Calcium Activity Research Articles

Brain state-dependent neocortico-hippocampal network dynamics are modulated by postnatal stimuli.

Neurons in the cerebral cortex and hippocampus discharge synchronously in brain state-dependent manner to transfer information. Published studies have highlighted the temporal coordination of neuronal activities between the hippocampus and a neocortical area, however, how the spatial extent of neocortical activity relates to hippocampal activity remains partially unknown. We imaged mesoscopic neocortical activity while recording hippocampal local field potentials in anesthetized and unanesthetized GCaMP-expressing transgenic mice. We found that neocortical activity elevates around hippocampal sharp wave ripples (SWR). SWR-associated neocortical activities occurred predominantly in vision-related regions including visual, retrosplenial and frontal cortex. While pre-SWR neocortical activities were frequently observed in awake and natural sleeping states, post-SWR neocortical activity decreased significantly in the latter. Urethane anesthetized mice also exhibited SWR-correlated calcium elevation, but in longer time scale than observed in natural sleeping mice. During hippocampal theta oscillation states, phase-locked oscillations of calcium activity were observed throughout the entire neocortical areas. In addition, possible environmental effects on neocortico-hippocampal dynamics were assessed in this study by comparing mice reared in ISO (isolated condition) and ENR (enriched environment). In both SWR and theta oscillations, mice reared in ISO exhibited clearer brain state-dependent dynamics than those reared in ENR. Our data demonstrate that the neocortex and hippocampus exhibit heterogeneous activity patterns that characterize brain states, and postnatal experience plays a significant role in modulating these patterns.Significant Statement The hippocampus is a center for memory formation. However, the memory formed in the hippocampus is not stored forever, but gradually transferred into the cerebral cortex synchronized activities between the neocortex and hippocampus has been hypothesized (for hippocampus-independent memory see (Sutherland and Rudy, 1989)). However, spatio-temporal dynamics between hippocampus and whole neocortical areas remains partially unexplored. We measured cortical calcium activities with hippocampal electroencephalogram (EEG) simultaneously and found that the activities of widespread neocortical areas are temporally associated with hippocampal EEG. The neocortico-hippocampal dynamics is primarily regulated by animal awake/sleep state. Even if similar EEG patters were observed, temporal dynamics between the neocortex and hippocampus exhibit distinct patterns between awake and sleep period. In addition, animals' postnatal experience modulates the dynamics.

Unveiling the functional connectivity of astrocytic networks with AstroNet, a graph reconstruction algorithm coupled to image processing

Astrocytes form extensive networks with diverse calcium activity, yet the organization and connectivity of these networks across brain regions remain largely unknown. To address this, we developed AstroNet, a data-driven algorithm that uses two-photon calcium imaging to map temporal correlations in astrocyte activation. By organizing individual astrocyte activation events chronologically, our method reconstructs functional networks and extracts local astrocyte correlations. We create a graph of the astrocyte network by tallying direct co-activations between pairs of cells along these activation pathways. Applied to the CA1 hippocampus and motor cortex, AstroNet reveals notable differences: astrocytes in the hippocampus display stronger connectivity, while cortical astrocytes form sparser networks. In both regions, smaller, tightly connected sub-networks are embedded within a larger, loosely connected structure. This method not only identifies astrocyte activation paths and connectivity but also reveals distinct, region-specific network patterns, providing new insights into the functional organization of astrocytic networks in the brain.

CalciumZero: a toolbox for fluorescence calcium imaging on iPSC derived brain organoids

Calcium plays an important role in regulating various neuronal activities in human brains. Investigating the dynamics of the calcium level in neurons is essential not just for understanding the pathophysiology of neuropsychiatric disorders but also as a quantitative gauge to evaluate the influence of drugs on neuron activities. Accessing human brain tissue to study neuron activities has historically been challenging due to ethical concerns. However, a significant breakthrough in the field has emerged with the advent of utilizing patient-derived human induced pluripotent stem cells (iPSCs) to culture neurons and develop brain organoids. This innovative approach provides a promising modeling system to overcome these critical obstacles. Many robust calcium imaging analysis tools have been developed for calcium activity analysis. However, most of the tools are designed for calcium signal detection only. There are limited choices for in-depth downstream applications, particularly in discerning differences between patient and normal calcium dynamics and their responses to drug treatment obtained from human iPSC-based models. Moreover, end-user researchers usually face a considerable challenge in mastering the entire analysis procedure and obtaining critical outputs due to the steep learning curve associated with these available tools. Therefore, we developed CalciumZero, a user-friendly toolbox to satisfy the unmet needs in calcium activity studies in human iPSC-based 3D-organoid/neurosphere models. CalciumZero includes a graphical user interface (GUI), which provides end-user iconic visualization and smooth adjustments on parameter tuning. It streamlines the entire analysis process, offering full automation with just one click after parameter optimization. In addition, it includes supplementary features to statistically evaluate the impact on disease etiology and the detection of drug candidate effects on calcium activities. These evaluations will enhance the analysis of imaging data obtained from patient iPSC-derived brain organoid/neurosphere models, providing a more comprehensive understanding of the results.

Reconstructing skeletal homeostasis through allogeneic hematopoietic stem cell transplantation in myelofibrosis

Myeloproliferative neoplasm-associated myelofibrosis is a clonal stem cell process characterized by pronounced bone marrow fibrosis associated with extramedullary hematopoiesis and splenomegaly. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment leading to bone marrow fibrosis regression. Here we provide an in-depth skeletal characterization of myelofibrosis patients before and after allo-HSCT utilizing clinical high-resolution imaging, laboratory analyses, and bone biopsy studies. Despite unimpaired bone microarchitecture at peripheral skeletal sites, we observe a marked increase in bone mineral density at the lumbar spine and proximal femur, which is histologically related to severe bone marrow fibrosis and osteosclerosis, fully normalizing after allo-HSCT. Importantly, the regression of fibrosis is accompanied by vanishing osteosclerosis along with restored osteoclastic resorption activity and whole-body calcium homeostasis. Together, our results provide evidence for an extensive reconstruction of skeletal homeostasis by allo-HSCT in MF, leading to rapid resolution of osteosclerosis.

In vivo Calcium Extrusion from Accumbal Astrocytes Reduces Anxiety-like Behaviors but Increases Compulsive-like Responses and Compulsive ethanol drinking in Mice.

The ventral striatum is crucially involved in reward processing. The present study investigates the behavioral effects of astrocyte-specific calcium extrusion virus "CalEx" on perseverative responses in the operant five-choice serial reaction time task and ethanol-conditioned place preference. Mice were injected with CalEx via the GfaABC1D promoter to extrude cytosolic calcium from astrocytes within the ventral striatum. We found that CalEx transfection in the ventral striatum reduced evoked response duration, the maximum amplitude, and the response frequency to 500 μM ATP as measured by ΔF/F fluorescence intensity of the genetically encoded calcium indicator targeting astrocytes GCaMP6f. During the five-choice serial reaction time task, CalEx mice persisted in perseverative responses compared to their counterparts. Additionally, during ethanol-conditioned place preference, CalEx mice showed increased place preference for a low ethanol concentration compared to control group. Furthermore, we found that accumbal astrocytic calcium extrusion increased quinine adulterated ethanol drinking. Our findings suggest that diminishing ventral striatum astrocyte calcium activity contributes to compulsive behaviors, ethanol drinking, and enhanced ethanol drug reward.

Activation of locus coeruleus noradrenergic neurons rapidly drives homeostatic sleep pressure.

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep for its revitalizing function, but the mechanism underlying sleep homeostasis remains poorly understood. Here, we show that optogenetic activation of locus coeruleus (LC) noradrenergic neurons immediately increased sleep propensity following a transient wakefulness, contrasting with many other arousal-promoting neurons whose activation induces sustained wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused a rapid reduction of calcium activity in LC neurons and steep declines in noradrenaline/norepinephrine (NE) release in both the LC and medial prefrontal cortex (mPFC). Knockdown of α2A adrenergic receptors in LC neurons mitigated the decline of NE release induced by repetitive stimulation and extended wakefulness, demonstrating an important role of α2A receptor-mediated auto-suppression of NE release. Together, these results suggest that functional fatigue of LC noradrenergic neurons, which reduces their wake-promoting capacity, contributes to sleep pressure.

CBD and the 5-HT1A receptor: A medicinal and pharmacological review.

Cannabidiol (CBD), a phytocannabinoid, has emerged as a promising candidate for addressing a wide array of symptoms. It has the ability to bind multiple proteins and receptors, including 5-HT1AR, transient receptor potential vanilloid 1 (TRPV1), and cannabinoid receptors. However, CBD's pharmacodynamic interaction with 5-HT1AR and its medicinal outcomes are still debated. This review explores recent literature to elucidate these questions, highlighting the neurotherapeutic outcomes of this pharmacodynamic interaction and proposing a signaling pathway underlying the mechanism by which CBD desensitizes 5-HT1AR signaling. A comprehensive survey of the literature underscores CBD's multifaceted neurotherapeutic effects, encompassing antidepressant, anxiolytic, neuroprotective, antipsychotic, antiemetic, anti-allodynic, anti-epileptic, anti-degenerative, and addiction-treating properties, attributable in part to its interactions with 5-HT1AR. Furthermore, evidence suggests that the pharmacodynamic interaction between CBD and 5-HT1AR is contingent upon dosage. Moreover, we propose that CBD can induce desensitization of 5-HT1AR via both homologous and heterologous mechanisms. Homologous desensitization involves the recruitment of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin, leading to receptor endocytosis. In contrast, heterologous desensitization is mediated by an elevated intracellular calcium level or activation of protein kinases, such as c-Jun N-terminal kinase (JNK), through the activity of other receptors.

Monitoring of inflammatory preterm responses via myometrial cell based multimodal electrophysiological and optical biosensing platform.

Preterm birth (PTB) remains a leading cause of neonatal morbidity and mortality, with inflammation-induced PTB posing a significant challenge due to its complex pathophysiology. To address this, we developed an in vitro platform utilizing hTERT-immortalized human myometrial (hTERT-HM) cells integrated with a multielectrode array (MEA) biosensing system and optical calcium imaging. Compared to primary uterine myometrial cells, hTERT-HM cells exhibit superior reproducibility, high scalability, and convenient manipulation, facilitating the consistent and large-scale investigations. This advanced system facilitates simultaneous real-time monitoring of electrophysiological activity and intracellular calcium transient, providing detailed insights into uterine cell behavior during inflammatory PTB. Our study revealed that oxytocin (OT) induces regular contractions in hTERT-HM cells, and the synergistic effect of OT and lipopolysaccharide (LPS) disrupts electrophysiological patterns and calcium signaling, closely mimicking the pathophysiology of inflammation-induced PTB. Meanwhile, magnesium sulfate is validated to effectively suppress OT-induced calcium release and mitigate LPS-triggered irregular electrophysiological signals. By integrating advanced biosensing technologies and advantages of hTERT-HM cells, this platform offers a reliable, reproducible model to investigate the mechanisms of inflammation-driven PTB and further develop targeted therapeutic interventions.

Polynorepinephrine nanoparticles activate vascular smooth muscle alpha-1 adrenergic receptors.

Biocompatible polymeric nanoparticles (NPs) as carriers for therapeutic agents with multifunctional activities have received unprecedented attention for a variety of bio-pharmaceutical applications. We describe the synthesis, the fluorescence properties, the bio-compatible nature and the alpha-1 adrenergic receptor bio-activity of engineered quantum dot-like polynorepinephrine (PNE) NPs. The spherical PNE NPs, which are internalized in smooth muscle cells via a receptor-selective mechanism, activate alpha-1-adrenoceptors in intact mouse aorta and aorta-derived cultured smooth muscle cells, leading to the activation of calcium signaling/contraction and stimulation of mitogen-activated protein kinase (MAPK), thereby displaying receptor-triggering biological activity, possibly acting both extracellularly and intracellularly. Our data indicate that NPs generated by the polymerization of pharmacologically active compounds like norepinephrine can retain receptor-selective biological activity coupled to inherent fluorescence.

Remimazolam alleviates sleep deprivation induced anxiety-like behaviors via regulating the STING pathway.

Sleep loss becomes a major problem in modern life and increases the incidence of anxiety disorders. Benzodiazepines are the most commonly used anxiolytic medications. Remimazolam is an ultra-short-acting benzodiazepine, which has been shown to reduce the preoperative anxiety levels in patients. However, the effects on anxiety-like behaviors caused by chronic sleep deprivation (CSD) and the underlying molecular mechanisms remain unclear. Here, we found that administration of remimazolam can effectively alleviate anxiety-like behaviors induced by CSD. Furthermore, remimazolam can significantly preserve the sleep deprivation-induced deficits in neuronal calcium activity in CA1 of the hippocampus. In addition, stimulator of interferon genes (STING) was activated in CA1 after CSD, while remimazolam was sufficient to block the activation of the STING pathway. Further study showed that inhibiting the activation of STING also effectively alleviates the anxiety symptoms induced by CSD. Overall, our research offers new insight and a promising therapeutic agent for the anxiety disorders caused by sleep deprivation.

LPL-RH suppresses bone loss in ovariectomised rat models

BackgroundEvidence has revealed that oestrogen deprivation-induced osteolysis is microbiota-dependent and can be treated by probiotics. However, the underlying mechanism require further investigation. This study aims to provide additional evidence supporting the use of probiotics as an adjuvant treatment and to explore the pathophysiology of oestrogen-deprived osteolysis.MethodsForty-five SD rats were randomly divided into five groups (n = 9). Rats from four groups were ovariectomised and treated with NS, calcium, probiotics, or calcium + probiotics, while one group underwent a sham operation and was treated with NS. The osteometabolic effects were evaluated, and the mechanistic role of the probiotic supplement was explored.ResultsIntragastric administration of Bifidobacterium animalis subsp. lactis LPL-RH (LPL-RH) markedly suppressed osteoclastic activation and bone calcium loss by downregulating TRAP enzymatic activity, the OPG/RANKL ratio, and the downstream signalling pathway RANKL/TRAF6/NF-κB/NFATc1/TRAP in ovariectomised SD rats. LPL-RH also reduced CD4+IL-17 A+ TH17 cells in the bone marrow, the pro-osteoclastogenic cytokine IL-17 A, pro-inflammatory molecules (LPS), and its binding protein (LBP) in the blood. LPL-RH restored intestinal ZO-1, occludin, claudin 2, claudin 12, and claudin 15, which improved ileal histopathology, reduced ileal oxidative stress, and attenuated the LPS-responsive TLR4/MyD88/NF-κB pathway. Furthermore, 16 S rRNA sequencing revealed that LPL-RH altered the faecal microbiome by reducing the relative abundance of S24-7 at the family level and promoting Prevotella and Bacteroides at the genus level.ConclusionCollectively, LPL-RH suppressed osteoclastogenesis and osteolysis by modulating type 17 immunity and gut microbiome.

Calcium Signalling in Neurological Disorders, with Insights from Miniature Fluorescence Microscopy

Neurological disorders (NDs), such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and schizophrenia, represent a complex and multifaceted health challenge that affects millions of people around the world. Growing evidence suggests that disrupted neuronal calcium signalling contributes to the pathophysiology of NDs. Additionally, calcium functions as a ubiquitous second messenger involved in diverse cellular processes, from synaptic activity to intercellular communication, making it a potential therapeutic target. Recently, the development of the miniature fluorescence microscope (miniscope) enabled simultaneous recording of the spatiotemporal calcium activity from large neuronal ensembles in unrestrained animals, providing a novel method for studying NDs. In this review, we discuss the abnormalities observed in calcium signalling and its potential as a therapeutic target for NDs. Additionally, we highlight recent studies that utilise miniscope technology to investigate the alterations in calcium dynamics associated with NDs.

Near-infrared light induces neurogenesis and modulates anxiety-like behavior

BackgroundThe hippocampus is associated with mood disorders, and the activation of quiescent neurogenesis has been linked to anxiolytic effects. Near-infrared (NIR) light has shown potential to improve learning and memory in human and animal models. Despite the vast amount of information regarding the effect of visible light, there is a significant gap in our understanding regarding the response of neural stem cells (NSCs) to NIR stimulation, particularly in anxiety-like behavior. The present study aimed to develop a new optical manipulation approach to stimulate hippocampal neurogenesis and understand the mechanisms underlying its anxiolytic effects.MethodsWe used 940 nm NIR (40 Hz) light exposure to stimulate hippocampal stem cells in C57BL/6 mice. The enhanced proliferation and astrocyte differentiation of NIR-treated NSCs were assessed using 5-ethynyl-2’-deoxyuridine (EdU) incorporation and immunofluorescence assays. Additionally, we evaluated calcium activity of NIR light-treated astrocytes using GCaMP6f recording through fluorescence fiber photometry. The effects of NIR illumination of the hippocampus on anxiety-like behaviors were evaluated using elevated plus maze and open-field test.ResultsNIR light effectively promoted NSC proliferation and astrocyte differentiation via the OPN4 photoreceptor. Furthermore, NIR stimulation significantly enhanced neurogenesis and calcium-dependent astrocytic activity. Moreover, activating hippocampal astrocytes with 40-Hz NIR light substantially improved anxiety-like behaviors in mice.ConclusionsWe found that flickering NIR (940 nm/40Hz) light illumination improved neurogenesis in the hippocampus with anxiolytic effects. This innovative approach holds promise as a novel preventive treatment for depression.

MAM-STAT3-Driven Mitochondrial Ca+2 Upregulation Contributes to Immunosenescence in Type A Mandibuloacral Dysplasia Patients.

Individuals with homozygous laminA/C p.R527C mutations manifest a severe form of Mandibuloacral dysplasia-(MAD) and exhibit overlapping progeroid symptoms, for which the underlying molecular pathology remains unknown. Herein, it is shown that MAD patients achieved inflammaging with different pro-inflammatory cytokines compared to progeria-(HGPS) patient. Characterization of MAD iPSC-derived Mesenchymal stem cells (MAD-iMSC) uncovers deregulated mitochondrial Ca+2 as the primary cause of inflammaging, mediated through inflammasome formation rather than the cGAS-STING pathway. Moreover, MAD-iMSCs extracellular vesicles (EVs) can also upregulate mitochondrial Ca+2 in healthy cells. This deregulated Ca+2 homeostasis is indirectly mediated by mitochondrial calcium mediator, signal transducer, and activator of transcription-3 (STAT3), situated on the mitochondrial associated membrane (MAM). Inflammaging is mitigated by various FDA-approved MAM-STAT3 upstream inhibitors, such as (Tocilizumab) or by correcting R527C mutation with CRISPR/CAS9. These results provide new insights into MAD disease and propose targeting defective mitochondrial Ca+2 homeostasis as a promising therapy for reversing immunosenescence.

Nutritional value and antioxidant activity of ora-pro-nobis, tamarillo and green banana biomasses

The consumption of unconventional food plants (UFP) can be a strategy to promote food and nutritional security, for which it is necessary to know their nutritional composition. The objective of this study was to analyze the nutritional value and antioxidant activity of ora-pro-nobis, tamarillo and bananas biomass (prata and nanica). Using standard methodologies (AOAC, 2005), the following compounds were analyzed in triplicate: protein, lipids, carbohydrate, calories, moisture, total fiber content, calcium, iron, vitamin C (by titrimetry), carotenoids (by High Performance Liquid Chromatography), phenolic compounds (by Folin-Ciocalteau) and antioxidant activities, by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis 3-ethylbenzothiacholine-6-sulfonic acid). Ora-pro-nobis stands out as a source of calcium, iron, carotenoids and protein, is low in fat and has good antioxidant activity. Tamarillo showed high antioxidant activity, is a protein source and low in fat. The two banana biomasses had similar results in most nutrients, but the “nanica” banana with more protein and the “prata” with greater antioxidant activity. The unconventional food plants analyzed are potential bioactive foods, because they have phenolic compounds and high antioxidant activity. Thus, UFP have the potential to promote a more diverse and nutritious diet for consumers in general.

An activating CaSR variant with biased signaling reveals a critical residue for Gα11 coupling.

Autosomal dominant hypocalcemia (ADH) is due to enhanced calcium-dependent signaling caused by heterozygous gain-of-function (GOF) variants in the CASR gene (ADH1) or in the GNA11 gene, encoding Gα11 (ADH2). Both ADH1 and ADH2 are associated with hypocalcemia and normal or inappropriately low levels of circulating PTH. ADH1 patients typically manifest hypercalciuria, while ADH2 is associated with short stature in approximately 42% of cases. We evaluated a 10-year-old boy with hypoparathyroidism and short stature. Biochemical analyses revealed hypocalcemia, hyperphosphatemia and inconsistent hypercalciuria. Genetic analyses revealed a de novo heterozygous p.Leu723Arg variant in CASR. We characterized the expression of recombinant wild type and Leu723Arg CaSR proteins in HEK293 cells and assessed G protein activation in vitro by CaSR using Bioluminescence Resonance Energy Transfer (BRET). Transient expression studies showed the Leu723Arg variant was normally expressed but resulted in a significantly lower EC50 for extracellular calcium activation of G11 but not other G proteins (i.e. Gi, Gq, Gs). The Leu723Arg substitution has a novel GOF phenotype that leads to biased CaSR activation of G11 signaling, suggesting that residue 723 specifies activation of G11 but not other G proteins. Similar studies of a previously described CaSR variant associated with hypoparathyroidism and short stature, Leu616Val, showed no changes in any G protein pathways, indicating it is likely to be a benign variant. Given the preferential activation of G11 by the Leu723Arg CaSR variant, we propose that the patient's short stature shares a similar basis to that in patients with ADH2 due to GOF variants in GNA11.

Differential timing of mitochondrial activation in rat dorsal striatum induced by procedural learning and swimming.

Stressful experiences form stronger memories due to enhanced neural plasticity mechanisms linked to glucocorticoid hormones (cortisol in humans, corticosterone in rats). Among other neural structures, the dorsal striatum plays a role in the corticosterone-induced consolidation of stressful memories, particularly in the cued water maze task. Neural plasticity is related to mitochondrial activity due to the relevance of energy production and signaling mechanisms for functional and morphological neuronal adaptations. Corticosterone has been shown to enhance brain mitochondrial activity by activating glucocorticoid receptors. In this context, striatum functions are susceptible to change in relation to mitochondrial responses. Based on this evidence, we hypothesized that training in the cued water maze would induce an increase in corticosterone levels and mitochondrial activity (mitochondrial membrane potential and calcium content) in the dorsal striatum, and that these adaptations might be related to memory consolidation of the task. We used an ELISA assay to evaluate plasma and striatal corticosterone levels; mitochondrial activity was determined with the florescent probes MitoTracker Red (mitochondrial membrane potential) and Rhod-2 (calcium content) in brain slices containing the dorsal striatum of rats trained in the cued water maze and euthanized at different times after training (0.5, 1.5, or 6.0 h). We also analyzed the effect of post-training inhibition of striatal mitochondrial activity by OXPHOS complex 1 inhibitor rotenone, on the consolidation of the cued water maze task. We found that cued water maze training induced an increase in corticosterone levels and a time-dependent elevation of mitochondrial membrane potential and mitochondrial calcium content in the dorsal striatum. Unexpectedly, rotenone administration facilitated the retention test. Altogether, our results suggest that enhanced mitochondrial activity in the dorsal striatum is relevant for cued water maze consolidation. The increase in mitochondrial activity was contextually associated with an elevation of corticosterone in plasma and the dorsal striatum. Additionally, our swimming groups also showed an increase in mitochondrial activity in the dorsal striatum, but with a different pattern, which could suggest a differential functional adaptation in this structure.

The background sodium leak channel NALCN is a major controlling factor in pituitary cell excitability.

The pituitary gland produces and secretes a variety of hormones that are essential to life, such as for the regulation of growth and development, metabolism, reproduction, and the stress response. This is achieved through an intricate signalling interplay between the brain and peripheral feedback signals that shape pituitary cell excitability by regulating the ion channel properties of these cells. In addition, endocrine anterior pituitary cells spontaneously fire action potentials to regulate the intracellular calcium ([Ca2+]i) level, an essential signalling conduit for hormonal secretion. To this end, pituitary cells must regulate their resting membrane potential (RMP) close to the firing threshold, but the molecular identity of the ionic mechanisms responsible for this remains largely unknown. Here, we revealed that the sodium leak channel NALCN, known to modulate neuronal excitability elsewhere in the brain, regulates excitability in the mouse anterior endocrine pituitary cells. Using viral transduction combined with powerful electrophysiology methods and calcium imaging, we show that NALCN forms the major Na+ leak conductance in these cells, appropriately tuning cellular RMP for sustaining spontaneous firing activity. Genetic depletion of NALCN channel activity drastically hyperpolarised these cells, suppressing their firing and [Ca2+]i oscillations. Remarkably, despite this profound function of NALCN conductance in controlling pituitary cell excitability, it represents a very small fraction of the total cell conductance. Because NALCN responds to hypothalamic hormones, our results also provide a plausible mechanism through which hormonal feedback signals from the brain and body could powerfully affect pituitary activity to influence hormonal function. KEY POINTS: Pituitary hormones are essential to life as they regulate important physiological processes, such as growth and development, metabolism, reproduction and the stress response. Pituitary hormonal secretion relies on the spontaneous electrical activity of pituitary cells and co-ordinated inputs from the brain and periphery. This appropriately regulates intracellular calcium signals in pituitary cells to trigger hormonal release. Using viral transduction in combination with electrophysiology and calcium imaging, we show that the activity of the background leak channel NALCN is a major controlling factor in eliciting spontaneous electrical activity and intracellular calcium signalling in pituitary cells. Remarkably, our results revealed that a minute change in NALCN activity could have a major influence on pituitary cell excitability. Our study provides a plausible mechanism through which the brain and body could intricately control pituitary activity to influence hormonal function.

Overexpression of Nogo-A changes nerve growth factor signaling dynamics in PC12 cells.

The nerve growth factor (NGF) receptor TrkA is a tightly regulated receptor tyrosine kinase that activates neuronal signaling pathways promoting cell survival in addition to axonal and dendritic outgrowth. Previously, we showed that NGF and TrkA signaling is altered in neuron-like PC12 cells that overexpress Nogo-A, a protein known to influence axonal outgrowth and dendritic arborization associated with neuronal plasticity. In the present report, we provide evidence for changes in NGF-mediated receptor-level and downstream signaling that occur in cells overexpressing Nogo-A. NGF stimulation increased the association of Nogo-A with TrkA, which corresponded to a decrease in sustained phosphorylation of TrkA and its downstream effectors Erk1/2, indicating that Nogo-A plays a role in the temporal regulation of this pathway. Furthermore, co-immunoprecipitation of the p75 neurotrophin receptor (p75NTR) with TrkA was significantly reduced in cells overexpressing Nogo-A, suggesting that Nogo-A blocked this interaction. Analysis of calcium and calmodulin involvement in NGF-induced activation of Erk1/2 revealed a calcium and calmodulin-dependent inhibition of sustained phosphorylation in Nogo-A-overexpressing cells but not in wild type cells, suggesting that Nogo-A facilitated the activation of calcium/calmodulin to alter NGF signaling. Taken together, these results provide evidence for Nogo-A regulation of NGF signaling, in part by modifying calcium and calmodulin-dependent mechanisms.

Long-Term Survival and Myocardial Function Following Systemic Delivery of Delandistrogene Moxeparvovec in DMDMDX Rats.

Delandistrogene moxeparvovec is a gene transfer therapy for Duchenne muscular dystrophy (DMD) that uses an adeno-associated viral vector to deliver a micro-dystrophin transgene to skeletal and cardiac muscle. This study evaluated the long-term survival and cardiac efficacy of delandistrogene moxeparvovec in a DMD-mutated (DMDMDX) rat model of DMD-related cardiomyopathy. DMDMDX male rats, aged 21-42 days, were injected with 1.33 × 1014 viral genomes/kilogram (vg/kg) delandistrogene moxeparvovec and followed for 12, 24, and 52 weeks. Ambulation was recorded via the Photobeam Activity System, whereas echocardiograms, cardiomyocyte contractility, calcium handling, and histological analysis of fibrosis were used to evaluate cardiac disease at 12-, 24-, and 52-weeks post-treatment. A separate cohort of rats was used to assess the impact of delandistrogene moxeparvovec on survival. Treatment with delandistrogene moxeparvovec extended median survival in DMDMDX rats to >25 months versus the 13-month median survival in saline-control-treated DMDMDX rats. Compared with saline control, delandistrogene moxeparvovec therapy elicited statistically significant improvements across cardiac parameters approaching wild-type values with additional benefits in mobility, histopathology, and fibrosis observed. Transgene expression was maintained up to >25 months and micro-dystrophin expression was broadly distributed across skeletal and cardiac muscle. Taken together, these findings demonstrate long-term cardiac efficacy and improved survival following delandistrogene moxeparvovec treatment in DMDMDX rats.