Journal articles: 'GCaMP6f'

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Chen, Yen Lin, Thomas M. Baker, Frank Lee, Bo Shui, Jane C. Lee, Petr Tvrdik, Michael I. Kotlikoff, and Swapnil K. Sonkusare. "Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice." Journal of Vascular Research 58, no. 3 (2021): 159–71. http://dx.doi.org/10.1159/000514210.

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Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. Methods: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. Results: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. Conclusion: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.

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Srienc, Anja I., Pei-Pei Chiang, Abby J. Schmitt, and Eric A. Newman. "Cortical spreading depolarizations induced by surgical field blood in a mouse model of neurosurgery." Journal of Neurosurgery 132, no. 6 (June 2020): 1820–28. http://dx.doi.org/10.3171/2018.12.jns181130.

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OBJECTIVECortical spreading depolarization (CSD) has been linked to poor clinical outcomes in the setting of traumatic brain injury, malignant stroke, and subarachnoid hemorrhage. There is evidence that electrocautery during neurosurgical procedures can also evoke CSD waves in the brain. It is unknown whether blood contacting the cortical surface during surgical bleeding affects the frequency of spontaneous or surgery-induced CSDs. Using a mouse neurosurgical model, the authors tested the hypothesis that electrocautery can induce CSD waves and that surgical field blood (SFB) is associated with more CSDs. The authors also investigated whether CSD can be reliably observed by monitoring the fluorescence of GCaMP6f expressed in neurons.METHODSCSD waves were monitored by using confocal microscopy to detect fluorescence increases at the cortical surface in mice expressing GCaMP6f in CamKII-positive neurons. The cortical surface was electrocauterized through an adjacent burr hole. SFB was simulated by applying a drop of tail vein blood to the brain through the same burr hole.RESULTSCSD waves were readily detected in GCaMP6f-expressing mice. Monitoring GCaMP6f fluorescence provided far better sensitivity and spatial resolution than detecting CSD events by observing changes in the intrinsic optical signal (IOS). Forty-nine percent of the CSD waves identified by GCaMP6f had no corresponding IOS signal. Electrocautery evoked CSD waves. On average, 0.67 ± 0.08 CSD events were generated per electrocautery episode, and multiple CSD waves could be induced in the same mouse by repeated cauterization (average, 7.9 ± 1.3 events; maximum number in 1 animal, 13 events). In the presence of SFB, significantly more spontaneous CSDs were generated (1.35 ± 0.37 vs 0.13 ± 0.16 events per hour, p = 0.002). Ketamine effectively decreased the frequency of spontaneous CSD waves (1.35 ± 0.37 to 0.36 ± 0.15 CSD waves per hour, p = 0.016) and electrocautery-stimulated CSD waves (0.80 ± 0.05 to 0.18 ± 0.08 CSD waves per electrocautery, p = 0.00002).CONCLUSIONSCSD waves are detected with far greater sensitivity and fidelity by monitoring GCaMP6f signals in neurons than by monitoring IOSs. Electrocautery reliably evokes CSD waves, and the frequency of spontaneous CSD waves is increased when blood is applied to the cortical surface. These experimental conditions recapitulate common scenarios in the neurosurgical operating room. Ketamine, a clinically available pharmaceutical agent, can block stimulated and spontaneous CSDs. More research is required to understand the clinical importance of intraoperative CSD.

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Ye, Liang, Mateen A. Haroon, Angelica Salinas, and Martin Paukert. "Comparison of GCaMP3 and GCaMP6f for studying astrocyte Ca2+ dynamics in the awake mouse brain." PLOS ONE 12, no. 7 (July 24, 2017): e0181113. http://dx.doi.org/10.1371/journal.pone.0181113.

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Asteriti, Sabrina, Che-Hsiung Liu, and Roger C. Hardie. "Calcium signalling in Drosophila photoreceptors measured with GCaMP6f." Cell Calcium 65 (July 2017): 40–51. http://dx.doi.org/10.1016/j.ceca.2017.02.006.

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Conti, Emilia, Anna Allegra Mascaro, and Francesco Pavone. "Large Scale Double-Path Illumination System with Split Field of View for the All-Optical Study of Inter-and Intra-Hemispheric Functional Connectivity on Mice." Methods and Protocols 2, no. 1 (January 29, 2019): 11. http://dx.doi.org/10.3390/mps2010011.

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Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a combination of recently developed optical tools to monitor neural population at the meso-scale level and to mould the cortical patterns of targeted neuronal population. Our goal was to investigate the propagation of neuronal activity over the mouse cortex that is triggered by optogenetic stimulation in the contralateral hemisphere. Towards this aim, we developed a wide-field fluorescence microscope that is characterized by a double illumination path allowing for the optogenetic stimulation of the transfected area in the left hemisphere and the simultaneous recording of cortical activity in the right hemisphere. The microscope was further implemented with a custom shutter in order to split the LED illumination path, resulting in a half-obscured field of view. By avoiding the spectral crosstalk between GCaMP6f and channelrhodopsin 2 (ChR2), this system offered the possibility of simultaneous “pumping and probing” of inter-hemispheric functional connectivity on Thy1-GCaMP6f mice.

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Park, Kicheon, Anuki C. Liyanage, Alan P. Koretsky, Yingtian Pan, and Congwu Du. "Optical imaging of stimulation-evoked cortical activity using GCaMP6f and jRGECO1a." Quantitative Imaging in Medicine and Surgery 11, no. 3 (March 2020): 998–1009. http://dx.doi.org/10.21037/qims-20-921.

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Carcaud, Julie, Marianne Otte, Bernd Grünewald, Albrecht Haase, Jean-Christophe Sandoz, and Martin Beye. "Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee." PLOS Biology 21, no. 1 (January 31, 2023): e3001984. http://dx.doi.org/10.1371/journal.pbio.3001984.

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Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities.

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Sathyanesan, Aaron, Panagiotis Kratimenos, and Vittorio Gallo. "Disruption of neonatal Purkinje cell function underlies injury-related learning deficits." Proceedings of the National Academy of Sciences 118, no. 11 (March 9, 2021): e2017876118. http://dx.doi.org/10.1073/pnas.2017876118.

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It is hypothesized that perinatal cerebellar injury leads to long-term functional deficits due to circuit dysmaturation. Using a novel integration of GCaMP6f fiber photometry with automated measurement of cerebellar behavior using the ErasmusLadder, we causally link cerebellar injury to altered Purkinje cell responses during maladaptive behavior. Chemogenetic inhibition of neonatal Purkinje cells is sufficient to phenocopy the effects of perinatal cerebellar injury. Our results uncover a direct link between perinatal cerebellar injury and activity-dependent maturation of cerebellar cortex.

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Chou, Allison Tzu-Han, Henry Hollis, Polina Fenik, Keelee Pullum, Michelle Slinger, Zachary Zamore, Yan Zhu, Ron Anafi, and Sigrid Veasey. "0288 Role of cofilin and calcium signaling in sleep-loss neural injury." Sleep 45, Supplement_1 (May 25, 2022): A130. http://dx.doi.org/10.1093/sleep/zsac079.286.

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Abstract Introduction Chronic sleep disruption (CSD) in young adult mice leads to phenotypes consistent with early (pre-plaque) Alzheimer’s Disease (AD), including increased Aβ and hippocampal neuron loss. Mechanisms underlying this injury are not known. Both acute sleep loss and AD activate cofilin, a regulator of actin dynamics. Activated cofilin (AC) in AD mouse models can impart neural injury, increase Aβ, and cofilin translocation to the mitochondria delays cytosolic Ca2+ clearance. We are critically testing the role of AC in chronic short sleep (CSS) and sleep fragmentation (SF) neural injury. Methods Synapse loss was studied using STED confocal microscopy and Imaris in CSS (n=9) and rested (n=10) mice. Synapses were identified as overlaps of pre- and postsynaptic densities. Percent area of cofilin was measured with FIJI. To further understand if and how wake-induced cofilin activation induces sleep-loss synapse and neural injury, we implanted AAV9CAMKII-GCaMP6f and then GRIN lenses, and later studied CAMKII calcium transients in CA1 of WT controls (n=4) and SF mice (n=4) by measuring GCaMP6f calcium transients. We developed a Shiny R application to analyze the frequency of Ca2+ spikes, ΔF/F0, and the rising and clearance patterns of spikes. To directly test cofilin’s role in delayed calcium clearance, we studied the calcium transients in hAPP mice (n=2) after injection of AAV-CAMKII-CofilinS3A to express AC and GCaMP6f. All data were analyzed with two-way ANOVA or unpaired t-tests. Results Results reveal significant synapse loss in CA1 of CSS mice (CSS=48.8±10.3; Rested=83.4±8.4), t(16)=2.63, p<0.02, and increased cofilin activation (AC=19.8±3.41; Rested=8.76±1.95), t(16)=8.43, p<0.0001. SF mice reveal an increase in NREM sleep firing rates, F(1,1)=22.0, p<0.001. In contrast, hAPP-AC mice show significantly increased ΔF/F0, F(1,1)=356, p<0.0001, prolonged calcium influx, F(1,1)=18.6, p<0.02, and prolonged calcium clearance duration, F(1,1)=23.9, p<0.01, but not increased firing frequencies. Conclusion CSS induces CA1 synapse loss and cofilin activation in WT mice. Increased CAMKII calcium ΔF/F0 occurs through different pathways in SF and AC, suggesting additional factors in CSD neural injury. Support (If Any) NIH AG054104; AG064231

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Cramer, Samuel, Laurentiu Popa, Samuel Haley, Sanjay Dhawan, Russell Carter, Jianfang Ning, Justin Aronson, Suhasa Kodandaramaiah, Timothy Ebner, and Clark Chen. "PATH-02. CHARACTERIZATION OF FUNCTIONAL NETWORK EFFECTS IN THE CEREBRAL CORTEX DURING BRAIN TUMORIGENESIS IN THE MOUSE." Neuro-Oncology 22, Supplement_2 (November 2020): ii164. http://dx.doi.org/10.1093/neuonc/noaa215.684.

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Abstract INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ flux imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bear pertinence to the pathogenesis of neuro-cognitive decline and tumor associated epilepsy.

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Cramer, Samuel, Laurentiu Popa, Samuel Haley, Sanjay Dhawan, Russell Carter, Jianfang Ning, Justin Aronson, Suhasa Kodandaramaiah, Timothy Ebner, and Clark Chen. "TMOD-10. EFFECT OF BRAIN TUMORIGENESIS ON CEREBRAL CORTICAL FUNCTIONAL CONNECTIVITY IN THE MOUSE." Neuro-Oncology 22, Supplement_2 (November 2020): ii229—ii230. http://dx.doi.org/10.1093/neuonc/noaa215.961.

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Abstract INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bears pertinence to the pathogenesis of neuro-cognitive decline and tumor-associated epilepsy.

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Aryal, Surya P., Mengfan Xia, Ebubechi Adindu, Caroline Davis, Pavel I. Ortinski, and Christopher I. Richards. "ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes." Analytical Chemistry 94, no. 4 (January 21, 2022): 2099–108. http://dx.doi.org/10.1021/acs.analchem.1c04321.

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Garg, Anupam K., Peichao Li, Mohammad S. Rashid, and Edward M. Callaway. "Color and orientation are jointly coded and spatially organized in primate primary visual cortex." Science 364, no. 6447 (June 27, 2019): 1275–79. http://dx.doi.org/10.1126/science.aaw5868.

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Previous studies support the textbook model that shape and color are extracted by distinct neurons in primate primary visual cortex (V1). However, rigorous testing of this model requires sampling a larger stimulus space than previously possible. We used stable GCaMP6f expression and two-photon calcium imaging to probe a very large spatial and chromatic visual stimulus space and map functional microarchitecture of thousands of neurons with single-cell resolution. Notable proportions of V1 neurons strongly preferred equiluminant color over achromatic stimuli and were also orientation selective, indicating that orientation and color in V1 are mutually processed by overlapping circuits. Single neurons could precisely and unambiguously code for both color and orientation. Further analyses revealed systematic spatial relationships between color tuning, orientation selectivity, and cytochrome oxidase histology.

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Shigetomi, Eiji, Sebastian Kracun, and Baljit S. Khakh. "Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters." Neuron Glia Biology 6, no. 3 (August 2010): 183–91. http://dx.doi.org/10.1017/s1740925x10000219.

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Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.

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Gu, Xiaochun, Wei Chen, Jiang You, Alan P. Koretsky, N. D. Volkow, Yingtian Pan, and Congwu Du. "Long-term optical imaging of neurovascular coupling in mouse cortex using GCaMP6f and intrinsic hemodynamic signals." NeuroImage 165 (January 2018): 251–64. http://dx.doi.org/10.1016/j.neuroimage.2017.09.055.

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Indersmitten, Tim, Tamara Berdyyeva, Leah Aluisio, Timothy Lovenberg, Pascal Bonaventure, and Ryan M. Wyatt. "Utilizing Miniature Fluorescence Microscopy to Image Hippocampal Place Cell Ensemble Function in Thy1.GCaMP6f Transgenic Mice." Current Protocols in Pharmacology 82, no. 1 (August 20, 2018): e42. http://dx.doi.org/10.1002/cpph.42.

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Ricci Signorini, Maria Elena, Monika Szepes, Anna Melchert, Mine Bakar, Sylvia Merkert, Alexandra Haase, Gudrun Göhring, Ulrich Martin, and Ina Gruh. "Generation of human induced pluripotent stem cell lines encoding for genetically encoded calcium indicators RCaMP1h and GCaMP6f." Stem Cell Research 60 (April 2022): 102697. http://dx.doi.org/10.1016/j.scr.2022.102697.

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Park, Dong-Wook, Jared P. Ness, Sarah K. Brodnick, Corinne Esquibel, Joseph Novello, Farid Atry, Dong-Hyun Baek, et al. "Electrical Neural Stimulation and Simultaneous in Vivo Monitoring with Transparent Graphene Electrode Arrays Implanted in GCaMP6f Mice." ACS Nano 12, no. 1 (January 8, 2018): 148–57. http://dx.doi.org/10.1021/acsnano.7b04321.

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Rosenthal, Zachary P., Ryan V. Raut, Ryan M. Bowen, Abraham Z. Snyder, Joseph P. Culver, Marcus E. Raichle, and Jin-Moo Lee. "Peripheral sensory stimulation elicits global slow waves by recruiting somatosensory cortex bilaterally." Proceedings of the National Academy of Sciences 118, no. 8 (February 17, 2021): e2021252118. http://dx.doi.org/10.1073/pnas.2021252118.

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Slow waves (SWs) are globally propagating, low-frequency (0.5- to 4-Hz) oscillations that are prominent during sleep and anesthesia. SWs are essential to neural plasticity and memory. However, much remains unknown about the mechanisms coordinating SW propagation at the macroscale. To assess SWs in the context of macroscale networks, we recorded cortical activity in awake and ketamine/xylazine-anesthetized mice using widefield optical imaging with fluorescent calcium indicator GCaMP6f. We demonstrate that unilateral somatosensory stimulation evokes bilateral waves that travel across the cortex with state-dependent trajectories. Under anesthesia, we observe that rhythmic stimuli elicit globally resonant, front-to-back propagating SWs. Finally, photothrombotic lesions of S1 show that somatosensory-evoked global SWs depend on bilateral recruitment of homotopic primary somatosensory cortices. Specifically, unilateral lesions of S1 disrupt somatosensory-evoked global SW initiation from either hemisphere, while spontaneous SWs are largely unchanged. These results show that evoked SWs may be triggered by bilateral activation of specific, homotopically connected cortical networks.

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Wong, Wen Mai, Jie Cao, Xingjian Zhang, Wayne I. Doyle, Luis L. Mercado, Laurent Gautron, and Julian P. Meeks. "Physiology-forward identification of bile acid–sensitive vomeronasal receptors." Science Advances 6, no. 22 (May 2020): eaaz6868. http://dx.doi.org/10.1126/sciadv.aaz6868.

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The mouse accessory olfactory system (AOS) supports social and reproductive behavior through the sensation of environmental chemosignals. A growing number of excreted steroids have been shown to be potent AOS cues, including bile acids (BAs) found in feces. As is still the case with most AOS ligands, the specific receptors used by vomeronasal sensory neurons (VSNs) to detect BAs remain unknown. To identify VSN BA receptors, we first performed a deep analysis of VSN BA tuning using volumetric GCaMP6f/s Ca2+ imaging. These experiments revealed multiple populations of BA-receptive VSNs with submicromolar sensitivities. We then developed a new physiology-forward approach for identifying AOS ligand-receptor interactions, which we call Fluorescence Live Imaging for Cell Capture and RNA sequencing, or FLICCR-seq. FLICCR-seq analysis revealed five specific V1R family receptors enriched in BA-sensitive VSNs. These studies introduce a powerful new approach for ligand-receptor matching and reveal biological mechanisms underlying mammalian BA chemosensation.

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Ukhanov, Kirill, Yuriy V. Bobkov, Jeffrey R. Martens, and Barry W. Ache. "Initial Characterization of a Subpopulation of Inherent Oscillatory Mammalian Olfactory Receptor Neurons." Chemical Senses 44, no. 8 (July 17, 2019): 583–92. http://dx.doi.org/10.1093/chemse/bjz052.

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Abstract Published evidence suggests that inherent rhythmically active or “bursting” primary olfactory receptor neurons (bORNs) in crustaceans have the previously undescribed functional property of encoding olfactory information by having their rhythmicity entrained by the odor stimulus. In order to determine whether such bORN-based encoding is a fundamental feature of olfaction that extends beyond crustaceans, we patch-clamped bORN-like ORNs in mice, characterized their dynamic properties, and show they align with the dynamic properties of lobster bORNs. We then characterized bORN-like activity by imaging the olfactory epithelium of OMP-GCaMP6f mice. Next, we showed rhythmic activity is not dependent upon the endogenous OR by patching ORNs in OR/GFP mice. Lastly, we showed the properties of bORN-like ORNs characterized in mice generalize to rats. Our findings suggest encoding odor time should be viewed as a fundamental feature of olfaction with the potential to be used to navigate odor plumes in animals as diverse as crustaceans and mammals.

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Degtyaruk, Oleksiy, Benedict Mc Larney, Xosé Deán-Ben, Shy Shoham, and Daniel Razansky. "Optoacoustic Calcium Imaging of Deep Brain Activity in an Intracardially Perfused Mouse Brain Model." Photonics 6, no. 2 (June 12, 2019): 67. http://dx.doi.org/10.3390/photonics6020067.

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One main limitation of established neuroimaging methods is the inability to directly visualize large-scale neural dynamics in whole mammalian brains at subsecond speeds. Optoacoustic imaging has advanced in recent years to provide unique advantages for real-time deep-tissue observations, which have been exploited for three-dimensional imaging of both cerebral hemodynamic parameters and direct calcium activity in rodents. Due to a lack of suitable calcium indicators excitable in the near-infrared window, optoacoustic imaging of neuronal activity at deep-seated areas of the mammalian brain has been impeded by the strong absorption of blood in the visible range of the light spectrum. To overcome this, we have developed and validated an intracardially perfused mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f that closely resembles in vivo conditions. By overcoming the limitations of hemoglobin-based light absorption, this new technique was used to observe stimulus-evoked calcium dynamics in the brain at penetration depths and spatio-temporal resolution scales not attainable with existing neuroimaging techniques.

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Éltes, Tímea, Miklos Szoboszlay, Katalin Kerti‐Szigeti, and Zoltan Nusser. "Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca 2+ ] transients in weakly GCaMP6f‐expressing hippocampal pyramidal cells." Journal of Physiology 597, no. 11 (May 6, 2019): 2925–47. http://dx.doi.org/10.1113/jp277681.

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Jeon, Bo-Hui, Yeong-Min Yoo, Eui-Man Jung, and Eui-Bae Jeung. "Dexamethasone Treatment Increases the Intracellular Calcium Level Through TRPV6 in A549 Cells." International Journal of Molecular Sciences 21, no. 3 (February 5, 2020): 1050. http://dx.doi.org/10.3390/ijms21031050.

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This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

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Celotto, Marco, Chiara De Luca, Paolo Muratore, Francesco Resta, Anna Letizia Allegra Mascaro, Francesco Saverio Pavone, Giulia De Bonis, and Pier Stanislao Paolucci. "Analysis and Model of Cortical Slow Waves Acquired with Optical Techniques." Methods and Protocols 3, no. 1 (January 31, 2020): 14. http://dx.doi.org/10.3390/mps3010014.

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Slow waves (SWs) are spatio-temporal patterns of cortical activity that occur both during natural sleep and anesthesia and are preserved across species. Even though electrophysiological recordings have been largely used to characterize brain states, they are limited in the spatial resolution and cannot target specific neuronal population. Recently, large-scale optical imaging techniques coupled with functional indicators overcame these restrictions, and new pipelines of analysis and novel approaches of SWs modelling are needed to extract relevant features of the spatio-temporal dynamics of SWs from these highly spatially resolved data-sets. Here we combined wide-field fluorescence microscopy and a transgenic mouse model expressing a calcium indicator (GCaMP6f) in excitatory neurons to study SW propagation over the meso-scale under ketamine anesthesia. We developed a versatile analysis pipeline to identify and quantify the spatio-temporal propagation of the SWs. Moreover, we designed a computational simulator based on a simple theoretical model, which takes into account the statistics of neuronal activity, the response of fluorescence proteins and the slow waves dynamics. The simulator was capable of synthesizing artificial signals that could reliably reproduce several features of the SWs observed in vivo, thus enabling a calibration tool for the analysis pipeline. Comparison of experimental and simulated data shows the robustness of the analysis tools and its potential to uncover mechanistic insights of the Slow Wave Activity (SWA).

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Barykina, Natalia V., Vladimir P. Sotskov, Anna M. Gruzdeva, You Kure Wu, Ruben Portugues, Oksana M. Subach, Elizaveta S. Chefanova, et al. "FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity." International Journal of Molecular Sciences 21, no. 8 (April 24, 2020): 3012. http://dx.doi.org/10.3390/ijms21083012.

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Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

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Weitz, Andrew C., Matthew R. Behrend, Nan Sook Lee, Ronald L. Klein, Vince A. Chiodo, William W. Hauswirth, Mark S. Humayun, James D. Weiland, and Robert H. Chow. "Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators." Journal of Neurophysiology 109, no. 7 (April 1, 2013): 1979–88. http://dx.doi.org/10.1152/jn.00852.2012.

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Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.

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Bancroft, Eric, Rahul Srinivasan, and Lee A. Shapiro. "Macrophage Migration Inhibitory Factor Alters Functional Properties of CA1 Hippocampal Neurons in Mouse Brain Slices." International Journal of Molecular Sciences 21, no. 1 (December 31, 2019): 276. http://dx.doi.org/10.3390/ijms21010276.

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Neuroinflammation is implicated in a host of neurological insults, such as traumatic brain injury (TBI), ischemic stroke, Alzheimer’s disease, Parkinson’s disease, and epilepsy. The immune response to central nervous system (CNS) injury involves sequelae including the release of numerous cytokines and chemokines. Macrophage migration inhibitory factor (MIF), is one such cytokine that is elevated following CNS injury, and is associated with the prognosis of TBI, and ischemic stroke. MIF has been identified in astrocytes and neurons, and some of the trophic actions of MIF have been related to its direct and indirect actions on astrocytes. However, the potential modulation of CNS neuronal function by MIF has not yet been explored. This study tests the hypothesis that MIF can directly influence hippocampal neuronal function. MIF was microinjected into the hippocampus and the genetically encoded calcium indicator, GCaMP6f, was used to measure Ca2+ events in acute adult mouse brain hippocampal slices. Results demonstrated that a single injection of 200 ng MIF into the hippocampus significantly increased baseline calcium signals in CA1 pyramidal neuron somata, and altered calcium responses to N-methyl-d-aspartate (NMDA) + D-serine in pyramidal cell apical dendrites located in the stratum radiatum. These data are the first to show direct effects of MIF on hippocampal neurons and on NMDA receptor function. Considering that MIF is elevated after brain insults such as TBI, the data suggest that, in addition to the previously described role of MIF in astrocyte reactivity, elevated MIF can have significant effects on neuronal function in the hippocampus.

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Henderson, Mark J., Heather A. Baldwin, Christopher A. Werley, Stefano Boccardo, Leslie R. Whitaker, Xiaokang Yan, Graham T. Holt, et al. "A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store." PLOS ONE 10, no. 10 (October 9, 2015): e0139273. http://dx.doi.org/10.1371/journal.pone.0139273.

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Luo, Jin, Lvli Chen, Feifei Huang, Ping Gao, Heping Zhao, Yingdian Wang, and Shengcheng Han. "Intraorganellar calcium imaging in Arabidopsis seedling roots using the GCaMP variants GCaMP6m and R-CEPIA1er." Journal of Plant Physiology 246-247 (March 2020): 153127. http://dx.doi.org/10.1016/j.jplph.2020.153127.

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Huerta, Tomas S., Bilal B. Haider, Richard Adamovich-Zeitlin, Sangeeta S. Chavan, Kevin J. Tracey, and Eric H. Chang. "Vagus nerve sensory neurons have distinct neural responses to inflammatory mediators." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 52.14. http://dx.doi.org/10.4049/jimmunol.208.supp.52.14.

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Abstract Cytokines are secreted signaling proteins that are important mediators of inflammation. While prior work has demonstrated that the level of cytokines can be regulated by nerve stimulation, the role of the nervous system in sensing these immune mediators is still poorly understood. During periods of inflammation, it has been shown that sensory signals travel up the vagus nerve to the brain. However, it is unclear how individual vagal sensory neurons encode specific immune information. Here we use in vivo calcium imaging of the nodose ganglion to monitor neural activity in individual vagus nerve sensory neurons in response to specific cytokines. Using mice that express the calcium indicator GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1-photon miniature microscope (Miniscope). During imaging, the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor (TNF) were applied directly to the vagus nerve. Fluorescence data was analyzed with a Python-based software CaImAn and a custom pipeline to output the single neuron activity as change in fluorescence: ΔF/F. Our results reveal that specific vagal sensory neurons respond differentially to specific immune mediators. The average amplitude, integral, and delay of TNF-responsive neurons was significantly higher than IL-1β-responsive cells (TNF vs IL-1β, p < 0.01), while having less number of peaks per response (TNF vs IL-1β, p < 0.05). This may suggest different patterns of transient neural activity associated with each particular cytokine. Further investigation into this type of neuro-immune signaling may identify novel neural targets for the treatment of inflammatory disorders. This study was funded in part by NIH/NIGMS

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Krogman, William, J. Alan Sparks, and Elison B. Blancaflor. "Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3." International Journal of Molecular Sciences 21, no. 17 (September 2, 2020): 6385. http://dx.doi.org/10.3390/ijms21176385.

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Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.

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Chen, Yingxiao, Xianqiang Song, Sheng Ye, Lin Miao, Yun Zhu, Rong-Guang Zhang, and Guangju Ji. "Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement." Protein & Cell 4, no. 4 (April 2013): 299–309. http://dx.doi.org/10.1007/s13238-013-2103-4.

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Haase, Alexandra, Tim Kohrn, Veronika Fricke, Maria Elena Ricci Signorini, Merlin Witte, Gudrun Göhring, Ina Gruh, and Ulrich Martin. "Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives." Stem Cell Research 52 (April 2021): 102206. http://dx.doi.org/10.1016/j.scr.2021.102206.

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Landsness, E. C., L. M. Brier, R. X. Hua, K. Chen, Z. P. Rosenthal, J. P. Culver, and J. Lee. "0126 Local Slow Wave Sleep and Post-Stroke Brain Repair." Sleep 43, Supplement_1 (April 2020): A50. http://dx.doi.org/10.1093/sleep/zsaa056.124.

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Abstract Introduction Recent evidence suggests that slow wave sleep (SWS) is important for synaptic plasticity and brain repair following stroke. Previous studies described a progressive increase in whole cortex and contralesional regional delta power during sleep after stroke, suggesting a global increase in SWS. However, these studies did not distinguish between the effects of global vs. local SWS. We hypothesized that local changes in SWS delta power would parallel changes in the functional remapping and circuit repair. Methods To study SWS in living mice we used Thy-1-GCaMP6f mice (n=12), serially imaged (baseline, 24 hours, weeks 1, 4,) during sleep following photothrombotic stroke of the left forepaw somatosensory cortex (S1FP). An optical fluorescence imaging system (OFI) was used to image whole-cortex neuronal activity. The evolution of local delta activity was compared across three ROIs: 1) infarct, 2) perilesional remapped, and 3) perilesional non-remapped left. Results The photothrombotic infarct encompassed the left S1FP stimulus map, resulting in significant attenuation of S1FP evoked responses at week 1; however, a small region of activation was retained in posterior left S1FP (peri-lesional remapped). The infarct region demonstrated a decrease in delta power during sleep; however, the perilesional region of future remapping exhibited a rebound in focal delta power at 1 week after an initial decline at 24 hours. In the perilesional non-remapped area delta power decreased, but did not increase until week 4. We also observed an early wide-spread increase in delta power at 24 hours and week 1 that decreased on week 4. Conclusion With the high spatial resolution of OFI, we find that SWS is disrupted throughout the brain following focal ischemia. These data suggest that local SWS selectively increases in the region of remapping prior to repair of that circuit and that local SWS may play a role in brain repair following stroke. Support AASM Foundation #183-PA-18, #201-BS-18

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Zamore, Zachary, Allison Tzu-Han Chou, Naomi Schifman, Polina Fenik, Keelee Pullum, Michelle Slinger, and Sigrid Veasey. "0300 Early Life Sleep Fragmentation Impairs Hippocampal-Dependent Learning and Sleep-Dependency in Hippocampal Calcium Transients." Sleep 45, Supplement_1 (May 25, 2022): A135. http://dx.doi.org/10.1093/sleep/zsac079.298.

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Abstract Introduction Sleep deprivation impairs hippocampal-dependent memory, and hippocampal-dependent memory impairments occur in some dementias, including Alzheimer’s disease. As our population continues to age, understanding the molecular basis for memory impairments is increasingly important. We hypothesized that early life sleep fragmentation would result in lasting increases in hippocampal calcium transient activity. Methods B6 mice were randomized to 12wk of sleep fragmentation or rested control conditions at age 8wk. Mice were microinjected with AAV9-CamKII-GCamp6F into the hippocampus and later implanted with a GRIN Lens into CA1 secured to a baseplate along with chronic EEG/EMG electrodes and recording connector. Calcium recordings were obtained two to three months after injection and recordings were obtained across sleep-wake cycles>4mins of wake and NREM sleep. Individual cells across animal were combined into sleep fragmented (n = 521 cells) or rested (n = 443 cells) groups during wake or sleep. Average FFx was analyzed by group and condition by T-tests, paired for within and unpaired across groups. A spatial object recognition assay was also performed on all mice (n=16 for both groups) and performance across group was analyzed by paired T-tests. Results Rested mice showed normal spatial object recognition (n = 16, p<0.05). In contrast, SF mice showed impaired spatial object recognition (n = 16, N.S.). There were no differences across sleep conditions in calcium transient FFx for waking (p>0.05). However, in sleep, cells in SF mice had significantly higher average FFx values than cells in rested mice (p<0.0001). Conclusion Early-life sleep fragmentation has long-lasting impacts on memory. Since spatial memory is dependent on hippocampal function, the calcium transient FFx data suggests that a driver of this hippocampal memory impairment may be higher firing rates in sleep and/or greater calcium exposure in hippocampal CamKII neurons in sleep, both of which may perturb microglial maintenance of synapses. Understanding the molecular drivers behind this calcium dysfunction will be essential in our understanding of neurodegeneration, dementia, and Alzheimer’s disease. Support (If Any) NIH AG054104; AG064231

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Krishnan, Vijai, Lauren C. Wade-Kleyn, Ron R. Israeli, and Galit Pelled. "Peripheral Nerve Injury Induces Changes in the Activity of Inhibitory Interneurons as Visualized in Transgenic GAD1-GCaMP6s Rats." Biosensors 12, no. 6 (June 1, 2022): 383. http://dx.doi.org/10.3390/bios12060383.

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Peripheral nerve injury induces cortical remapping that can lead to sensory complications. There is evidence that inhibitory interneurons play a role in this process, but the exact mechanism remains unclear. Glutamate decarboxylase-1 (GAD1) is a protein expressed exclusively in inhibitory interneurons. Transgenic rats encoding GAD1–GCaMP were generated to visualize the activity in GAD1 neurons through genetically encoded calcium indicators (GCaMP6s) in the somatosensory cortex. Forepaw denervation was performed in adult rats, and fluorescent Ca2+ imaging on cortical slices was obtained. Local, intrahemispheric stimulation (cortical layers 2/3 and 5) induced a significantly higher fluorescence change of GAD1-expressing neurons, and a significantly higher number of neurons were responsive to stimulation in the denervated rats compared to control rats. However, remote, interhemispheric stimulation of the corpus callosum induced a significantly lower fluorescence change of GAD1-expressing neurons, and significantly fewer neurons were deemed responsive to stimulation within layer 5 in denervated rats compared to control rats. These results suggest that injury impacts interhemispheric communication, leading to an overall decrease in the activity of inhibitory interneurons in layer 5. Overall, our results provide direct evidence that inhibitory interneuron activity in the deprived S1 is altered after injury, a phenomenon likely to affect sensory processing.

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Zaelzer, Cristian, Claire Gizowski, Christopher K. Salmon, Keith K. Murai, and Charles W. Bourque. "Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells." Journal of Neurophysiology 120, no. 3 (September 1, 2018): 1386–96. http://dx.doi.org/10.1152/jn.00467.2017.

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Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured “sniffer cells” can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

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Chen, Zhiyong, Chi Zhang, Xiaodan Song, Xiang Cui, Jing Liu, Neil C. Ford, Shaoqiu He, et al. "BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo." Cells 11, no. 15 (July 23, 2022): 2280. http://dx.doi.org/10.3390/cells11152280.

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The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

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Kravets, Vira, JaeAnn M. Dwulet, Wolfgang E. Schleicher, David J. Hodson, Anna M. Davis, Laura Pyle, Robert A. Piscopio, Maura Sticco-Ivins, and Richard K. P. Benninger. "Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response." PLOS Biology 20, no. 9 (September 13, 2022): e3001761. http://dx.doi.org/10.1371/journal.pbio.3001761.

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Insulin-secreting β-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine “first responder” cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified “hub” and “leader” cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (β-GCamP6s) that show β-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of β-cells in the islet was spatially organized, dependent on the cell’s distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder β-cell state, critical for the islet first-phase response to glucose.

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Ivashkina, Olga I., Anna M. Gruzdeva, Marina A. Roshchina, Ksenia A. Toropova, and Konstantin V. Anokhin. "Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory." International Journal of Molecular Sciences 22, no. 15 (July 31, 2021): 8244. http://dx.doi.org/10.3390/ijms22158244.

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The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

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Pushchina, Evgeniya V., Ilya A. Kapustyanov, Ekaterina V. Shamshurina, and Anatoly A. Varaksin. "A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, Oncorhynchus keta, Using Mouse Adeno-Associated Viral Vectors." International Journal of Molecular Sciences 22, no. 11 (May 26, 2021): 5661. http://dx.doi.org/10.3390/ijms22115661.

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To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.

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Oláh, Attila, Mihály Ruppert, Tamás István Orbán, Ágota Apáti, Balázs Sarkadi, Béla Merkely, and Tamás Radovits. "Hemodynamic characterization of a transgenic rat strain stably expressing the calcium sensor protein GCaMP2." American Journal of Physiology-Heart and Circulatory Physiology 316, no. 5 (May 1, 2019): H1224—H1228. http://dx.doi.org/10.1152/ajpheart.00074.2019.

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A novel transgenic rat strain has recently been generated that stably expresses the genetically engineered calcium sensor protein GCaMP2 in different cell types, including cardiomyocytes, to investigate calcium homeostasis. To investigate whether the expression of the GCaMP2 protein itself affects cardiac function, in the present work we aimed at characterizing in vivo hemodynamics in the GCaMP2 transgenic rat strain. GCaMP2 transgenic rats and age-matched Sprague-Dawley control animals were investigated. In vivo hemodynamic characterization was performed by left ventricular (LV) pressure-volume analysis. Postmortem heart weight data showed cardiac hypertrophy in the GCaMP2 group (heart-weight-to-tibial-length ratio: 0.26 ± 0.01 GCaMP2 vs. 0.23 ± 0.01 g/cm Co, P < 0.05). We detected elevated mean arterial pressure and increased total peripheral resistance in transgenic rats. GCaMP2 transgenesis was associated with prolonged contraction and relaxation. LV systolic function was not altered in transgenic rats, as indicated by conventional parameters and load-independent, sensitive indices. We found a marked deterioration of LV active relaxation in GCaMP2 animals (τ: 16.8 ± 0.7 GCaMP2 vs. 12.2 ± 0.3 ms Co, P < 0.001). Our data indicated myocardial hypertrophy, arterial hypertension, and impaired LV active relaxation along with unchanged systolic performance in the heart of transgenic rats expressing the GCaMP2 fluorescent calcium sensor protein. Special caution should be taken when using transgenic models in cardiovascular studies. NEW & NOTEWORTHY Genetically encoded Ca2+-sensors, like GCaMP2, are important tools to reveal molecular mechanisms for Ca2+-sensing. We provided left ventricular hemodynamic characterization of GCaMP2 transgenic rats and found increased afterload, cardiac hypertrophy, and prolonged left ventricular relaxation, along with unaltered systolic function and contractility. Special caution should be taken when using this rodent model in cardiovascular pharmacological and toxicological studies.

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Ding, JingJin, Andrew F. Luo, LiYan Hu, DaCheng Wang, and Feng Shao. "Structural basis of the ultrasensitive calcium indicator GCaMP6." Science China Life Sciences 57, no. 3 (January 4, 2014): 269–74. http://dx.doi.org/10.1007/s11427-013-4599-5.

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Chen, Jiakun, Li Xia, Michael R. Bruchas, and Lilianna Solnica-Krezel. "Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish." Developmental Biology 430, no. 2 (October 2017): 385–96. http://dx.doi.org/10.1016/j.ydbio.2017.03.010.

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Liang, Zhifeng, Yuncong Ma, Glenn D. R. Watson, and Nanyin Zhang. "Simultaneous GCaMP6-based fiber photometry and fMRI in rats." Journal of Neuroscience Methods 289 (September 2017): 31–38. http://dx.doi.org/10.1016/j.jneumeth.2017.07.002.

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Hartung, J., and M. Gold. "(210) Considerations when Using GCaMP6 in Trigeminal Nociceptive Afferents." Journal of Pain 20, no. 4 (April 2019): S27—S28. http://dx.doi.org/10.1016/j.jpain.2019.01.131.

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Steinmetz, Nicholas A., Christina Buetfering, Jerome Lecoq, Christian R. Lee, Andrew J. Peters, Elina A. K. Jacobs, Philip Coen, et al. "Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines." eneuro 4, no. 5 (September 2017): ENEURO.0207–17.2017. http://dx.doi.org/10.1523/eneuro.0207-17.2017.

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Dana, Hod, Tsai-Wen Chen, Amy Hu, Brenda C. Shields, Caiying Guo, Loren L. Looger, Douglas S. Kim, and Karel Svoboda. "Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo." PLoS ONE 9, no. 9 (September 24, 2014): e108697. http://dx.doi.org/10.1371/journal.pone.0108697.

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Fletcher, Max L., Arjun V. Masurkar, Junling Xing, Fumiaki Imamura, Wenhui Xiong, Shin Nagayama, Hiroki Mutoh, Charles A. Greer, Thomas Knöpfel, and Wei R. Chen. "Optical Imaging of Postsynaptic Odor Representation in the Glomerular Layer of the Mouse Olfactory Bulb." Journal of Neurophysiology 102, no. 2 (August 2009): 817–30. http://dx.doi.org/10.1152/jn.00020.2009.

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Abstract:

Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca2+-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca2+ indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.

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