Genetically Encoded Calcium Indicators Research Articles

Cellular resolution contributions to ictal population signals.

The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. This work provides a critical link between single neuron activity and population measures of seizure activity.

Expression of GCaMP6s in the dentate gyrus induces tonic–clonic seizures

GCaMP is a genetically encoded calcium indicator (GECI) widely used in neuroscience research. It measures intracellular Ca2+ level by fluorescence changes as it directly binds to Ca2+. In this process, the effect of this calcium buffer on the intracellular calcium signaling and cell physiology is often not taken into consideration. However, growing evidence from calcium imaging studies shows GCaMP expression under certain conditions can generate aberrant activity, such as seizures. In this study, we examined the effect of GCaMP6 expression in the dentate gyrus (DG) on epileptogenesis. We found that viral expression of GCaMP6s but not GCaMP6f in the DG induces tonic–clonic seizures several weeks after viral injection. Cell-type specific expression of GCaMP6s revealed the granule cells (GCs) as the key player in GCaMP6s-induced epilepsy. Finally, by using slice electrophysiology, we demonstrated that GCaMP6s expression increases neuronal excitability in the GCs. Together, this study highlights the ability of GCaMP6s in DG-associated epileptogenesis.

Removing crosstalk signals in neuron activity by time multiplexed excitations in a two-photon all-optical physiology system.

The two-photon all-optical physiology system has attracted great interest in deciphering neuronal circuits in vivo, benefiting from its advantages in recording and modulating neuronal activities at single neuron resolutions. However, the interference, or crosstalk, between the imaging and photostimulation beams introduces a significant challenge and may impede the future application of voltage indicators in two-photon all-optical physiology system. Here, we propose the time multiplexed excitation method to distinguish signals from neuronal activities and crosstalks from photostimulation. In our system, the laser pulses of the imaging beam and photostimulation beam are synchronized, and a time delay is introduced into these pulses to separate the fluorescence signal generated by these two beams. We demonstrate the efficacy of our system in eliminating crosstalk signals from photostimulation and evaluate its influence on both genetically encoded calcium indicators (GECIs) and genetically encoded voltage indicators (GEVIs) through in vivo experiments.

A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput Screening of hTRPV1 Agonists.

Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists. The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared. A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) < nonivamide (67 ± 3.05 nM) < piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists. The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.

Whole-Brain Calcium Imaging in Drosophila during Sleep and Wake.

Genetically encoded calcium indicators (GECIs) allow for the noninvasive evaluation of neuronal activity in vivo, and imaging GECIs in Drosophila has become commonplace for understanding neural functions and connectivity in this system. GECIs can also be used as read-outs for studying sleep in this model organism. Here, we describe a methodology for tracking the activity of neurons in the fly brain using a two-photon (2p) microscopy system. This method can be adapted to perform functional studies of neural activity in Drosophila under both spontaneous and evoked conditions, as well as during spontaneous or induced sleep. We first describe a tethering and surgical procedure that allows survival under the microscopy conditions required for long-term recordings. We then outline the steps and reagents required for optogenetic activation of sleep-promoting neurons while simultaneously recording neural activity from the fly brain. We also describe the procedure for recording from two different locations-namely, the top of the head (e.g., to record mushroom body calyx activity) or the back of the head (e.g., to record central complex activity). We also provide different strategies for recording from GECIs confined to the cell body versus the entire neuron. Finally, we describe the steps required for analyzing the multidimensional data that can be acquired. In all, this protocol shows how to perform calcium imaging experiments in tethered flies, with a focus on acquiring spontaneous and induced sleep data.

Development of a microelectrode for simultaneous &lt;i&gt;in vivo&lt;/i&gt; calcium and electrophysiological recording of hippocampal neuronal activity

Calcium (Ca2+) imaging is a commonly utilized neuroscience technique for in vivo recording of neuronal activity. It involves the optical measurement of calcium concentration using genetically encoded calcium indicators (GECI) [1]. However, the kinetics of changes in the fluorescence of GECI are relatively slow and limited by the biophysics of the calcium binding [2]. In response to single action potentials (APs) in pyramidal neurons, most of the widely used GECI have a fluorescence half-life of approximately 100 ms [3]. As a result, GECI cannot provide complete information about the dynamics of neural ensembles. To address this issue, new variants of GECI, such as jGCaMP7 [3], and jGCaMP8 [4], have been developed, or genetically encoded voltage indicators (GEVI), such as JEDI-2P [5], have been utilized. However, the speed of GECI or GEVI is still lower than that of electrophysiological registration methods. Thus, we have designed a microelectrode that can be utilized with a gradient lens for in vivo calcium imaging with a miniscope. The miniscope is a miniature microscope for single photon epifluorescence Ca2+ imaging, which enables recording of neuronal activity in freely moving laboratory animals, unlike the traditionally used two-photon imaging technique. The miniscope utilizes gradient-index (GRIN) lenses that are implanted directly into the brain of a laboratory animal, instead of a conventional lens. The gradient lens is a transparent cylinder with a diameter of 1.8 mm and a length of 3.8 mm. To facilitate electrophysiological recording, we developed a microelectrode that can be aligned with a GRIN lens. The microelectrode is a three-layer structure consisting of: 1) a polyimide film, 2) conductive copper tracks deposited through thermoforming, and 3) a polyimide film with cutouts for pads. On one side of the microelectrode, there are 12 gold-plated conductive contacts for registering local field potentials, while on the other side, a similar number of conductive tracks are present for connecting to a connector that transmits data to the processing board. The flexible microelectrode is wrapped around a gradient lens and fixed using thermoforming, after which it is implanted in the animal's brain. Using the developed microelectrode, our aim is to perform a comparative analysis of the calcium and electrophysiological activity of hippocampal neurons in freely moving wild-type mice and in a mouse model of Alzheimer's disease. This study will enable the identification of any abnormalities in Alzheimer's disease at the level of neural ensembles and may suggest new treatment approaches or mechanisms for the development of the progressive memory loss pathology associated with this disease. We would like to express our gratitude to Anastasia Viktorovna Bolshakova for her administrative assistance, and to the staff of the Laboratory of Molecular Neurodegeneration for their invaluable help and advices.

Distinct and Targetable Role of Calcium and Calcium Sensing Receptor in Leukaemia

Calcium, the most abundant mineral in the body, is a key component of bones and is released by bone remodelling. Calcium ions play a role in the localisation, engraftment and the adhesion of normal haematopoietic stem cells (HSC) to extracellular matrix (ECM) proteins in the bone marrow microenvironment (BMM) via the calcium sensing receptor (CaSR), thereby maintaining normal haematopoiesis. However, the role of this heterotrimeric G-protein-coupled receptor and its associated pathways in the local BMM for the development of leukaemia is poorly understood. We hypothesized that calcium ions, subject to a fine balance between osteoblasts and osteoclasts and released from bone, and/or CaSR contribute to development, progression and response to therapy in leukaemia and might be targetable via CaSR. By imaging of a genetically-encoded calcium indicator using intravital microscopy (IVM), we have shown that the local calcium concentration forms a gradient in the BMM with the highest concentrations close to the endosteal area. Furthermore, calcium levels differ between different leukaemias, with the calcium concentration of mice and human patients being highest in acute myeloid leukaemia (AML). Additionally, CaSR was expressed at a higher level on AML compared to chronic myeloid leukaemia (CML) cells. Using genetic deletion or overexpression or pharmacological inhibition of CaSR, we revealed that this receptor influences the localization of CML and AML cells in the BMM. Furthermore, we showed that the secondary messenger intracellular calcium (iCa 2+) can be regulated by CaSR in different leukaemia cell lines which have differential sensitivity to extracellular calcium (eCa 2+). Exposure of the human cell lines K562 (BCR-ABL1 +) and THP1 (MLL-AF9 +) to increasing calcium concentrations revealed that CaSR expression, migration and adhesion to the extracellular matrix (ECM) protein fibronectin were not altered in CML, but significantly increased in AML. Genetic deletion or overexpression of Casr in leukaemia-initiating cells in murine models revealed that CaSR acts as tumour suppressor in BCR-ABL1-driven CML and B-cell acute lymphoblastic leukaemia (B-ALL) and as oncogene in AML. Conversely, overexpression of CaSR in CaSR-deficient AML-initiating cells ‘rescued’ the disease. Limiting dilution transplantation of CaSR-deficient AML-initiating cells to assess leukaemic stem cell number and/or function revealed a 6.5-fold reduction of leukemic stem cells. Treatment of mice with the CaSR agonist cinacalcet and imatinib prolonged survival of mice with CML, while treatment with a CaSR antagonist NPS-2143 significantly reduced tumour burden and prolonged survival of mice with AML in syngeneic and xenogeneic transplantation experiments. In summary, our results suggest that calcium ions, likely originating from the BMM and/or CaSR strongly and differentially influence leukaemia localisation and progression. As an adjunct to existing treatment strategies, targeting of CaSR with specific pharmacologic agonists may be beneficial in CML and B-ALL, while CaSR antagonists may prolong survival of patients with AML.

An expanded GCaMP reporter toolkit for functional imaging in Caenorhabditis elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMPs, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters is also well suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

GCaMP – a Family of Single-Fluorophore Genetically Encoded Calcium Indicators

Single fluorophore genetically encoded calcium indicators (GECIs) such as GCaMP are widely utilized tools for investigating neuronal activity. Their primary advantage lies in their capacity to provide real-time and highly sensitive responses to fluctuations in intracellular calcium ion concentrations. This characteristic is of significant importance when studying neuronal processes and ensembles, wherein calcium signals play a crucial role in information transmission. This comprehensive review focuses on the GCaMP family, encompassing an analysis of their various types, distinctive features, and potential applications for visualizing neuronal activity. Special attention is dedicated to the ongoing advancements in GCaMP technology, particularly the endeavors to expand their spectral properties and enhance their capability to detect high-frequency spike activity.

Assessing CaMPARI as new approach methodology for evaluating neurotoxicity

Developmental exposure to environmental toxicants has been linked to the onset of neurological disorders and diseases. Despite substantial advances in the field of neurotoxicology, there remain significant knowledge gaps in our understanding of cellular targets and molecular mechanisms that mediate the neurotoxicological endpoints associated with exposure to both legacy contaminants and emerging contaminants of concern. Zebrafish are a powerful neurotoxicological model given their high degree sequence conservation with humans and the similarities they share with mammals in micro- and macro-level brain structures. Many zebrafish studies have effectively utilized behavioral assays to predict the neurotoxic potential of different compounds, but behavioral phenotypes are rarely able to predict the brain structures, cell types, or mechanisms affected by chemical exposures. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI), a recently developed genetically-encoded calcium indicator, undergoes a permanent green to red switch in the presence of elevated intracellular Ca2+ concentrations and 405-nm light, which allows for a “snapshot” of brain activity in freely-swimming larvae. To determine whether behavioral results are predictive of patterns of neuronal activity, we assessed the effects of three common neurotoxicants, ethanol, 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95), and monoethylhexyl phthalate (MEHP), on both brain activity and behavior by combining the behavioral light/dark assay with CaMPARI imaging. We demonstrate that brain activity profiles and behavioral phenotypes are not always concordant and, therefore, behavior alone is not sufficient to understand how toxicant exposure affects neural development and network dynamics. We conclude that pairing behavioral assays with functional neuroimaging tools such as CaMPARI provides a more comprehensive understanding of the neurotoxic endpoints of compounds while still offering a relatively high throughput approach to toxicity testing.

High fidelity sensory-evoked responses in neocortex after intravenous injection of genetically encoded calcium sensors.

Two-photon imaging of genetically-encoded calcium indicators (GECIs) has traditionally relied on intracranial injections of adeno-associated virus (AAV) or transgenic animals to achieve expression. Intracranial injections require an invasive surgery and result in a relatively small volume of tissue labeling. Transgenic animals, although they can have brain-wide GECI expression, often express GECIs in only a small subset of neurons, may have abnormal behavioral phenotypes, and are currently limited to older generations of GECIs. Inspired by recent developments in the synthesis of AAVs that readily cross the blood brain barrier, we tested whether an alternative strategy of intravenously injecting AAV-PHP.eB is suitable for two-photon calcium imaging of neurons over many months after injection. We injected C57BL/6 J mice with AAV-PHP.eB-Synapsin-jGCaMP7s via the retro-orbital sinus. After allowing 5 to 34 weeks for expression, we performed conventional and widefield two-photon imaging of layers 2/3, 4 and 5 of the primary visual cortex. We found reproducible trial-by-trial neural responses and tuning properties consistent with known feature selectivity in the visual cortex. Thus, intravenous injection of AAV-PHP.eB does not interfere with the normal processing in neural circuits. In vivo and histological images show no nuclear expression of jGCaMP7s for at least 34 weeks post-injection.

Calcium imaging and analysis of the jugular-nodose ganglia enables identification of distinct vagal sensory neuron subsets

Objective. Sensory nerves of the peripheral nervous system (PNS) transmit afferent signals from the body to the brain. These peripheral nerves are composed of distinct subsets of fibers and associated cell bodies, which reside in peripheral ganglia distributed throughout the viscera and along the spinal cord. The vagus nerve (cranial nerve X) is a complex polymodal nerve that transmits a wide array of sensory information, including signals related to mechanical, chemical, and noxious stimuli. To understand how stimuli applied to the vagus nerve are encoded by vagal sensory neurons in the jugular-nodose ganglia, we developed a framework for micro-endoscopic calcium imaging and analysis. Approach. We developed novel methods for in vivo imaging of the intact jugular-nodose ganglion using a miniature microscope (Miniscope) in transgenic mice with the genetically-encoded calcium indicator GCaMP6f. We adapted the Python-based analysis package Calcium Imaging Analysis (CaImAn) to process the resulting one-photon fluorescence data into calcium transients for subsequent analysis. Random forest classification was then used to identify specific types of neuronal responders. Results. We demonstrate that recordings from the jugular-nodose ganglia can be accomplished through careful surgical dissection and ganglia stabilization. Using a customized acquisition and analysis pipeline, we show that subsets of vagal sensory neurons respond to different chemical stimuli applied to the vagus nerve. Successful classification of the responses with a random forest model indicates that certain calcium transient features, such as amplitude and duration, are important for encoding these stimuli by sensory neurons. Significance. This experimental approach presents a new framework for investigating how individual vagal sensory neurons encode various stimuli on the vagus nerve. Our surgical and analytical approach can be applied to other PNS ganglia in rodents and other small animal species to elucidate previously unexplored roles for peripheral neurons in a diverse set of physiological functions.

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G+QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

CNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Calcium Imaging of Anopheles coluzzii Mosquito Antennae Expressing the Calcium Indicator GCaMP6f.

Calcium imaging is a technique used to measure functional neuronal activities in response to stimuli. It has been used for years to study odorant-induced responses in insects (i.e., honeybees, Drosophila, and moths) and was recently introduced into mosquitoes. Traditionally, calcium imaging in mosquitoes was performed using nonspecific calcium indicator dyes to examine neuronal responses in whole insect brain regions, but the development of genetically encoded calcium indicators (GECIs) has facilitated the ability to perform functional calcium imaging on specific tissues. For example, by specifically expressing a GECI in olfactory neurons, the odor-induced responses of these neurons in peripheral organs can be examined. Calcium imaging of mosquito antennae further provides an advantageous method for simultaneously visualizing the activity of several antennal neurons in a single experiment. In this protocol, we describe a calcium imaging method to study odor-evoked responses in Anopheles coluzzii antennae expressing the calcium indicator GCaMP6f. This method requires imaging equipment (compound microscope, light sources, and camera), an odorant delivery system, and image acquisition software. The mosquito preparation is straightforward but requires practice to minimize mosquito movement during imaging. Recorded videos can be analyzed using Fiji software to generate heatmaps and activity traces for odorant-evoked responses. This protocol can also be used, with some modifications, to study other peripheral organs (such as labella, palps, and tarsi).

The GEM-GECO Calcium Indicator Is Useable in Ogataea parapolymorpha Yeast, but Aggravates Effects of Increased Cytosolic Calcium Levels.

Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse using a Large Cranial Window.

Wide-field calcium imaging from the mouse's neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

&lt;em&gt;In Vivo&lt;/em&gt; Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse's neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator.

Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.

PyZebrascope: An Open-Source Platform for Brain-Wide Neural Activity Imaging in Zebrafish.

Understanding how neurons interact across the brain to control animal behaviors is one of the central goals in neuroscience. Recent developments in fluorescent microscopy and genetically-encoded calcium indicators led to the establishment of whole-brain imaging methods in zebrafish, which record neural activity across a brain-wide volume with single-cell resolution. Pioneering studies of whole-brain imaging used custom light-sheet microscopes, and their operation relied on commercially developed and maintained software not available globally. Hence it has been challenging to disseminate and develop the technology in the research community. Here, we present PyZebrascope, an open-source Python platform designed for neural activity imaging in zebrafish using light-sheet microscopy. PyZebrascope has intuitive user interfaces and supports essential features for whole-brain imaging, such as two orthogonal excitation beams and eye damage prevention. Its camera module can handle image data throughput of up to 800 MB/s from camera acquisition to file writing while maintaining stable CPU and memory usage. Its modular architecture allows the inclusion of advanced algorithms for microscope control and image processing. As a proof of concept, we implemented a novel automatic algorithm for maximizing the image resolution in the brain by precisely aligning the excitation beams to the image focal plane. PyZebrascope enables whole-brain neural activity imaging in fish behaving in a virtual reality environment. Thus, PyZebrascope will help disseminate and develop light-sheet microscopy techniques in the neuroscience community and advance our understanding of whole-brain neural dynamics during animal behaviors.