Nerve Terminals Research Articles

Prognostic Value of Neurological Manifestations Related to COVID-19 Positive Patients: Observational Cross-Sectional Study

Abstract Background COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus. Corona virus affects the central nervous system in various different ways either through a retrograde transfer via the olfactory epithelium or through the cribriform bone and reaches the brain directly in seven days’ time or due to disruption of blood brain barrier which occurs during the viremia phase of illness and the virus will enter the brain directly. Another postulated mechanism is the invasion of peripheral nerve terminals by COVID-19 which then gains entry to the CNS through the synapse connected route. Objective To determine the prevalence of neurological symptoms and complications in COVID-19 patients and if we can use the neurological symptoms and its relevance as prognostic factors for disease severity. Patients and Methods This was a cross sectional study that was conducted on COVID-19 positive patients admitted to isolation ICU Ain Shams University Hospital through 4 months. Results The present study aimed to provide a comprehensive overview of neurologic manifestations associated with SARS-CoV-2 infection and to describe the clinical course and outcomes of COVID-19 patients with neurologic manifestations. Our results highlight the broad spectrum of neurological manifestations associated with SARS-CoV- 2 infection: the majority of neurologic manifestations were altered mental status (27.7%), anosmia (13.8%) followed by headache (12.5%). Conclusion the neurological manifestations are common in Covid-19 patients. There is an association between the presence of neurological manifestations and mortality.

Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

The flower-spray nerve endings are afferent nerve terminals in the carotid sinus that arise from carotid sinus nerve of glossopharyngeal nerve. However, the three-dimensional ultrastructural characteristics of flower-spray nerve endings and spatial relationships between the terminal parts and other cellular elements have not been fully understood. To elucidate their detailed relationship, backscattered electron imaging of serial sections was performed with a scanning electron microscope to produce a three-dimensional reconstruction of the flower-spray endings. The terminal parts of flower-spray endings were distributed horizontally approximately 5µm outside the external elastic membrane in the tunica adventitia of the internal carotid artery. The three-dimensional reconstruction showed that the terminal parts of flower-spray endings were flat with irregular contours and were partially covered by the thin cytoplasmic processes of Schwann cells. The complex consisting of the nerve terminals and associated Schwann cells was surrounded by a multilayered basement membrane. The terminal parts of the endings were also surrounded by fibroblasts with elastic fibers and collagen fibrils. Secretory vesicles without an electron-dense core were observed in the terminal parts of the endings. The accumulation of vesicles just below the axonal membrane was observed in terminal parts not covered by Schwann cell cytoplasmic processes on both the luminal and basal sides. Swollen mitochondria, concentric membranous structures, and glycogen granule-like electron-dense materials were often noted in some of the terminal parts of the endings and the parent axon. Collectively, the present results suggest that flower-spray endings are baroreceptors because their morphology was similar to other mechanoreceptors. Furthermore, flower-spray endings may be affected by glutamate secreted in an autocrine manner.

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Constitutive and Conditional Epitope Tagging of Endogenous G-Protein-Coupled Receptors in Drosophila.

To visualize the cellular and subcellular localization of neuromodulatory G-protein-coupled receptors in Drosophila, we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Oct β 1R, Oct β 2R, two isoforms of OAMB, and mGluR The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show expression patterns for these receptors in female brains and that 5-HT1A and 5-HT2B localize to the mushroom bodies (MBs) and central complex, respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Octβ1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their functions at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the MBs as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor.

Identification of adrenergic presynaptic and postsynaptic protein locations at neuromuscular junctions, their decrease during aging, and recovery by nicotinamide mononucleotide administration.

Neuromuscular junctions are innervated by motor and sympathetic nerves. The sympathetic modulation of motor innervation shows functional decline during aging, but the cellular and molecular mechanism of this change is not fully known. This study aimed to evaluate the effect of aging on sympathetic nerves and synaptic proteins at mouse neuromuscular junctions. Sympathetic nerves, presynaptic, and postsynaptic proteins of sympathetic nerves at neuromuscular junctions were visualized using immunohistochemistry, and aging-related changes were compared between adult-, aged-, and nicotinamide mononucleotide (NMN) administered aged mice. Sympathetic nerves were detected by anti-tyrosine hydroxylase antibody, and presynaptic protein vesicular monoamine transporter 2 colocalized with the sympathetic nerves. These two signals surrounded motor nerve terminals and acetylcholine receptor clusters. Postsynaptic neurotransmitter receptor β2-adrenergic receptors colocalized with motor nerve terminals and resided in reduced density at extrasynaptic sarcolemma. The signal intensity of the sympathetic nerve marker did not show a significant difference at neuromuscular junctions between 8.5-month-old adult mice and 25-month-old aged mice. However, the signal intensity of vesicular monoamine transporter 2 and β2-adrenergic receptors showed age-related decline at neuromuscular junctions. Interestingly, both age-related declines reverted to the adult level after 1 month of oral administration of NMN by drinking water. In contrast, NMN administration did not alter the expression level of sympathetic marker tyrosine hydroxylase at neuromuscular junctions. The results suggest a functional decline of sympathetic nerves at aged neuromuscular junctions due to decreases in presynaptic and postsynaptic proteins, which can be reverted to the adult level by NMN administration.

Aromatase inhibitors evoke periorbital allodynia in mice via calcitonin gene-related peptide and its receptors in Schwann cells

Background: Treatment with the currently recommended aromatase inhibitors (AIs) for adjuvant endocrine treatment of estrogen receptorpositive breast cancer is associated with debilitating musculoskeletal pain symptoms (AIMS) and headache. Recent evidence suggests that the proalgesic channel transient receptor potential ankyrin 1 (TRPA1) is implicated in AIMS. Here, we investigated the cellular and molecular mechanisms, including TRPA1, implicated in periorbital mechanical allodynia (PMA), a surrogate of headache-like pain, evoked by AIs in mice. Methods: C57BL6/J mice were treated with intragastric letrozole (0.05-0.5 mg/kg), exemestane (1-5 mg/kg) or anastrozole (0.02-0.2 mg/kg) and were evaluated by applying von Frey filaments to the periorbital region over the rostral portion of the eye. Some mice were pretreated (subcutaneous in the periorbital area) with receptor, channel, or enzyme inhibitors. PMA was also investigated in mice with selective silencing of Trpa1 and receptor activity modifying protein 1 [Ramp1, the component of calcitonin gene related peptide (CGRP) receptor required for its functioning] in Schwann cells (Plp-Cre+-Trpa1fl/fl and Plp-Cre+-Ramp1fl/fl mice, respectively) or trigeminal neurons (Adv-Cre+- Trpa1fl/fl and Adv- Cre+- Ramp1fl/fl mice, respectively). Results: Letrozole dose-dependently produced PMA that was attenuated by a TRPA1 antagonist (A967079) or a CGRP receptor antagonist (olcegepant), whereas indomethacin was ineffective. Selective silencing of Trpa1 in both Schwann cells and trigeminal neurons reduced letrozole- evoked PMA. Silencing of Ramp1 in Schwann cells, but not in trigeminal neurons, attenuated PMA. Inhibition of the intracellular pathway known to promote PMA by CGRP action in Schwann cells, including adenylyl cyclase (SQ-22536), nitric oxide synthase (L-NG-Nitro arginine methyl ester), and oxidative stress (N-tert-butyl-a-phenylnitrone) inhibitors reduced letrozole-evoked PMA. PMA evoked by exemestane (1, 5, 10 mg/kg i.g.) or anastrozole (0.02, 0.1, 0.2 mg/kg i.g.) Was also markedly reduced in mice with selective silencing of TRPA1 in Schwann cells and nociceptors. Conclusions: Data indicate that letrozole, targeting TRPA1 in peptidergic nerve terminals, releases CGRP that engages its receptor in adjacent Schwann cells to trigger a complex intracellular pathway that results in TRPA1 activation and the ensuing ROS release to sustain PMA. Should these mechanisms be present in patients, their inhibition may ameliorate cephalic mechanical allodynia associated with aromatase inhibitors-induced headaches.

Frankincense: A neuronutrient to approach Parkinson's disease treatment.

Parkinson's disease (PD), characterized by tremor, slowness of movement, stiffness, and poor balance, is due to a significant loss of dopaminergic neurons in the substantia nigra pars compacta and dopaminergic nerve terminals in the striatum with deficit of dopamine. To date the mechanisms sustaining PD pathogenesis are under investigation; however, a solid body of experimental evidence involves neuroinflammation, mitochondrial dysfunction, oxidative stress, and apoptotic cell death as the crucial factors operating in the pathogenesis of PD. Nutrition is known to modulate neuroinflammatory processes implicated in the pathogenesis and progression of this neurodegenerative disorder. Consistent with this notion, the Burseraceae family, which includes the genera Boswellia and Commiphora, are attracting emerging interest in the treatment of a wide range of pathological conditions, including neuroinflammation and cognitive decline. Bioactive components present in these species have been shown to improve cognitive function and to protect neurons from degeneration in in vitro, animal, as well as clinical research. These effects are mediated through the anti-inflammatory, antiamyloidogenic, anti-apoptotic, and antioxidative properties of bioactive components. Although many studies have exploited possible therapeutic approaches, data from human studies are lacking and their neuroprotective potential makes them a promising option for preventing and treating major neurodegenerative disorders.

Inhibitory actions of melanin-concentrating hormone in the lateral septum.

Melanin-concentrating hormone (MCH) neurons can co-express several neuropeptides or neurotransmitters and send widespread projections throughout the brain. Notably, there is a dense cluster of nerve terminals from MCH neurons in the lateral septum (LS) that innervate LS cells by glutamate release. The LS is also a key region integrating stress- and anxiety-like behaviours, which are also emerging roles of MCH neurons. However, it is not known if or where the MCH peptide acts within the LS. We analysed the projections from MCH neurons in male and female mice anteroposteriorly throughout the LS and found spatial overlap between the distribution pattern of MCH-immunoreactive (MCH-ir) fibres with MCH receptor Mchr1mRNA hybridization or MCHR1-ir cells. This overlap was most prominent along the ventral and lateral border of the rostral part of the LS (LSr). Most MCHR1-labelled LS neurons lay adjacent to passing MCH-ir fibres, but some MCH-ir varicosities directly contacted the soma or cilium of MCHR1-labelled LS neurons. We thus performed whole-cell patch-clamp recordings from MCHR1-rich LSr regions to determine if and how LS cells respond to MCH. Bath application of MCH to acute brain slices activated a bicuculline-sensitive chloride current that directly hyperpolarized LS cells. This MCH-mediated hyperpolarization was blocked by calphostin C, which suggested that the inhibitory actions of MCH were mediated by protein kinase C-dependent activation of GABAA receptors. Taken together, these findings define potential hotspots within the LS that may elucidate the contributions of MCH to stress- or anxiety-related feeding behaviours. KEY POINTS: Melanin-concentrating hormone (MCH) neurons have dense nerve terminals within the lateral septum (LS), a key region underlying stress- and anxiety-like behaviours that are emerging roles of the MCH system, but the function of MCH in the LS is not known. We found spatial overlap between MCH-immunoreactive fibres, Mchr1 mRNA, and MCHR1 protein expression along the lateral border of the LS. Within MCHR1-rich regions, MCH directly inhibited LS cells by increasing chloride conductance via GABAA receptor activation in a protein kinase C-dependent manner. Electrophysiological MCH effects in brain slices have been elusive, and few studies have described the mechanisms of MCH action. Our findings demonstrated, to our knowledge, the first description of MCHR1 Gq-coupling in brain slices, which was previously predicted in cell or primary culture models only. Together, these findings defined hotspots and mechanistic underpinnings for MCH effects such as in feeding and anxiety-related behaviours.

The mucocutaneous junction and its associated nerves, immunoreactive cells and changes with age: a review with novel observations from human fetal and cadaveric specimens

The mucocutaneous junction (MCJ) is a specific site located at body orifices such as the mouth, nose, eyelid, vulva, prepuce, and anus. The present study was conducted to reassess the MCJ in terms of 1) cytokeratin expression, 2) the distribution of immunoreactive cells, 3) nerves and their terminals, and 4) differences between near-term fetuses and adults. For cadaveric specimens after long-term preservation, cytokeratin 14 was useful for identification of the MCJ. In near-term fetuses, routine histology clearly demonstrated the putative MCJ due to transient, thick stratification of the squamous epithelium. This was most evident in the oral mucosa and less evident in the palpebral skin. In the anus and eyelid, the MCJ developed from highly proliferative epithelia of the anal sinus and meibomian gland, independent of the growing skin and mucosa. In elderly individuals, the MCJ was expanded in the nose and anus possibly as a result of mechanical stimuli, and was apparently connected to a hemorrhoid venous plexus. At the vulva and prepuce, a morphology intermediate between pseudostratified columnar and stratified squamous epithelia was evident. Except for the vulva and prepuce, corpusclelike nerve terminals were sandwiched between the dermal papillae and epidermis, whereas nerves arranged in parallel exhibited free ends beneath the mucosa. At the anus, the myenteric autonomic nerve plexus disappeared slightly orad to the MCJ. Langerhans cells were richly distributed in the skin stratum spinosum, while suppressive T lymphocyte clusters were present in the lamina propria mucosae. General rules and site-specificity of the MCJ were reviewed.

Organization of the Reserve Pool of Synaptic Vesicles in Nerve Terminals Lacking Protein Liquid Phase Components

Organization of the Reserve Pool of Synaptic Vesicles in Nerve Terminals Lacking Protein Liquid Phase Components

Characterization of β-Hydroxybutyrate as a Cell Autonomous Fuel for Active Excitatory and Inhibitory Neurons: β-Hydroxybutyrate as a Fuel for Active Neurons.

The ketogenic diet is an effective treatment for drug-resistant epilepsy, but the therapeutic mechanisms are poorly understood. Although ketones are able to fuel the brain, it is not known whether ketones are directly metabolized by neurons on a time scale sufficiently rapid to fuel the bioenergetic demands of sustained synaptic transmission. Here, we show that nerve terminals can use the ketone β-hydroxybutyrate in a cell- autonomous fashion to support neurotransmission in both excitatory and inhibitory nerve terminals and that this flexibility relies on Ca2+ dependent upregulation of mitochondrial metabolism. Using a genetically encoded ATP sensor, we show that inhibitory axons fueled by ketones sustain much higher ATP levels under steady state conditions than excitatory axons, but that the kinetics of ATP production following activity are slower when using ketones as fuel compared to lactate/pyruvate for both excitatory and inhibitory neurons.

Oxytocin Receptors on Calvarial Periosteal Innervation: Therapeutic Target for Post-Traumatic Headache?

Following a mild traumatic brain injury (mTBI), the most prevalent and profoundly debilitating occurrence is the emergence of an acute and persistent post-traumatic headache (PTH), for which there are presently no approved treatments. A crucial gap in knowledge exists regarding the consequences of an mTBI, which could serve as a foundation for the development of therapeutic approaches. The activation of trigeminal sensory nerve terminals that innervate the calvarial periosteum (CP)-a densely innervated tissue layer covering the calvarial skull-has been implicated in both migraines and PTHs. We have previously shown that trigeminal oxytocin receptors (OTRs) may provide a therapeutic target for PTHs. This study examined the expression of oxytocin receptors on trigeminal nerves innervating the periosteum and whether these receptors might serve as a therapeutic target for PTHs using a direct application of oxytocin to the periosteum in a rodent model of PTH. We used retrograde tracing and immunohistochemistry to determine if trigeminal ganglion (TG) neurons innervating the periosteum expressed OTRs and/or CGRPs. To model the impact of local inflammation that occurs following an mTBI, we applied chemical inflammatory mediators directly to the CP and assessed for changes in immediate-early gene expression as an indication of neuronal activation. We also determined whether mTBI would lead to expression changes to OTR levels. To determine whether these OTRs could be a viable therapeutic target, we assessed the impact of oxytocin injections into the CP in a mouse model of PTH-induced periorbital allodynia. The results of these experiments demonstrate the following: (1) the cell bodies of CP afferents reside in the TG and express both OTRs and CGRPs; (2) inflammatory chemical stimulation of the periosteum leads to rapid activation of TG neurons (phospho-ERK (p-ERK) expression), (3) mTBI-induced inflammation increased OTR expression compared to the sham group; and (4) administration of oxytocin into the periosteum on day 2 and day 40 blocked cutaneous allodynia for up to one hour post-administration for both acute and persistence phases in the PTH model-an effect that was preventable by the administration of an OTR antagonist. Taken together, our observations suggest that periosteal trigeminal afferents contribute to post-TBI craniofacial pain, and that periosteum tissue can be used as a potential local target for therapeutics such as oxytocin.

Role of autonomic nervous system in BDE-209 maternal exposure induced immunotoxicity in female offspring.

Decabrominated diphenyl ether (BDE-209) is a typical persistent organic pollutant that can cross the placental barrier, increasing the exposure risk for offspring. Norepinephrine (NE) from nerve terminals and acetylcholine (Ach) can bind to specific receptors on immune cells, inhibit the immune function of the body then cause immunotoxicity. However, whether maternal exposure to BDE-209 could lead to immunotoxicity in the offspring by acting on the sympathetic and parasympathetic nervous systems remains unclear. In view of this, the pregnancy and lactation rat BDE-209 exposure model was established and the results demonstrated that pregnancy and lactation BDE-209 exposure could induce immunotoxicity to female offspring via affecting immunopathology (hematological and biochemical parameters, organ indices, and spleen histopathological), decreasing humoral immunity (serum hemolysin, immunoglobulins, and cytokine productions), damaging cellular immunity (splenic lymphocytes and spleen cytokine productions), and restraining nonspecific immunity. Moreover, a dramatically significant correlation was observed between spleen nerve indices and immunity indices. Additionally, the mechanism revealed that maternal BDE-209 exposure caused offspring immunotoxicity through (1) activating MHC/PKCθ/NF-κB pathway; (2) promoting sympathetic nervous pathway, by upregulating the expression of β2AR protein, which in turn elevating cAMP, following activate PKA and phosphorylate CREB, ultimately leading to immunotoxicity;(3) activating parasympathetic nerve pathway by reducing the binding with Ach and α7nAchR, upregulating the expression of JAK2 and phosphorylating STAT3, induced immunotoxicity of female offspring.

Cardiac Uptake of the Adrenergic Imaging Agent meta-Iodobenzylguanidine (mIBG) Is Mediated by Organic Cation Transporter 3 (Oct3).

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 μg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

Anatomical variations of the brachial plexus in adult cadavers: A descriptive study and clinical significance.

Brachial plexus injury is recognized as one of the most severe clinical challenges due to the complex anatomical configuration of the brachial plexus and its propensity for variation, which complicates safe clinical interventions. This study aimed to ascertain the prevalence and characterize the types of brachial plexus variations, and to elucidate their clinical implications. We conducted meticulous dissections of 60 formalin-fixed cadavers' upper arm, axilla and lower neck to reveal and assess the roots, trunks, divisions, cords, and branches of the brachial plexus. The pattern of branching was noted by groups of dissecting medical students and confirmed by the senior anatomists. The variations discovered were record and photographed using a digital camera for further analysis. Variations in the brachial plexus were identified in 40 of the 60 cadavers, yielding a prevalence rate of 66.7%. These variations were classified into root anomalies (2.1%), trunk anomalies (8.5%), division anomalies (2.1%), and cord anomalies (4.3%). Notably, anomalies in communicating branches were observed in 39 cadavers (83.0%): 14 with bilateral anomalies, 14 with anomalies on the left side, and 11 on the right side. These communicating branches formed connections between the roots and other segments, including trunks, cords, and terminal nerves, and involved the median, musculocutaneous, and ulnar nerves. The frequency and diversity of brachial plexus variations, particularly in communicating branches, are significant in cadavers. It is imperative that these variations are carefully considered during the diagnostic process, treatment planning, and prior to procedures such as supraclavicular brachial plexus blocks and nerve transfers, to mitigate the risk of iatrogenic complications.

Physiological and Pathophysiological Relevance of Nitric Oxide Synthases (NOS) in Retinal Blood Vessels.

Nitric oxide synthases (NOS) are essential regulators of vascular function, and their role in ocular blood vessels is of paramount importance for maintaining ocular homeostasis. Three isoforms of NOS-endothelial (eNOS), neuronal (nNOS), and inducible (iNOS)-contribute to nitric oxide production in ocular tissues, exerting multifaceted effects on vascular tone, blood flow, and overall ocular homeostasis. Endothelial NOS, primarily located in endothelial cells, is pivotal for mediating vasodilation and regulating blood flow. Neuronal NOS, abundantly found in nerve terminals, contributes to neurotransmitter release and vascular tone modulation in the ocular microvasculature. Inducible NOS, expressed under inflammatory conditions, plays a role in response to pathological stimuli. Understanding the distinctive contributions of these NOS isoforms in retinal blood vessels is vital to unravel the mechanisms underlying various ocular diseases, such diabetic retinopathy. This article delves into the unique contributions of NOS isoforms within the complex vascular network of the retina, elucidating their significance as potential therapeutic targets for addressing pathological conditions.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

IATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Differential Modulation of Catecholamine and Adipokine Secretion by the Short Chain Fatty Acid Receptor FFAR3 and α2-Adrenergic Receptors in PC12 Cells.

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α2-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α2-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gβγ-activated phospholipase C (PLC)-β/Ca2+ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action.

Sleep fragmentation exacerbates myocardial ischemia‒reperfusion injury by promoting copper overload in cardiomyocytes

Sleep disorders increase the risk and mortality of heart disease, but the brain-heart interaction has not yet been fully elucidated. Cuproptosis is a copper-dependent type of cell death activated by the excessive accumulation of intracellular copper. Here, we showed that 16 weeks of sleep fragmentation (SF) resulted in elevated copper levels in the male mouse heart and exacerbated myocardial ischemia–reperfusion injury with increased myocardial cuproptosis and apoptosis. Mechanistically, we found that SF promotes sympathetic overactivity, increases the germination of myocardial sympathetic nerve terminals, and increases the level of norepinephrine in cardiac tissue, thereby inhibits VPS35 expression and leads to impaired ATP7A related copper transport and copper overload in cardiomyocytes. Copper overload further leads to exacerbated cuproptosis and apoptosis, and these effects can be rescued by excision of the sympathetic nerve or administration of copper chelating agent. Our study elucidates one of the molecular mechanisms by which sleep disorders aggravate myocardial injury and suggests possible targets for intervention.