Cell Ca2 Research Articles

Cholinergic Stimulation Modulates the Functional Composition of CA3 Cell Types in the Hippocampus.

The functional heterogeneity of hippocampal CA3 pyramidal neurons has emerged as a key aspect of circuit function. Here, we explored the effects of long-term cholinergic activity on the functional heterogeneity of CA3 pyramidal neurons in organotypic slices obtained from male rat brains. Application of agonists to either AChRs generally, or mAChRs specifically, induced robust increases in network activity in the low-gamma range. Prolonged AChR stimulation for 48 h uncovered a population of hyperadapting CA3 pyramidal neurons that typically fired a single, early action potential in response to current injection. Although these neurons were present in control networks, their proportions were dramatically increased following long-term cholinergic activity. Characterized by the presence of a strong M-current, the hyperadaptation phenotype was abolished by acute application of either M-channel antagonists or the reapplication of AChR agonists. We conclude that long-term mAChR activation modulates the intrinsic excitability of a subset of CA3 pyramidal cells, uncovering a highly plastic cohort of neurons that are sensitive to chronic ACh modulation. Our findings provide evidence for the activity-dependent plasticity of functional heterogeneity in the hippocampus.SIGNIFICANCE STATEMENT The large heterogeneity of neuron types in the brain, each with its own specific functional properties, provides the rich cellular tapestry needed to account for the vast diversity of behaviors. By studying the functional properties of neurons in the hippocampus, a region of the brain involved in learning and memory, we find that exposure to the neuromodulator acetylcholine can alter the relative number of functionally defined neuron types. Our findings suggest that the heterogeneity of neurons in the brain is not a static feature but can be modified by the ongoing activity of the circuits to which they belong.

Associations between hippocampal subfield volume and cognitive performance

AbstractBackgroundIt is well demonstrated that hippocampal atrophy is associated with cognitive impairment. Existing studies have associated smaller hippocampal volumes with dementia, and hippocampal volume reduction has been established as a biomarker for the conversion of mild cognitive impairment to Alzheimer’s Disease. Although some studies have examined correlations between hippocampal subfield volumes and cognitive domains such as memory, further research is needed to clarify the associations between the volume of each subfield and the various cognitive domains. So far a few studies have been conducted among Asian populations. A more specific understanding of these relationships is crucial given the functional and histological differences in the subfields and their distinct patterns of atrophy, which imply that the hippocampal subfields may affect cognition differentially. This study aimed to investigate the associations between the volumes of 12 hippocampal subfields on cognitive performance in a Singaporean sample.Method448 patients at a memory clinic in Singapore (97 with no cognitive impairment, 177 with CIND, and 174 with dementia) underwent magnetic resonance imaging (MRI) and a neuropsychological assessment. MRI was processed through free surfer (v.6.1) to extract volumes of 12 hippocampal subfields—the tail, subiculum, presubiculum, parasubiculum, CA1, CA3, CA4, fimbria, fissure, molecular layer, the molecular and granule cell layers of the of the dentate gyrus (GCMLDG), and the hippocampus‐amygdala transition area (HATA). Cognition was assessed using NINDS–Canadian Stroke Network harmonization neuropsychological battery.ResultGlobal cognition was associated with the subiculum, presubiculum, CA1, CA3, CA4, molecular layer, and GCMLDG (all p‐values < .0005). Except for the fissure, all hippocampal subfields were associated with visuospatial skills (all p‐values < .05) and memory (all p‐values < .035). The fimbria was associated with the most cognitive domains, including executive dysfunction (p = .011), language (p = .006), visuomotor function (p = .003), visuospatial skills (p < .001), and memory (p < .001).ConclusionThe current study builds on existing research on the associations between hippocampal subfield volumes and cognitive dysfunction. Longitudinal studies are required to investigate the volumetric reduction of hippocampal subfields as a biomarker of cognitive decline.

Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca2+ Influx in Lung Microvascular Endothelial Cells.

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca2+ ([Ca2+]i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca2+-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca2+]i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca2+ imaging. We found that histamine increased [Ca2+]i was maintained at a static elevated level after a transient peak. The elevated Ca2+ plateau was vanished when extracellular Ca2+ was removed, indicating Ca2+ influx from extracellular mediated the histamine-induced Ca2+ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca2+ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca2+ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

Endogenous Amyloid-formed Ca2+-permeable Channels in Aged 3xTg AD Mice.

Alzheimer's disease (AD), the leading cause of dementia, is characterized by the accumulation of beta-amyloid peptides (Aβ). However, whether Aβ itself is a key toxic agent in AD pathogenesis and the precise mechanism of Aβ-elicited neurotoxicity are still debated. Emerging evidence demonstrates that the Aβ channel/pore hypothesis could explain Aβ toxicity, because Aβ oligomers are able to disrupt membranes and cause edge-conductivity pores that may disrupt cell Ca2+ homeostasis and drive neurotoxicity in AD. However, all available data to support this hypothesis have been collected from "in vitro" experiments using high concentrations of exogenous Aβ. It is still unknown whether Aβ channels can be formed by endogenous Aβ in AD animal models. Here, we report an unexpected finding of the spontaneous Ca2+ oscillations in aged 3xTg AD mice but not in age-matched wild-type mice. These spontaneous Ca2+ oscillations are sensitive to extracellular Ca2+, ZnCl2, and the Aβ channel blocker Anle138b, suggesting that these spontaneous Ca2+ oscillations in aged 3xTg AD mice are mediated by endogenous Aβ-formed channels.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

Estrogen receptor subtype mediated anti-inflammation and vasorelaxation via genomic and nongenomic actions in septic mice.

Sepsis is a life-threatening disease with high mortality worldwide. Septic females have lower severity and mortality than the males, suggesting estrogen exerts a protective action, but nothing is known about the role of vascular endothelial estrogen receptor subtypes in this process. In the present study, we aimed to study the estrogen receptors on mesenteric arterioles in normal and sepsis mice and to elucidate the underlying mechanisms. Sepsis was induced in mice by intraperitoneal injection of LPS. The changes in the expression and release of the serum and cell supernatant proinflammatory cytokines, including TNF-α, IL-1β and IL-6, were measured by qPCR and ELISA, and the functions of multiple organs were analyzed. The functional activities of mouse mesenteric arterioles were determined by a Mulvany-style wire myograph. The expression of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in endothelial cells were examined by Western blot and their functions were characterized by cell Ca2+ imaging. Septic female mice had higher survival rate than the male mice, and pretreatment with E2 for 5 days significantly improved the survival rate and inhibited proinflammatory cytokines in septic male mice. E2 ameliorated pulmonary, intestinal, hepatic and renal multiple organ injuries in septic male mice; and ER subtypes inhibited proinflammatory cytokines in endothelial cells via PLC/IP3R/Ca2+ pathway. E2/ER subtypes immediately induced endothelial-derived hyperpolarization (EDH)-mediated vasorelaxation via PLC/IP3R/Ca2+ pathway, which was more impaired in septic male mice. E2/ER subtypes could rescue the impaired acetylcholine (ACh)-induced EDH-mediated vasorelaxation in septic male mice. E2 through ER subtypes mediates anti-inflammation and vasorelaxation via genomic and nongenomic actions in sepsis. Mechanistically, activation of endothelial ER subtypes reduces proinflammatory cytokines and induces EDH-mediated vasorelaxation via PLC/IP3R/Ca2+ pathway, leading to amelioration of sepsis-induced organ injury and survival rate.

Neuroprotective effects of photobiomodulation by hormesis on scopolamine induced neurodegenerative diseases of memory disorders in rats a paradigm shift.

The loss and progressive dysfunction of neurons are hallmarks of neurodegenerative diseases. The aim of the current study is to explore the effects of photobiomodulation at 460-660 nm (100-1000 lux units) on the progression of scopolamine-induced cognitive dysfunctions in Wistar male rats. Photobiomodulation (PBM) is defined as "the use of monochromatic or quasi-monochromatic light from a low-power laser or light-emitting diode (LED) source to modify or modulate biological functions." Neuroprotective activity was assessed by in vivo models such as the Morris water maze, the elevated plus maze (EPM), and the T-maze. After using scopolamine (1 mg/kg/day) as a dementia induction model for 21 days, the induction was primarily due to impairments in cholinergic transmission, oxidative stress, and inflammation. The in vitro determinations, including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), Interleukin 1 beta (IL-1β), and alkaline phosphatase (ALP), were assessed biochemicals and biomarkers. The structural and morphological integrity of the cortex and hippocampus was investigated through histopathology. In vivo studies of exteroceptive behavior models such as the Morris water maze, the EPM, and the T-maze revealed that administration of scopolamine resulted in enhanced escape latency time (ELT), transfer latency (TL), and decreased percentage alternation, respectively. The levels of AChE, BChE, reduced, GSH, SOD, TNF-α, IL-1β and ALP were increased, while MDA level was decreased. In contrast to normal and control groups with treatment groups, histopathology of the cortex and hippocampus examination revealed the maintenance of structural integrity and densities of CA1 and CA3 neuronal cells. However, network pharmacology predicted Ca+2 modulation of various pathways, among the treatments with red LED light showed highly significant amelioration compared with normal and control groups. Photobiomodulation by hormesis, chromophores in cells, and tissues excitation can influence neuroprotective effect mainly by scavenging of ROS, variation in the level of GSH MDA and SOD mitochondrial electron transfer, the improved abscopal effects on improved in gut microbiome by resembles the of fecal ALP level correlation of intestinal microbiome, cholinergic neurotransmissions, anti-inflammatory, and antioxidant activities.

Probiotic Treatment Ameliorates Exercise Intolerance, Deficits in Fear Learning and Systemic and Neuro-inflammation in a Mouse Model of Gulf War Illness

Veterans of the Persian Gulf War (GW) continue to suffer from Gulf War Illness (GWI) characterized by cognitive deficit and fatigue. Our group and others have suggested possible involvement of the microbiota-gut-brain axis in GWI pathology (Kozlova et a., 2021a; 2021b). The current study tested the hypothesis that probiotic treatment (P) prevents GWI symptoms, and systemic and neuro-inflammation. Adult male C57Bl/6N mice were separated into 4 groups (n=16/group): GW group was exposed to 8.7 mg/kg/d pyridostigmine bromide (PB) in saline (150uL/30g bw, oral gavage), 1.3 mg/kg PER in 100% DMSO (dermal), and 33% DEET in 70% EtOH (dermal) for 28 days (5 days/week). CON/S group was sham-treated. CON/S+P and GW+P groups were treated with a probiotic (P) cocktail of L. reuteri, L. rhamnosus, L. casei, B. longum (10 8 CFU/mL, oral gavage) 3 times/week for 2 weeks prior to and during sham/GW agent treatment and until sacrifice (post-treatment (PT) 150). All groups were subjected to restraint stress (5 min/d for 28 d). RT-qPCR on fecal pellets showed bacteria colonization of all strains except L. rhamnosus and B. longum after administration of 6-7 doses. At PT1, all strains showed increased colonization relative to baseline, which persisted to PT150, except B. longum. Body weight, fat, lean mass at PT150 showed no changes among groups. An exercise endurance (EE) test was performed to measure differences in fatigue. GW mice tired faster relative to CON/S at PT56 which persisted at PT150. Probiotic normalized exercise endurance to CON/S levels at PT56. On the passive avoidance test all groups behaved normally at PT50 but GW showed deficits on day 1 acquisition trial of fear learning when tested again 14 weeks later at PT150. GW+P behaved similarly to CON/S. Strength and depression-like behavior was examined before and after exercise stress to simulate post-exertional malaise. Mean percent time spent mobile on tail suspension test (TST) were significantly lower for GW but not GW+P at PT50. All groups were normal at PT150. There was no effect of GW agents or probiotics on the hanging wire test. Systemic and neuroinflammatory markers: C-reactive protein (CRP), high mobility group box 1 (HMGB1), and interleukin-6 (IL-6) were examined using ELISA. Liver and plasma CRP was increased in GW group but not GW+P in liver (p=0.06 in plasma). Plasma HMGB1 was reduced and brain IL-6 was elevated in GW mice but not GW+P. Immunofluorescence analysis in fixed hippocampal sections showed more Iba-1 positive cells in CA1 and DG in GW vs CON/S, while GW+P showed no significant changes vs CON/S. These results show that GW mice can be used to model long-lasting fatigue and other GWI symptoms as well as systemic and neuro-inflammation concurrently. After exercise stress GW agents produced depression. Importantly, probiotic treatment improved exercise endurance, depression, systemic and neuro-inflammation in GW mice, indicating its potential therapeutic use in GWI. Clara Berdasco and Elena Kozlova are co-first authors. Supported by DoD #W81XWH-19-1-0802 (MCC). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Two distinct calcium signaling behaviors are present in interstitial cells of Cajal of the gastroesophageal junction

Background/Objectives: The lower esophageal sphincter (LES) is located within the gastroesophageal junction (GEJ) and generates tone to prevent gastric reflux into the esophagus. Previously, we demonstrated that the generation and maintenance of tone in the LES clasp muscle involves intramuscular interstitial cells of Cajal (ICC-IM) (Drumm et al., 2022; PMID: 35229888). The aim of the current study was to further characterize Ca2+ signaling in ICC-IM within the GEJ. We hypothesize that Ca2+ release from the endoplasmic reticulum (ER) activates Ca2+-activated Cl- channels (ANO1) in ICC leading to depolarization that is conducted to smooth muscle cells (SMCs) via gap junctions; this in turn leads to Ca2+ influx via L-type Ca2+ channels (CavL) and the development of tone. Methods: Confocal spinning disc microscopy was used to image Ca2+ transients in the LES and distal esophagus of mice expressing GCaMP6f in ICC (Kit-GCaMP6f). Muscles of the GEJ imaged with the submucosal surface upwards at 20x. Ani9 (3mM), nifedipine (1mM) and CPA (10mM) were used to inhibit ANO1, CavL and SERCA, respectively. Pinacidil (10mM) was used to activate KATP channels. Results: ICC-IM within the GEJ displayed two types of Ca2+ signaling behaviors. Ca2+ transients in ICC-IM of the LES clasp muscle were stochastic, arising from multiple firing sites within the same cell. These Ca2+ transients were not synchronized within the same cell or between adjacent cells. In contrast, two Ca2+ signaling patterns were observed in ICC-IM of the distal esophagus where smooth muscle transitions to skeletal muscle. One ICC-IM population (Type I) shared the Ca2+ signaling behaviors of ICC-IM in the LES clasp. Another ICC population (Type II) exhibited rhythmic whole-cell Ca2+ transients that were synchronized between adjacent Type II ICC. Whole cell Ca2+ transients in Type II ICC were abolished by Ani9, nifedipine or pinacidil, revealing underlying Ca2+ transients similar to those observed in Type I ICC. In contrast, Ca2+ transients in Type I ICC were resistant to Ani9, nifedipine and pinacidil. Ca2+ transients were abolished in both types of ICC by removal of extracellular Ca2+, however the activity in Type I ICC persisted longer than that of Type II ICC. Discussion/Conclusions: This study demonstrates two distinct Ca2+ signaling behaviors in ICC-IM of the GEJ with differing pharmacology. This is similar to what has been previously described within the internal anal sphincter (IAS) (Hannigan et al., 2020; PMID: 32587396). In the IAS, it has been suggested that Type II ICC-IM generate slow waves however their role in the GEJ remains unclear as slow waves have not been described within this region of the mouse. Thus, the functional role of ICC-IM in the GEJ, particularly Type II ICC, warrants further investigation. NIH DK129528 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pressure induced changes in endothelial cell morphology and Ca2+ signaling in ex vivo pressurized resistance arteries revealed using 4D diSPIM myography and AI deep-learning

The vascular endothelium is critical in promoting vasodilation of resistance-size arteries through Ca 2+ dependent pathways. Vascular endothelial cells (ECs) are exposed to mechanical stimulation through changing intravascular pressure and flow, yet it is unclear how these forces impact EC morphology and Ca 2+ signaling in pressurized arteries. Visualizing how the endothelium of an intact resistance-size vessel responds to changing physical forces requires isometric, sub-micron, resolution. Conventional confocal fluorescent microscope systems are limited by poor axial resolution. 4D dual inverted selective plane of illumination microscopy (diSPIM; 3i) uniquely provides isometric resolution at ~330 nm with high temporal resolution and minimal photobleaching or phototoxicity. We designed custom pressure myography chambers to allow simultaneous 4D diSPIM fluorescent imaging and vessel diameter tracking measurements via transmitted IR light (diSPIM Myography). We captured fluorescent images of ex vivo pressurized resistance-size mouse mesenteric arteries at 80, 60, 40, and 20 mm Hg before and after the generation of myogenic tone and in the absence of extracellular Ca 2+ . Representative images were annotated for ECs, nuclei, myoendothelial projections (MEPS), and Ca 2+ signals. Annotations were used as ground truth for CNN AI model generation and validation using Apeer (Zeiss). Arivis Vision4D (Zeiss) was used to run the AI model and quantify data. As static intravascular pressure is increased in arteries without tone (with or without Ca 2+ ), ECs increase in width as the vessel dilates. Myogenic tone minimizes pressure dilation and changes in EC width, however, ECs compress by up to 1 micron as pressure increases from 20 to 80 mm Hg. Additionally, increasing intravascular pressure in arteries with tone decreases local EC Ca 2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca 2+ signaling in intact resistance arteries under conditions of physiologic temperature, pressure, pH, and tone. diSPIM myography provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function. AI image annotation reduces analysis bias and increases the breadth of quantifiable microscopy data. DMC is supported by NIH R00HL133451 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Perturbation of the host cell Ca2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the β-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.

Spike-based coupling between single neurons and populations across rat sensory cortices, perirhinal cortex, and hippocampus.

Cortical computations require coordination of neuronal activity within and across multiple areas. We characterized spiking relationships within and between areas by quantifying coupling of single neurons to population firing patterns. Single-neuron population coupling (SNPC) was investigated using ensemble recordings from hippocampal CA1 region and somatosensory, visual, and perirhinal cortices. Within-area coupling was heterogeneous across structures, with area CA1 showing higher levels than neocortical regions. In contrast to known anatomical connectivity, between-area coupling showed strong firing coherence of sensory neocortices with CA1, but less with perirhinal cortex. Cells in sensory neocortices and CA1 showed positive correlations between within- and between-area coupling; these were weaker for perirhinal cortex. All four areas harbored broadcasting cells, connecting to multiple external areas, which was uncorrelated to within-area coupling strength. When examining correlations between SNPC and spatial coding, we found that, if such correlations were significant, they were negative. This result was consistent with an overall preservation of SNPC across different brain states, suggesting a strong dependence on intrinsic network connectivity. Overall, SNPC offers an important window on cell-to-population synchronization in multi-area networks. Instead of pointing to specific information-coding functions, our results indicate a primary function of SNPC in dynamically organizing communication in systems composed of multiple, interconnected areas.

The temporal and contextual stability of activity levels in hippocampal CA1 cells

Recent long-term optical imaging studies have demonstrated that the activity levels of hippocampal neurons in a familiar environment change on a daily to weekly basis. However, it is unclear whether there is any time-invariant property in the cells' neural representations. In this study, using miniature fluorescence microscopy, we measured the neural activity of the mouse hippocampus in four different environments every 3d. Although the activity level of hippocampal neurons fluctuated greatly in each environment across days, we found a significant correlation between the activity levels for different days, and the correlation was higher for averaged activity levels across multiple environments. When the number of environments used for averaging was increased, a higher activity correlation was observed. Furthermore, the number of environments in which a cell showed activity was preserved. Cells that showed place cell activity in many environments had greater spatial information content and more stable spatial representation, and thus carried more abundant and stable information about the current position. In contrast, cells that were active only in a small number of environments provided sparse representation for the environment. These results suggest that each cell has not only an inherent activity level but also play a characteristic role in the coding of space.

Synthetic Data Resource and Benchmarks for Time Cell Analysis and Detection Algorithms.

Hippocampal CA1 cells take part in reliable, time-locked activity sequences in tasks that involve an association between temporally separated stimuli, in a manner that tiles the interval between the stimuli. Such cells have been termed time cells. Here, we adopt a first-principles approach to comparing diverse analysis and detection algorithms for identifying time cells. We generated synthetic activity datasets using calcium signals recorded in vivo from the mouse hippocampus using two-photon (2-P) imaging, as template response waveforms. We assigned known, ground truth values to perturbations applied to perfect activity signals, including noise, calcium event width, timing imprecision, hit trial ratio and background (untuned) activity. We tested a range of published and new algorithms and their variants on this dataset. We find that most algorithms correctly classify over 80% of cells, but have different balances between true and false positives, and different sensitivity to the five categories of perturbation. Reassuringly, most methods are reasonably robust to perturbations, including background activity, and show good concordance in classification of time cells. The same algorithms were also used to analyze and identify time cells in experimental physiology datasets recorded in vivo and most show good concordance.

Drought priming mechanisms in wheat elucidated by in-situ determination of dynamic stomatal behavior.

Stomata play a critical role in balancing photosynthesis and transpiration, which are essential processes for plant growth, especially in response to abiotic stress. Drought priming has been shown to improve drought tolerance. Lots of studies have been done with the response of stomatal behavior to drought stress. However, how the stomatal dynamic movement in intact wheat plants response to drought priming process is not known. Here, a portable microscope was used to take microphotographs in order to in-stiu determination of stomatal behavior. Non-invasive micro-test technology was used for measurements of guard cell K+, H+ and Ca2+ fluxes. Surprisingly, the results found that primed plants close stomatal much faster under drought stress, and reopening the stomatal much quicker under recovery, in relation to non-primed plants. Compared with non-primed plants, primed plants showed higher accumulation of ABA and Ca2+ influx rate in guard cells under drought stress. Furthermore, genes encoding anion channels were higher expressed and K+ outward channels activated, leading to enhanced K+ efflux, resulting in faster stomatal closure in primed plants than non-primed plants. During recovery, both guard cell ABA and Ca2+ influx of primed plants were found to be significantly reducing K+ efflux and accelerating stomatal reopening. Collectively, a portable non-invasive stomatal observation of wheat found that priming promoted faster stomatal closure under drought stress and faster reopening during post-drought recovery in relation to non-primed plants, thereby enhancing overall drought tolerance.

Constraints of vigilance-dependent noradrenergic signaling to mouse cerebellar Bergmann glia.

Behavioral state plays an important role in determining astroglia Ca2+ signaling. In particular, locomotion-mediated elevated vigilance has been found to trigger norepinephrine-dependent whole cell Ca2+ elevations in astroglia throughout the brain. For cerebellar Bergmann glia it has recently been found that locomotion-induced transient Ca2+ elevations depend on their α1A -adrenergic receptors. With increasing availability and implementation of locomotion as behavioral parameter it becomes important to understand the constraints of noradrenergic signaling to astroglia. Here we evaluated the effect of speed, duration and interval of locomotion on Ca2+ signals in Bergmann glia as well as cerebellar noradrenergic axon terminals. We found almost no dependence on locomotion speed, but following the initial Ca2+ transient prolonged locomotion events revealed a steady-state Ca2+ elevation. Comparison of time course and recovery of transient Bergmann glia and noradrenergic terminal Ca2+ dynamics suggested that noradrenergic terminal Ca2+ activity determines Bergmann glia Ca2+ activation and does not require noradrenergic receptor desensitization to account for attenuation during prolonged locomotion. Further, analyzing the correlation among Ca2+ dynamics within regions within the field of observation we found that coordinated activity among noradrenergic terminals accounts for fluctuations of steady-state Bergmann glia Ca2+ activity. Together, our findings will help to better understand astroglia Ca2+ dynamics during less controlled awake behavior and may guide the identification of behavioral contexts preferably dependent on astroglia Ca2+ signaling.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signalingnor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

Heterogeneity of network and coding states in mouse CA1 place cells.

Theta sequences and phase precession shape hippocampal activity and are considered key underpinnings of memory formation. Theta sequences are sweeps of spikes from multiple cells, tracing trajectories from past to future. Phase precession is the correlation between theta firing phase and animal position. Here, we reconsider these temporal processes in CA1 and the computational principles that they are thought to obey. We find stronger heterogeneity than previously described: we identify cells that do not phase precess but reliably express theta sequences. Other cells phase precess only when medium gamma (linked to entorhinal inputs) is strongest. The same cells express more sequences, but not precession, when slow gamma (linked to CA3 inputs) dominates. Moreover, sequences occur independently in distinct cell groups. Our results challenge the view that phase precession is the mechanism underlying the emergence of theta sequences, suggesting a role for CA1 cells in multiplexing diverse computational processes.

Genetic knockout of prestin limits cardiac hypertrophy in a murine pressure overload model.

Up until recently, Prestin (Slc26a5) was considered to be exclusively expressed in the outer hair cells of the inner ear, where it serves as a voltage-to-force convertor and mediates electromotility. Our recent studies demonstrate that prestin is expressed in the heart, where it amplifies cardiac motor function. Due to prestin's ability to modulate cardiac contractility, we hypothesize that it plays a pivotal role in pathological cardiac hypertrophy. We subjected wild-type (WT) and Slc26a5-/- mice to sham operation or chronic pressure overload induced by transverse aortic constriction (TAC) surgery. Cardiac structure and function were assessed by echocardiography. We performed single cell Ca2+ transients and cell shortening recordings on isolated cardiomyocytes. Atomic force microscopy, a high-resolution technique, was also used to quantify the Young's modulus. After 8 weeks of TAC, the expression of prestin was markedly elevated in WT TAC mice relative to WT sham mice. To test whether the increased expression is maladaptive or cardioprotective, Slc26a5-/- mice underwent the same surgeries. Slc26a5-/- sham mice had depressed cardiac function relative to WT sham mice. In contrast and unexpectedly, 4 weeks after TAC, Slc26a5-/- mice exhibited blunted cardiac structural remodeling and more robust cardiac function as compared to WT TAC mice. Moreover, we found that cardiomyocytes from Slc26a5-/- mice showed more normalized Ca2+ transients and cellular shortening relative to cardiomyocytes from WT TAC mice. Our findings demonstrate that knockout of Slc26a5 is cardioprotective in a murine model of pressure overload, highlighting its crucial role in both physiological and pathological conditions. Overexpression of prestin in cardiac hypertrophy may significantly alter the mechanical properties of cardiomyocytes, leading to worsening in hypertrophy-induced cardiac dysfunction. Our study suggests that targeting prestin may represent a viable therapeutic avenue for cardiac hypertrophy.

Influence of Different Sevoflurane Concentrations on Postoperative Cognitive Function in Aged Rats.

Postoperative cognitive dysfunction may be associated with neuroinflammation, and sevoflurane suppresses surgery-induced inflammation. We hypothesized that low concentrations of sevoflurane would result in more impaired postoperative cognitive function compared to high concentrations. Aged male Sprague-Dawley rats (n = 21, 17-22 months) were randomly assigned to 1 of 3 groups: control (C), sevoflurane 2% (S2), and sevoflurane 4% (S4). Rats in the S2 and S4 groups underwent open femoral fracture and intramedullary fixation of the left hind limb under 2 hours of sevoflurane anesthesia. Neurological outcomes were evaluated using the Morris water maze (MWM) test, and histopathological outcomes were assessed 28 days after surgery. The S2 group showed prolonged swimming latency compared to S4 on day 7 (difference of means, 34.4; 95% confidence interval [CI], 2.57-66.3; P = .031) and compared to the C group on day 9 (difference of means, -33.4; 95% CI, -65.3 to -1.55; P = .037). The intact CA1 cells in the S2 group were significantly less than those in the C and S4 groups (H statistic, 10.87; P = .006 versus C; P = .033 versus S4). We found that low concentrations of sevoflurane prolonged the swimming latency of the MWM compared to high concentrations and reduced intact CA1 hippocampal neurons in aged rats. These results suggest that low-concentration sevoflurane anesthesia may be more detrimental than high concentration for spatial cognitive function and postoperative impairment of hippocampal CA1 cells in aged rats.