Modality-Specific Impairment of Hippocampal CA1 Neurons of Alzheimer's Disease Model Mice.

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 ß (Aß) 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 Aß 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 Aß aggregates. These results indicate the differential impact of Aß aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

A Critical Neurodevelopmental Role for L-Type Voltage-Gated Calcium Channels in Neurite Extension and Radial Migration.

Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na+ channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Cav1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies.SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.

Rational design of a high-affinity, fast, red calcium indicator R-CaMP2.

Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-ß in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.

Principal component analysis of odor coding at the level of third-order olfactory neurons in Drosophila.

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 Ca(2+) 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 Ca(2+) imaging. Principal component analysis of the population activity patterns clearly differentiated the responses to distinct odors.

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.

Low computational-cost cell detection method for calcium imaging data.

The rapid progress of calcium imaging techniques has reached a point where the activity of thousands to tens of thousands of cells can be recorded simultaneously with single-cell resolution in a field-of-view (FOV) of about ten mm2. Consequently, there is a pressing need for developing automatic cell detection methods for large-scale image data. Several research groups have proposed automatic cell detection algorithms. Almost all algorithms can solve large-scale optimization problems for data, including hundreds of cells recorded from a conventional FOV at a resolution of 512 × 512 pixels, but the solution becomes more difficult as the data size increases beyond that. To handle large-scale data acquired with the latest large FOV microscopes, we propose a method called low computational cost cell detection (LCCD) that is based on filtering and thresholding. We compared LCCD with two other methods, constrained non-negative matrix factorization (CNMF) and Suite2P. We found that LCCD makes it possible to detect cells in artificial and actual data showing a high number density of cells within a shorter time and with an accuracy comparable to or better than those of CNMF and Suite2P. Moreover, LCCD succeeded in detecting more than 20,000 active cells from data acquired with the latest microscopy, called FASHIO-2PM, with a FOV of 3.0 mm × 3.0 mm.

Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.

Derived (mutated)-types of TRPV6 channels elicit greater Ca²+ influx into the cells than ancestral-types of TRPV6: evidence from Xenopus oocytes and mammalian cell expression system.

The frequency of the allele containing three derived nonsynonymous SNPs (157C, 378M, 681M) of the gene encoding calcium permeable TRPV6 channels expressed in the intestine has been increased by positive selection in non-African populations. To understand the nature of these SNPs, we compared the properties of Ca²+ influx of ancestral (in African populations) and derived-TRPV6 (in non-African populations) channels with electrophysiological, Ca²+-imaging, and morphological methods using both the Xenopus oocyte and mammalian cell expression systems. Functional electrophysiological and Ca²+-imaging analyses indicated that the derived-TRPV6 elicited more Ca²+ influx than the ancestral one in TRPV6-expressing cells where both channels were equally expressed in the cells. Ca²+-inactivation properties in the ancestral- and derived-TRPV6 were almost the same. Furthermore, fluorescence resonance energy transfer (FRET) analysis showed that both channels have similar multimeric formation properties, suggesting that derived-TRPV6 itself could cause higher Ca²+ influx. These findings suggest that populations having derived-TRPV6 in non-African areas may absorb higher Ca²+ from the intestine than ancestral-TRPV6 in the African area.

Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona intestinalis type A (Ciona robusta) larva.

Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In Ciona intestinalis type A larvae, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. Tunicate larvae occupy a non-reproductive dispersal stage, yet the role of their GnRH system remains elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in Ciona larvae and visualized the activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons and dopaminergic cells express gnrh2, suggesting that GnRH plays a role in controlling swimming behavior. However, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in motor control is also suggested by a relationship between the activity of gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that active intercellular signaling occurs in the glia cells of the nerve cord.

Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice.

Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease.

Distinct Mechanisms of Over-Representation of Landmarks and Rewards in the Hippocampus.

In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.

Inhibitory effect of lomerizine, a prophylactic drug for migraines, on serotonin-induced contraction of the basilar artery.

We examined the effects of lomerizine on serotonin (5-hydroxytryptamine, 5-HT)-induced contraction of the basilar artery and compared them with those of nifedipine. Although both lomerizine and nifedipine completely blocked K(+)-induced vasoconstriction, 5-HT-induced vasoconstriction was more strongly inhibited by lomerizine than nifedipine. A 5-HT(2A) antagonist inhibited the 5-HT-induced vasoconstriction, but a 5-HT(1B) antagonist did not. Lomerizine, but not nifedipine, suppressed 5-HT-induced Ca(2+) release in 5-HT(2A)-expressing HEK293 cells. Moreover, neither antagonist affected ATP-induced Ca(2+) release. These results suggest that lomerizine may inhibit not only voltage-dependent Ca(2+) channels but also 5-HT(2A) receptors and so inhibit 5-HT-induced contraction in the basilar artery.

Wide and Deep Imaging of Neuronal Activities by a Wearable NeuroImager Reveals Premotor Activity in the Whole Motor Cortex.

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.

Orchestrated ensemble activities constitute a hippocampal memory engram.

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 Ca2+ 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.

ELKS/Voltage-Dependent Ca2+ Channel-ß Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic ß Cells.

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

Both 5-hydroxytryptamine 5-HT2A and 5-HT1B receptors are involved in the vasoconstrictor response to 5-HT in the human isolated internal thoracic artery.

1. The 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for 5-HT-induced contraction of the human ITA. 2. The contractile responses to 5-HT of endothelium-denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5-HT(2A) and 5-HT(1B) receptors, respectively, on the 5-HT-induced vasoconstriction. Finally, 5-HT(2A) and 5-HT(1B) receptors in the human ITA were immunolabelled. 3. 5-Hydroxytryptamine (1 nmol/L-10 micromol/L) caused vasoconstriction in a concentration-dependent manner. Both sarpogrelate (1 micromol/L) and SB224289 (1 micromol/L) significantly, but not completely, inhibited 5-HT-induced vasoconstriction. 4. Conversely, simultaneous pretreatment with supramaximum concentrations (1 micromol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. 5. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the human ITA. 6. These results demonstrate that, in human ITA, 5-HT-induced vasoconstriction is mediated by activation of both 5-HT(2A) and 5-HT(1B) receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced vasospasm.

Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans.

BACKGROUND: Tyramine, known as a "trace amine" in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans (C. elegans), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior. RESULTS: Using calcium imaging in freely moving C. elegans, we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns. CONCLUSION: We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans.

Super-wide-field two-photon imaging with a micro-optical device moving in post-objective space.

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 wide-field calcium imaging of mouse thalamocortical synapses with an 8 K ultra-high-definition camera.

In vivo wide-field imaging of neural activity with a high spatio-temporal resolution is a challenge in modern neuroscience. Although two-photon imaging is very powerful, high-speed imaging of the activity of individual synapses is mostly limited to a field of approximately 200 µm on a side. Wide-field one-photon epifluorescence imaging can reveal neuronal activity over a field of ≥1 mm2 at a high speed, but is not able to resolve a single synapse. Here, to achieve a high spatio-temporal resolution, we combine an 8 K ultra-high-definition camera with spinning-disk one-photon confocal microscopy. This combination allowed us to image a 1 mm2 field with a pixel resolution of 0.21 µm at 60 fps. When we imaged motor cortical layer 1 in a behaving head-restrained mouse, calcium transients were detected in presynaptic boutons of thalamocortical axons sparsely labeled with GCaMP6s, although their density was lower than when two-photon imaging was used. The effects of out-of-focus fluorescence changes on calcium transients in individual boutons appeared minimal. Axonal boutons with highly correlated activity were detected over the 1 mm2 field, and were probably distributed on multiple axonal arbors originating from the same thalamic neuron. This new microscopy with an 8 K ultra-high-definition camera should serve to clarify the activity and plasticity of widely distributed cortical synapses.