveness of imaging is an important strategy to reveal the intact brain processes active in cognition and memory.

Background: Real-time recording and manipulation of neural activity in freely behaving animals can greatly advance our understanding of how neural circuits regulate behavior. Ca 2+ imaging and optogenetic manipulation with optical probes are key technologies for this purpose. However, integrating the two optical approaches with behavioral analysis has been technically challenging., New Method: Here, we developed a new imaging system, ICaST (Integrated platform for Ca 2+ imaging, Stimulation, and Tracking), which combines an automatic worm tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. We optimized different excitation wavelengths for the concurrent use of channelrhodopsin-2 and G-CaMP, a green fluorescent protein (GFP)-based, genetically encoded Ca 2+ indicator., Results: Using ICaST in conjunction with an improved G-CaMP7, we successfully achieved long-term tracking and Ca 2+ imaging of the AVA backward command interneurons while tracking the head of a moving animal. We also performed all-optical manipulation and simultaneous recording of Ca 2+ dynamics from GABAergic motor neurons in conjunction with behavior monitoring., Comparison With Existing Method(s): Our system differs from conventional systems in that it does not require fluorescent markers for tracking and can track any part of the worm's body via bright-field imaging at high magnification. Consequently, this approach enables the long-term imaging of activity from neurons or nerve processes of interest with high spatiotemporal resolution., Conclusion: Our imaging system is a powerful tool for studying the neural circuit mechanisms of C. elegans behavior and has potential for use in other small animals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The level of wakefulness is one of the major factors affecting nociception and pain. Stress-induced analgesia supports an animal's survival via prompt defensive responses against predators or competitors. Previous studies have shown the pharmacological effects of orexin peptides on analgesia. However, orexin neurons contain not only orexin but also other co-transmitters such as dynorphin, neurotensin and glutamate. Thus, the physiological importance of orexin neuronal activity in nociception is unknown. Here we show that adult-stage selective ablation of orexin neurons enhances pain-related behaviors, while pharmacogenetic activation of orexin neurons induces analgesia. Additionally, we found correlative activation of orexin neurons during nociception using fiber photometry recordings of orexin neurons in conscious animals. These findings suggest an integrative role for orexin neurons in nociceptive perception and pain regulation.

Understanding the information processing performed by a single neuron requires the monitoring of physiological dynamics from a variety of subcellular compartments including dendrites and axons. In this study, we showed that the expression of a fusion protein, consisting of a Ca²⁺ indicator protein (G-CaMP6) and a cytoskeleton protein (actin), enabled large-scale recording of Ca²⁺ dynamics from hundreds of postsynaptic spines and presynaptic boutons in a cortical pyramidal cell. At dendritic spines, G-CaMP6-actin had the potential to detect localized Ca²⁺ activity triggered by subthreshold synaptic inputs. Back-propagating action potentials reliably induced Ca²⁺ fluorescent increases in all spines. At axonal boutons, G-CaMP6-actin reported action potential trains propagating along axonal collaterals. The detectability of G-CaMP6-actin should contribute toward a deeper understanding of neural network architecture and dynamics at the level of individual synapses.

Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.

Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparative in vivo analysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of the Drosophila larval neuromuscular junction. Robust neural activity did not result in any or noteworthy fluorescence changes when Flash-Pericam, Camgaroo-1, and Camgaroo-2 were expressed. However, calculated on the raw data, fractional fluorescence changes up to 18% were reported by synapto-pHluorin, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, GCaMP1.3, GCaMP1.6, and the troponin C-based calcium sensor TN-L15. The response characteristics of all of these indicators differed considerably from each other, with GCaMP1.6 reporting high rates of neural activity with the largest and fastest fluorescence changes. However, GCaMP1.6 suffered from photobleaching, whereas the fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible, with TN-L15 showing the fastest rise of the signals at lower activity rates. We show for GCaMP1.3 and YC3.3 that an expanded range of neural activity evoked fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR). The expression level of the indicator biased the signal kinetics and SNR, whereas the signal amplitude was independent. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.

The insular cortex (IC) participates in diverse complex brain functions, including social function, yet their cellular bases remain to be fully understood. Using microendoscopic calcium imaging of the agranular insular cortex (AI) in mice interacting with freely moving and restrained social targets, we identified 2 subsets of AI neurons—a larger fraction of "Social-ON" cells and a smaller fraction of "Social-OFF" cells—that change their activity in opposite directions during social exploration. Social-ON cells included those that represented social investigation independent of location and consisted of multiple subsets, each of which was preferentially active during exploration under a particular behavioral state or with a particular target of physical contact. These results uncover a previously unknown function of AI neurons that may act to monitor the ongoing status of social exploration while an animal interacts with unfamiliar conspecifics. The insular cortex participates in diverse complex brain functions, including social function, but their cellular basis remains unclear. This study uses microendoscopic calcium imaging in mice interacting with conspecifics to identify "social cells" in the agranular insular cortex; multiple subsets of neurons encode distinct aspects of ongoing social behavior. [ABSTRACT FROM AUTHOR]

Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6. [ABSTRACT FROM AUTHOR]

Olfactory information in Drosophila is conveyed by projection neurons from olfactory sensory neurons to Kenyon cells ( KCs) in the mushroom body ( MB). A subset of KCs responds to a given odor molecule, and the combination of these KCs represents a part of the neuronal olfactory code. KCs are also thought to function as coincidence detectors for memory formation, associating odor information with a coincident punishment or reward stimulus. Associative conditioning has been shown to modify KC output. This plasticity occurs in the vertical lobes of MBs containing α/α' branches of KCs, which is shown by measuring the average Ca2+ levels in the branch of each lobe. We devised a method to quantitatively describe the population activity patterns recorded from axons of >1000 KCs at the α/α' branches using two-photon Ca2+ imaging. Principal component analysis of the population activity patterns clearly differentiated the responses to distinct odors. [ABSTRACT FROM AUTHOR]

Genetically encoded Ca2+ indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca2+ transients triggered by single action potentials (APs) with a probability of 95% and a signal-tonoise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca2+ transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The coexpression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca2+ transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution. [ABSTRACT FROM AUTHOR]

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca2+] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17±10% ⊿F/F [mean±SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302±50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca2+ accumulations evoked by activation of synaptic NMDA receptors. We observed robust ⊿F/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo. [ABSTRACT FROM AUTHOR]

Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5′ regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl- d-aspartate receptors. [ABSTRACT FROM AUTHOR]