Search Results (63)

Genetically encoded voltage indicators (GEVIs) have long promised optical access to membrane potential, yet their adoption has lagged significantly behind genetically encoded calcium indicators. A central but underappreciated reason is that the metrics used to evaluate and compare GEVIs—fractional fluorescence change (ΔF/F), kinetics, and signal-to-noise ratio—rest on an assumption that is frequently violated: that GEVI fluorescence reflects a single underlying process. In this perspective, we argue that GEVI signals are composite optical measurements, arising from the superposition of voltage-dependent fluorescence, intracellular and nonresponsive signal, background, and contributions from neighboring cells. Under these conditions, ΔF/F is not a measure of sensor sensitivity but a contrast metric whose value depends on baseline fluorescence composition, optical sampling, and imaging configuration. This reinterpretation has two key consequences. First, it explains a substantial source of variability in GEVI performance that is currently attributed to noise or experimental inconsistency. Second, and more importantly, it reveals that the complexity of GEVI signals is not a limitation to be minimized but a resource to be exploited. By resolving composite signal components, GEVIs can report multiplexed physiological variables, expose hidden conformational states of voltage-sensing domains, probe membrane organization, and reveal intracellular and intercellular electrical coupling. We propose that realizing the full potential of GEVIs requires treating ΔF/F not as a gold standard for sensor performance, but as one interpretable component of a richer optical measurement whose structure encodes multiple layers of cellular physiology. Full article

Macrophages are among the earliest responders to tissue injury and remain associated with the wound throughout the healing process. Calcium (Ca²⁺) signaling regulates many immune cell behaviors, yet its role in macrophage responses to injury in vivo remains poorly defined. Here, we used transgenic zebrafish (Danio rerio) and Danionella cerebrum lines that specifically express the genetically encoded Ca²⁺ indicator, GCaMP, in macrophages. Live confocal imaging was used to monitor macrophage Ca²⁺ dynamics during the early wound response. We found that injury triggers macrophage recruitment to the wound site, where cells exhibit robust and repetitive intracellular Ca²⁺ transients that persist for several hours. Pharmacological perturbation revealed that endoplasmic reticulum Ca²⁺ stores contribute to sustaining these transients, while additional Ca²⁺ sources likely participate in macrophage Ca²⁺ signaling in vivo. Functionally, these Ca²⁺ transients do not appear to be required for chemotaxis, phagocytosis, or TNFα activation during the early stages of wound healing. Together, these findings uncover a previously uncharacterized macrophage Ca²⁺ signaling behavior and highlight the complexity of Ca²⁺ regulation during tissue injury responses in vivo. Full article

KCa3.1 encodes the intermediate-conductance calcium-activated potassium channel KCa3.1, a regulator of membrane potential and calcium-dependent signalling in cardiovascular and immune cells. Increasing evidence indicates that KCa3.1 is a shared driver of vascular remodelling, inflammation, fibrosis, and electrical instability across multiple cardiovascular diseases. In ischaemic heart disease (IHD), KCa3.1 is upregulated in endothelial cells, vascular smooth muscle cells, macrophages, and T lymphocytes, where it promotes smooth muscle proliferation, neointimal formation, and chronic vascular inflammation. Genetic deletion or pharmacological blockade of KCa3.1 reduces atherosclerotic plaque burden and restenosis in animal models. In atrial fibrillation (AF), KCa3.1 contributes to electrical remodelling by shortening atrial action potential duration and to structural remodelling by driving fibroblast activation and collagen deposition. KCa3.1 also regulates macrophage polarisation and pro-inflammatory cytokine release in atrial tissue, linking immune activation to arrhythmogenic substrate formation. Inhibition of KCa3.1 prolongs atrial refractoriness, attenuates atrial fibrosis, and reduces AF inducibility in multiple preclinical models. Emerging data in valvular heart disease suggest that KCa3.1 is upregulated in valvular interstitial cells and regions of active calcification, where it supports myofibroblast differentiation, osteogenic signalling, and inflammatory crosstalk, implicating the channel in fibrocalcific valve degeneration. Collectively, these findings position KCa3.1 as a central molecular integrator of electrical, fibrotic, and inflammatory pathways in cardiovascular disease. The availability of selective KCa3.1 inhibitors with established human safety profiles supports the feasibility of therapeutic translation. Targeting KCa3.1 may enable disease-modifying strategies that extend beyond symptom control to suppress maladaptive cardiovascular remodelling. Full article

This review addresses the challenges of obtaining high-quality quantitative data in the optical imaging of membrane voltage and calcium dynamics. The paper provides a comprehensive overview and systematization of recent studies that analyze factors limiting signal fidelity and propose strategies to enhance data quality. The primary sources of signal degradation in biological optical imaging, with an emphasis on membrane voltage and calcium imaging, are systematically explored across four major indicator classes: voltage-sensitive dyes (VSDs), genetically encoded voltage indicators (GEVIs), calcium-sensitive dyes (CSDs), and genetically encoded calcium indicators (GECIs). Common mechanisms that compromise data quality are classified into three main categories: fundamental photon shot noise, device-related errors, and sample-related measurement errors. For each class of limitation, its physical or biological origin and characteristic manifestations are described, which are followed by an analysis of available mitigation strategies, including hardware optimization, choice of sensors, sample preparation and experimental design, post-processing and computational correction methods. Full article

Astrocytic calcium signaling is a central mechanism of neuron-glia communication that operates across multiple spatial and temporal scales. Traditionally, research has focused on intracellular Ca²⁺ oscillations that regulate gliotransmitter release, ion homeostasis, and metabolic support. Recent evidence, however, reveals that extracellular calcium ([Ca²⁺]o) is not a passive reservoir but a dynamic signaling mediator capable of influencing neuronal excitability within milliseconds. Through mechanisms such as calcium-sensing receptor (CaSR) activation, ion channel modulation, surface charge effects, and ephaptic coupling, astrocytes emerge as active partners in both slow and rapid modes of communication. This dual perspective reshapes our understanding of brain physiology and disease. Disrupted Ca²⁺ signaling contributes to network instability in epilepsy, synaptic dysfunction in Alzheimer’s and Parkinson’s disease, and impaired maturation in neurodevelopmental disorders. Methodological advances, including Ca²⁺-selective microelectrodes, genetically encoded extracellular indicators, and computational modeling, are beginning to uncover the richness of extracellular Ca²⁺ dynamics, though challenges remain in achieving sufficient spatial and temporal resolution. By integrating classical intracellular pathways with emerging insights into extracellular signaling, this review highlights astrocytes as central architects of the ionic landscape. Recognizing calcium as both an intracellular messenger and an extracellular signaling mediator provides a unifying framework for neuron–glia interactions and opens new avenues for therapeutic intervention. Full article

Fusarium oxysporum-induced root rot severely threatens global soybean production, yet limited understanding of resistance mechanisms constrains breeding progress. This study conducted comparative transcriptomic analysis between highly resistant (Xiaoheiqi) and susceptible (L83-4752) soybean accessions following pathogen inoculation across four time points (8–17 days post-infection). RNA-seq analysis identified 1496 differentially expressed genes following pathogen challenge. KEGG pathway enrichment analysis revealed significant enrichment in MAPK signaling pathway (12 genes) and plant–pathogen interaction pathway (13 genes). Eight genes co-occurred in both pathways, with GmCML (Glyma.10G178400) exhibiting the most dramatic differential expression among these candidates. This gene encodes a 151-amino acid calmodulin-like protein showing 185-fold higher expression in resistant plants at 17 days post-inoculation, confirmed by qRT-PCR validation. Functional validation through transgenic hairy root overexpression demonstrated that GmCML significantly enhanced disease resistance by coordinately activating antioxidant defense systems. Overexpression of GmCML in transgenic soybean enhanced resistance to F. oxysporum by modulating the activity of antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT) and the accumulation of osmoregulatory substances (proline and soluble sugars). Population genetic analysis of 295 diverse soybean accessions revealed three GmCML haplotypes based on promoter region polymorphisms. Two favorable variants (Hap2 and Hap3) conferred significantly lower disease indices and exhibited evidence of positive selection during domestication, indicating evolutionary importance in disease resistance. This research provides the first comprehensive characterization of GmCML’s role in soybean–Fusarium interactions, establishing this calmodulin-like protein as a regulatory hub linking calcium signaling to coordinated defense responses. The identified natural variants and functional mechanisms offer validated targets for both marker-assisted breeding and genetic engineering approaches to enhance soybean disease resistance. Full article

Metatropic dysplasia is an autosomal dominant skeletal disorder characterized by progressive kyphoscoliosis, severe platyspondyly, pronounced metaphyseal enlargement, and shortening of the long bones. This condition is caused by pathogenic variants in the TRPV4 (Transient Receptor Potential Vanilloid 4) gene, which encodes a non-selective calcium channel involved in bone homeostasis. Variants in TRPV4 have been associated with two major disease groups: skeletal dysplasias and neuropathies, with recent findings indicating an overlap in their clinical features. We report three patients with metatropic dysplasia, each presenting a distinct severity profile. All exhibited a bell-shaped thorax, significant platyspondyly, and shortened long bones with broad metaphyses. Notably, patients 1 and 3 had more complex clinical courses, including seizures and global developmental delay. Genetic analysis revealed two different TRPV4 variants: p.Asn796del (patient 1) and p.Pro799Leu (patients 2 and 3). These cases illustrate variability in extra-skeletal manifestations, complications, and prognosis. In our patients with TRPV4-related disorders, the co-occurrence of neurological symptoms and skeletal abnormalities suggests a clinically heterogeneous spectrum consistent with a single disease rather than distinct entities. A comprehensive, multidisciplinary approach is essential to optimize management and improve the quality of life for patients. Full article

This study investigated the phylogenetic relationships in the pear calcium-dependent protein kinase (CDPK) pan-gene family and elucidated its role in the resistance to scab disease caused by Venturia nashicola. By integrating data from eight genomic sets from five cultivated pear species, Pyrus bretschneideri, P. ussuriensis, P. sinkiangensis, P pyrifolia, and P. communis, along with P. betulifolia and interspecific hybrids, 63 PyCDPK family members were identified. Among these, P. communis possessed the highest number of CDPK genes, whereas P. bretschneiderilia had the fewest. These genes encode proteins ranging from 459 to 810 amino acids in length, and are predominantly localized to the cell membrane. Six genes, PyCDPK9, PyCDPK11, PyCDPK12, PyCDPK14, PyCDPK16, and PyCDPK19, were classified as core members of the pan-genome, and PyCDPK19 showed evidence of positive selection pressure. Clustering analysis and transcriptomic expression profiling of disease-resistance-related CDPKs identified PyCDPK19 as a key candidate associated with scab resistance. Promoter analysis revealed that the regulatory region of PyCDPK19 contains multiple cis-acting elements involved in defense responses and methyl jasmonate signaling. Transient overexpression of PyCDPK19 in tobacco leaves induced hypersensitive cell necrosis, accompanied by significant increases in hydrogen peroxide (H₂O₂) accumulation and malondialdehyde (MDA) content. Similarly, overexpression in pear fruit callus tissue followed by pathogen inoculation resulted in elevated levels of both H₂O₂ and MDA. Collectively, these findings indicate that PyCDPK19 mediates defense responses through the activation of the reactive oxygen species pathway in both tobacco and pear plants, providing a promising genetic target for enhancing scab resistance in pears. Full article

In breast cancer, progression from localized stage I to distant metastatic stage IV disease is associated with a reduction of 5-year survival from nearly 100% to 23.2%. Expression of the calcium-activated protease isoforms calpain-1 and calpain-2 has been correlated with cell migration and invasion in vitro, metastatic potential in preclinical mouse models of cancer, and breast cancer prognosis in patients. It is unclear which of these two calpain isoforms is responsible for the apparent metastatic potential of cancer cells. Here, we demonstrate that while individual CRISPR-Cas9 knockouts of either CAPN1 or CAPN2 genes (encoding the catalytic subunits of calpain-1 and -2, respectively) reduce in vitro migration and marginally suppress in vivo metastasis, genetic disruption of both calpain-1 and calpain-2 through knockout of the CAPNS1 gene (encoding the common regulatory subunit of calpain-1 and -2) diminishes metastasis by 83.4 ± 13.6% in a mouse xenograft model of human triple-negative breast cancer. The effect of calpain-1/2 deficiency was replicated in vitro with a modified cell-permeable calpastatin (CAST)-based peptide inhibitor (cell migration reduced to 53.5 ± 11.0% of vehicle control). However, this peptide inhibitor was not effective in vivo at reducing metastasis under the conditions used (vehicle vs. CAST, 1.12 ± 1.35 lung metastases per mm² vs. 0.34 ± 0.20 metastases per mm²), likely due to rapid clearance, as indicated by the short serum half-life. This work demonstrates that calpain-1/2 disruption effectively abrogates metastasis and provides rationale for development of effective calpain inhibitors. Full article

Accumulating studies have shown that astrocytes are essential for regulating neurons at both synaptic and circuit levels. The main mechanism of brain astrocytic intracellular Ca²⁺ activity is through the release of Ca²⁺ via the inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) from the endoplasmic reticulum (ER). Studies using IP3R2 knockout mouse models (Itpr2^−/−) have shown that eliminating IP3R2 leads to a significant reduction in astrocytic Ca²⁺ activity However, there is ongoing controversy regarding the effect of this IP3R2-dependent reduction in astrocytic Ca²⁺ transients on neuronal activity. In our study, we employed dual-color two-photon Ca²⁺ imaging to study astrocytes and neurons simultaneously in vibrissa somatosensory cortex (vS1) in awake-behaving wild-type and Itpr2^−/− mice. We systematically characterized and compared both recorded astrocytic and neuronal Ca²⁺ activities in wild-type and Itpr2^−/− mice during various animal behaviors, particularly during the transition period from stillness to locomotion. We report that vS1 astrocytic Ca²⁺ elevation in both wild-type and Itpr2^−/− mice was significantly modulated by free whisking and locomotion. However, vS1 neurons were only significantly modulated by locomotion in wild-type mice, but not in Itpr2^−/− mice. Our study suggests a non-synaptic modulatory mechanism on functions of astrocytic IP3R2-dependent Ca²⁺ transients to local neurons. Full article

The neurobiology of chronic pain is complex and multifaceted, intertwining with the mesocorticolimbic system to regulate the behavioral and perceptional response to adverse stimuli. Specifically, the ventral tegmental area (VTA), the dopaminergic hub of the reward pathways located deep within the midbrain, is crucial for regulating the release of dopamine (DA) throughout the central nervous system (CNS). To better understand the nuances among chronic pain, VTA response, and therapeutics, implementing progressive approaches for mapping and visualizing the deep brain in real time during nociceptive stimulation is crucial. In this study, we utilize a fluorescence imaging platform with a genetically encoded calcium indicator (GCaMP6s) to directly visualize activity in the VTA during acute nociceptive stimulation in both healthy adult mice and adult mice with partial nerve ligation (PNL)-induced neuropathic pain. We also investigate the visualization of the analgesic properties of morphine. Deep brain imaging using our self-fabricated µ-complementary metal–oxide–semiconductor (CMOS) imaging device allows the tracking of the VTA’s response to adverse stimuli. Our findings show that nociceptive stimulation is associated with a reduction in VTA fluorescence activity, supporting the potential of this platform for visualizing pain-related responses in the central nervous system. Additionally, treatment with morphine significantly reduces the neuronal response caused by mechanical stimuli and is observable using the CMOS imaging platform, demonstrating a novel way to potentially assess and treat neuropathic pain. Full article

(1) Background: Darier disease (DD) is a rare autosomal dominant disorder caused by mutations in ATP2A2, a gene that encodes the sarco(endo)plasmic reticulum calcium-ATPase 2 enzyme, which disrupts calcium homeostasis in keratinocytes. Pruritus, a frequently overlooked symptom in DD, can lead to physical and emotional complications, especially in patients with DD who are genetically predisposed to psychiatric comorbidities. (2) Methods: This study aimed to analyze pruritus and other related symptoms in patients with DD and explore their correlation with neuropsychiatric conditions, psychological challenges, disease severity, and body surface area (BSA) involvement through a retrospective review of a tertiary center. (3) Results: Data from 76 patients (equal gender distribution, mean age 44 years) revealed a prevalence of pruritus of 90.8%, surpassing symptoms such as pain (34.3%) and malodor (43.4%). Burning sensations due to DD lesions were significantly correlated with the diagnosis of comorbid neuropsychiatric conditions (p = 0.047) and psychiatric medication use (p = 0.019). While pruritus correlated with disease severity and %BSA involvement, the findings were not statistically significant. Patients reporting pruritus had a significantly higher Dermatology Life Quality Index symptom score (2.4 ± 1.0), which is defined as the presence of itch, soreness, pain, or stinging, than those who did not (1.5 ± 0.6), indicating accurate symptom reporting. (4) Conclusions: In conclusion, a striking majority of patients with DD experience pruritus, with higher prevalence among those with neuropsychiatric challenges, severe Darier disease, and greater %BSA skin involvement. Clinicians should recognize pruritus as a key therapeutic target and adopt comprehensive treatment approaches that both address the neuropsychiatric comorbidities and the added psychological burden of pruritus in patients with DD. Full article

Astrocytes from different brain regions respond with Ca²⁺ elevations to the catecholamine norepinephrine (NE). However, whether this noradrenergic-mediated signaling is present in astrocytes from the ventral tegmental area (VTA), a dopaminergic circuit receiving noradrenergic inputs, has not yet been investigated. To fill in this gap, we applied a pharmacological approach along with two-photon microscopy and an AAV strategy to express a genetically encoded calcium indicator in VTA astrocytes. We found that VTA astrocytes from both female and male young adult mice showed a strong Ca²⁺ response to NE at both soma and processes. Our results revealed that Gq-coupled α1 adrenergic receptors, which elicit the production of IP3, are the main mediators of the astrocyte response. In mice lacking the IP3 receptor type-2 (IP3R2^−/− mice), we found that the astrocyte response to NE, even if reduced, is still present. We also found that in IP3R2^−/− astrocytes, the residual Ca²⁺ elevations elicited by NE depend on the release of Ca²⁺ from the endoplasmic reticulum, through IP3Rs different from IP3R2. In conclusion, our results reveal VTA astrocytes as novel targets of the noradrenergic signaling, opening to new interpretations of the cellular and molecular mechanisms that mediate the NE effects in the VTA. Full article

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium’s function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells. Specifically, genetically encoded calcium indicators (GECIs) are the most in-demand molecular tools in their class. In this work, we address two issues of calcium imaging by designing indicators based on the successful GCaMP6 backbone and the fluorescent protein BrUSLEE. The first indicator variant (GCaMP6s-BrUS), with a reduced, calcium-insensitive fluorescence lifetime, has potential in monitoring calcium dynamics with a high temporal resolution in combination with advanced microscopy techniques, such as light beads microscopy, where the fluorescence lifetime limits acquisition speed. Conversely, the second variant (GCaMP6s-BrUS-145), with a flexible, calcium-sensitive fluorescence lifetime, is relevant for static measurements, particularly for determining absolute calcium concentration values using fluorescence lifetime imaging microscopy (FLIM). To identify the structural determinants of calcium sensitivity in these indicator variants, we determine their spatial structures. A comparative structural analysis allowed the optimization of the GCaMP6s-BrUS construct, resulting in an indicator variant combining calcium-sensitive behavior in the time domain and enhanced molecular brightness. Our data may serve as a starting point for further engineering efforts towards improved GECI variants with fine-tuned fluorescence lifetimes. Full article

The reinforcement of drug-seeking motivation following drug withdrawal is recognized as a significant factor contributing to relapse. The ventral pallidum (VP) plays a crucial role in encoding and translating motivational aspects of reward. However, current research lacks a clear understanding of how the VP mediates drug-seeking motivation and the feedback modulation between the VP and the nucleus accumbens (NAc) following drug withdrawal. Therefore, utilizing a rat model of cocaine self-administration, we investigated the circuitry mechanisms underlying drug-seeking behavior post-drug withdrawal involving the NAc-VP pathway. Initially, we observed a significant enhancement in drug-seeking behavior 14 days after cocaine withdrawal. Subsequently, we identified the feedback mechanism through which the NAc-VP regulates this behavior. Immunofluorescence results indicated an increase in c-Fos expression levels in the ventral pallidum ventromedial (VPvm) and ventrolateral ventral pallidum (VPvl) following drug withdrawal. Calcium fiber photometry further elucidated that during the expression of high motivational drug-seeking behavior, there was a specific enhancement in VPvm neuronal activity, and retrograde tracing techniques suggested a weakened transmission function in the NAc-VPm pathway. Additionally, chemical genetic techniques demonstrated that inhibiting the activity of the NAc-VP pathway could increase the motivational level of drug-seeking behavior. These findings indicate that the reduced inhibitory function of the NAc-VP pathway following prolonged cocaine withdrawal forms the basis for heightened reactivity in VPvm neurons, thus regulating the expression of high motivational behavior triggered by drug-related cues. Our study results suggest that maintaining normal NAc-VP pathway functionality may decrease drug-seeking motivation post long-term drug withdrawal, offering new insights for interventions targeting relapse. Full article