More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell “spread” of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca2+ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca2+ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca2+ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca2+ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.Excitotoxicity is classically attributed to Ca However, recent studies have identified several additional complexities that challenge this classical view and identify novel ways to suppress excitotoxic neuronal death. These complexities arise from interactions between superoxide and nitric oxide, the sources of superoxide formation, and the newly appreciated role of non-ionotropic NMDA receptor (NMDAr) signaling.The term “excitotoxicity” was first used in reference to rapid neuronal death caused by glutamate receptor activation . The ter2–), by contrast, is polar, largely lipid impermeable, and has much shorter half-life and mean diffusion distance. Nitric oxide is generated in a subset of neurons by neuronal nitric oxide synthase. It is considered a reactive oxygen species (ROS) because it contains an unpaired electron, but it is not intrinsically a highly reactive molecule in biological systems. Nevertheless, studies using both pharmacological and genetic abrogation of nitric oxide production showed near-complete suppression of excitotoxic neuronal death (An intriguing aspect of excitotoxic cell death is that it requires the combined effects of two independently generated “gaseous” reactive oxygen species: nitric oxide and superoxide. Nitric oxide (NO) is non-polar, lipid permeable, and has a relatively long half-life and diffusion distance in brain . Superoxal death , 1996.–)  . Peroxyn–) . Though –) . Increas–) . Only wi+ by electrons from mitochondrial dehydrogenases or electron transport complexes . NOX is a membrane-associated enzyme that transfers an electron from NADPH on one side of the membrane to molecular oxygen on the other side, forming O2– . NOX was2– . NOX is  subunit . Electro subunit .phox subunit . Accordingly, NMDAr-induced superoxide production and cell death can be blocked with the PI3K inhibitor wortmannin, and this blockade can be circumvented with either exogenous phosphoinositol-3-phosphate or with a constitutively active form of PKCζ  was originally designed to block NMDAr-induced nitric oxide production by displacing GluN2B from the PSD95, with which neuronal nitric oxide synthase is associated . Measure2+ dependent remains unresolved. Other Ca2+-dependent NMDAr signaling events have been shown to be mediated by calcium/calmodulin-dependent protein kinases  do not produce nitric oxide production, superoxide production, or excitotoxic cell death , thus suggesting a mechanism coupling non-ionotropic signaling to NOX2 activation.“Non-ionotropic” receptor signaling refers to signal transduction events triggered by transmembrane conformational changes rather than by ion flux. Non-ionotropic signaling by NMDAr was first identified by Westbrook and colleagues, who found that agonist binding independent of ion flux could downregulate GluN1/GluN2A receptor function through a process involving dephosphorylation of GluN1 tyrosine residues . Subsequmulation . Crucial+ influx . Ligand +, Ca2+, and other ions and molecules (2+ and Na+ influx essentially amplify the elevations caused by ion flux through the NMDAr itself. A synthetic peptide that prevents pannexin-1 channel activation by preventing its phosphorylation by Src kinase attenuates NMDAr-induced Ca2+ elevations and neuronal death (A second and apparently independent role of metabotropic signaling in excitotoxicity involves pannexin channel opening. When activated, pannexin ion channels become permeable to Naolecules . Thompsoal death , 2016. T2+, superoxide, and nitric oxide in excitotoxicity have stood the test of time, but subsequent observations have identified complexities that both expand our understanding of this process and open additional questions. Among these complexities is that elevations in either superoxide or nitric oxide levels can drive production of peroxynitrite, such that it is rarely possible to ascertain which one of these ROS is driving excitotoxic injury. It has also been demonstrated that superoxide production induced by NMDAr stimulation is generated primarily by NOX2, rather than mitochondria, but it remains uncertain whether this superoxide signal may be amplified by resultant mitochondrial dysfunction. The identification of NOX2 as the primary source of superoxide and provides an explanation for the extracellular actions of superoxide in excitoxicity, and also indicates the existence of a signaling cascade linking NMDAr to NADPH oxidase. This signaling cascade has been shown to include Ca2+ influx, PI3K activation, and PKCζ activation, but the nature of the Ca2+-dependent step remains to be established. Notably, Ca2+ influx alone is not sufficient to induce superoxide formation or excitotoxicity, and recent studies suggest that these processes also require concomitant non-ionotropic signaling induced by agonist binding to NMDAr.The early observations that identified key roles for CaRS and JW both contributed to the literature research and writing for this review. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TSPAN5 were associated with plasma serotonin (5-HT) concentrations which were themselves associated with selective serotonin reuptake inhibitor treatment outcomes in patients with major depressive disorder (MDD). TSPAN5 SNPs were also associated with alcohol consumption and alcohol use disorder (AUD) risk. The present study was designed to explore the biological function of TSPAN5 with a focus on 5-HT and kynurenine concentrations in the tryptophan pathway. Ethanol treatment resulted in decreased 5-HT concentrations in human induced pluripotent stem cell (iPSC)-derived neuron culture media, and the downregulation of gene expression of TSPAN5, DDC, MAOA, MAOB, TPH1, and TPH2 in those cells. Strikingly, similar observations were made when the cells were treated with acamprosate—an FDA approved drug for AUD therapy. These results were replicated in iPSC-derived astrocytes. Furthermore, TSPAN5 interacted physically with proteins related to clathrin and other vesicle-related proteins, raising the possibility that TSPAN5 might play a role in vesicular function in addition to regulating expression of genes associated with 5-HT biosynthesis and metabolism. Downregulation of TSPAN5 expression by ethanol or acamprosate treatment was also associated with decreased concentrations of kynurenine, a major metabolite of tryptophan that plays a role in neuroinflammation. Knockdown of TSPAN5 also influenced the expression of genes associated with interferon signaling pathways. Finally, we determined that TSPAN5 SNPs were associated with acamprosate treatment outcomes in AUD patients. In conclusion, TSPAN5 can modulate the concentrations of 5-HT and kynurenine. Our data also highlight a potentially novel pharmacogenomic mechanism related to response to acamprosate.We previously reported that SNPs near  TSPAN5 eQTL SNPs on chromosome 4 were associated with variation in plasma serotonin (5-HT) concentrations which were themselves correlated with selective serotonin reuptake inhibitor treatment outcomes in patients with major depressive disorder (MDD) [trans-eQTLs in the brain for TSPAN5 which maps to chromosome 4. When the UK Biobank study results were stratified by sex, the rs114026228 SNP in TSPAN5 (p = 3.60E−13) was the top signal associated with alcohol consumption in men [TSPAN5 SNPs were associated with alcohol use disorder (AUD) risk in an African–American population [TSPAN5 rs11947402 SNP which was originnally identified from our GWAS for baseline plasma 5-HT concentrations in MDD patients was also associated with AUD risk (p = 0.017) in that same AUD GWAS meta-analysis [TSPAN5 with a focus on the tryptophan pathway using human iPSC-derived CNS cells exposed to either ethanol (EtOH) or acamprosate—an FDA approved medication for the treatment of AUD [We previously reported that er (MDD) . We alsoer (MDD) . It shouer (MDD) –4. A rect of AUD .TSPAN5 is one of the 33 members of the tetraspanin gene family [TSPAN5 is widely expressed in the brain based on the GTEx database [TSPAN5 in AUD is unknown. Since our previous report demonstrated that TSPAN5 is associated with plasma concentrations of 5-HT—a metabolite of tryptophan—the present study places a focus on the impact of TSPAN5 on the tryptophan metabolic pathway, one branch of which leads to the formation of 5-HT, with the other main branch resulting in the formation of kynurenine  related pathways. Those experiments were followed by a series of functional genomic studies using astrocytes and microglia which showed that TSPAN5 might have a role in CNS immune response. Finally, we found that several SNPs that are cis-eQTLs for TSPAN5 were associated with acamprosate treatment outcomes in patients with AUD.e family . TSPAN5 database . Howeverine Fig. . During TSPAN5 and plasma 5-HT regulation [TSPAN5 can modulate and influence major metabolites of tryptophan and the CNS immune response—both of which have been implicated in neuropsychiatric disorders, including AUD [TSPAN5 in acamprosate treatment response. As a result, they have expanded and broadened our understanding of acamprosate’s mechanism of action.In summary, the present study greatly extends our original observations with regard to gulation , and serding AUD . Taken tThe Mayo Clinic Center for the Individualized Treatment of Alcoholism recruited 442 AUD subjects with associated clinical data, and DNA samples were obtained for genotyping –16. SpecFibroblasts from skin biopsies for all subjects were utilized for iPSC reprogramming using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit . Patient-derived iPSCs were characterized as previously described , 19. ThoRNA-seq was performed by GENEWIZ using an Illumina HiSeq 4000 platform. Fastq files containing paired RNASeq reads were aligned with STAR  against DDC, TPH1, TPH2, MAOA, and MAOB, all of which play roles in 5-HT biosynthesis and metabolism  after the knockdown of TSPAN5 Fig. . Those fTSPAN5 might be associated with the length of abstinence until the first drink of alcohol during 3 months of acamprosate treatment for AUD patients enrolled in the Mayo Clinic Center for the Individualized Treatment of Alcoholism clinical trial  were in tight linkage disequilibrium (LD) (R2 > 0.98) and were associated with length of abstinence during 3 months of acamprosate treatment. Those same three SNPs were also associated with a different but related clinical phenotype, i.e. abstinence length until heavy drinking or complete abstinence during 3 months of acamprosate treatment  which includes 134 brains from individuals free of neurodegenerative disorders. These results, taken together, indicate that genetic variants that are associated with TSPAN5 expression might be biomarkers for abstinence length in AUD patients treated with acamprosate.We next set out to determine whether SNPs within ent Fig. . In addient Fig. . FinallyTSPAN5 SNP genotypes appeared to be associated with acamprosate treatment outcomes in AUD patients—although those observations will require replication. This series of results represent a potentially important step in the process of obtaining functional insight into molecular mechanisms underlying the role of TSPAN5 in the regulation of two major tryptophan pathway metabolites, 5-HT and kynurenine, as well as individualized treatment outcomes for AUD patients treated with acamprosate.In summary, the results of this series of experiments suggest that TSPAN5 may play a significant role in 5-HT and kynurenine regulation and metabolism. Both of these tryptophan metabolites have been reported to play important roles in neuropsychiatric disorders , 31–34. TSPAN5 genetic variation might be associated with acamprosate treatment outcomes in AUD patients. AUD is the most prevalent substance use disorder [The present study provides the first evidence that disorder . Howeverdisorder , 36, 37.Acamprosate, an NMDA glutamate receptor antagonist, is a synthetic compound with a chemical structure similar to those of the neurotransmitter gamma-aminobutyric acid (GABA) and the amino acid taurine . The AUDTSPAN5 is an alcohol responsive gene that plays a role in the regulation of 5-HT and kynurenine concentrations. Both EtOH and acamprosate decreased TSPAN5 expression, ultimately leading to decreased 5-HT concentrations in cell culture medium. These molecular mechanism(s) could be multifactorial in nature as a result of acamprosate’s effects on neurotransmission, neuroinflammation and/or intracellular signaling in AUD patients. The functional genomic data from our studies of iPSC-derived CNS cells have opened a new avenue for understanding the biological role of TSPAN5 in AUD. We utilized iPSC-derived astrocytes which are expandable to perform a series of functional genomic studies, studies that required a large number of cells. Specifically, we performed gene expression profiling before and after TSPAN5 knockdown in iPSC-derived astrocytes and found that genes associated with changes in expression after the knockdown of TSPAN5 were enriched in IFN signaling pathways. We also determined the possible influence of TSPAN5 on levels of 5-HT and kynurenine, two major tryptophan metabolites that have been implicated in a variety of neuropsychiatric disorders [isorders , 32, 34.isorders . HoweverIt should be pointed out that the concentrations of acamprosate used in our cell culture studies were within the range of acamprosate concentrations observed in AUD patients treated with acamprosate . The celThe present study used human iPSC-derived CNS-like cells to perform functional genomic studies. Obviously, iPSC-derived cell lines, like any cell lines, have limitations. For example, it should be emphasized that iPSC-derived CNS cells are “region-specific”. The present study utilized forebrain-specific glial and neural cells, cells that have been implicated in the pathophysiology of AUD , 48. HowTSPAN5 were enriched in IFN signaling pathways. Our data also raise the possibility that TSPAN5, which appears to play a functional role in CNS immune response, might also contribute to individualized acamprosate treatment outcomes through a novel pharmacogenomic mechanism.In summary, the present study has highlighted the fact that TSPAN5 can regulate the two major metabolites of the tryptophan metabolic pathway as well as CNS immune responses. Specifically, genes that were modulated by Supplementary TextSupplementary Figure and Table legendsSupplementary FiguresSupplementary Tables


Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression  The symptoms and markers for CAVD manifest via both degenerative (apoptotic) and active (osteogenic) mechanisms6. Aortic valve endothelial and interstitial cells differentiate into an osteoblast-like phenotype, the extracellular matrix becomes thicker and stiffer and calcium mineralization occurs throughout the tissue8. Aortic stenosis and sclerosis have an increased prevalence in the elderly and contribute to a 50% elevated risk of infarction and other potentially fatal cardiovascular pathologies10. Currently, valve replacement is the preferred treatment method, as other strategies, such as the retardation of calcific progression, prevention and early diagnosis, are non-existent11. Diagnostic techniques like echocardiography, cardiac MRI and cardiac CT are widely used, but are only sensitive during later stages of the disease once there is tissue mineralization, and hemodynamic and geometric impairment13.Calcific aortic stenosis or calcific aortic valve disease (CAVD) is a progressive disease involving multiple signaling pathways, endothelial dysfunction, cytokine infiltration, collagen remodeling, as well as lipid and calcium deposition14. VICs exist in a quiescent state under healthy conditions and are activated due to injury or disease15, potentially differentiating into an osteogenic-like phenotype to potentiate calcification2. Current in vitro biochemical techniques to assess CAVD are typically destructive as they involve cell lysis or fixation and do not facilitate the longitudinal assessment of CAVD progression over time. That is, there is a dearth of nondestructive, label-free mechanisms to study the structural functional and phenotypic changes occurring in VICs during CAVD progression. Gaining deeper insights into the optical metabolic changes in VICs during disease pathogenesis would thus aid in the development of potential non-invasive tools to track CAVD progression in vitro.Aortic valve interstitial cells (VICs) are the primary cells in the heart valves and are involved in tissue maintenance, repair and remodeling17, which can be detected and quantified using two-photon excited fluorescence (TPEF) microscopy to determine cellular metabolic state through an optical redox ratio (ORR) of FAD/(NADH + FAD)19. NADH autofluorescence can also be used to assess the mitochondrial organization by determining mitochondrial fractal dimension (FD)20. Previously, TPEF was used to monitor the progression of osteogenic differentiation in human mesenchymal stem cells (MSCs)22 and ORR and mitochondrial FD was shown to correlate with the osteogenic differentiation of human MSCs18. Recently, TPEF imaging was utilized in vitro and on ex vivo tissue explants to detect mineralization, a key hallmark of CAVD23. In the context of VICs, we have previously shown that ORR correlated with cellular proliferative potential when VICs were cultured under different media conditions24. We have also previously reported that an increase in pathological stretch reduced the ORR in VICs, suggesting that the involved signaling pathways and VIC pathological function are closely linked to the overall cellular metabolic state24. However, TPEF imaging metrics - ORR and mitochondrial clustering have not yet been shown to predict or correlate with the pathological changes in VICs during early CAVD progression.Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are metabolic cofactors that play a key role in carrying electrons from the tricarboxylic acid cycle (TCA cycle) to the electron transport chain in mitochondria. These two molecules exhibit natural autofluorescencein vitro.The objective of this study was thus to investigate the potential of ORR and mitochondrial organization as label-free markers for tracking early CAVD progression. We seeded porcine aortic VICs as monolayers in quiescent versus osteogenic media on two dimensional soft or stiff substrates. We examined these samples using TPEF microscopy to quantify ORR and mitochondrial FD and simultaneously characterized the CAVD progression in our model using traditional end-point biomarkers, such as calcific nodule quantification, gene expression, cell proliferation and apoptosis. We then correlated ORR and FD with the aforementioned VIC structural and biological metrics. Our results showed that TPEF metrics correlated with the early markers of CAVD progression and thus suggest that TPEF microscopy can be utilized as a label-free non-destructive tool for assessing CAVD progression in vitro9. In our study, VICs cultured under quiescent conditions showed little to no calcium-rich nodules  and these correlations were found to be stronger in stiffer matrices especially in the osteogenic conditions and at later time points  for all conditions and time points but showed the strongest correlations at in early osteogenic VICs   and osteocalcin (OCN)2. ORR also correlated with TGFβR1 , OPN , OCN  and RUNX2  gene expression in early osteogenic VICs. Though RUNX2 had stronger correlation with ORR on stiffer substrates while not showing any correlations on compliant substrates, OCN correlated with ORR much stronger on compliant substrates especially in the osteogenic conditions , OPN , OCN  and RUNX2  gene expression for days 1–7 and with ACTA2 , RHOA  and OCN  gene expression for days 14–28 for all groups  Table . Additions Table . Finallyps Table . FD showps Table .Figure 333. We have also shown that VIC aspect ratio under normal and osteogenic culture conditions correlates with nuclear aspect ratio33. In the current study, ORR maps were utilized to assess nuclear area . It has been previously demonstrated that changes in ORR can occur before changes in mitochondrial organization within the cell18, and our results support this concept as well. Additionally, we also reported a correlation between osteogenic gene expression and ORR and FD as seen in prior studies18, suggesting the possibility of using TPEF metrics to predict the CAVD disease process.ORR and FD were previously shown to correlate with the osteogenic differentiation of mesenchymal stem cells25, and thus correlated well with ORR during early timepoints. Additionally, RUNX2 and OCN correlating with ORR based on stiffness, further supported the mechanosensitive nature of RUNX2 signaling in VICs. RUNX2 and OCN also correlated with VIC proliferation index, suggesting that osteogenic cells tended to be more proliferative. In the context of the above results, it is also important to note that while our quiescent media and osteogenic media formulations contain differing amounts of FBS, previous reports have rigorously characterized these specific media formulations to maintain quiescent and osteogenic cells, respectively39. Our results also agree with those accounted previously that demonstrated that quiescent VICs are less proliferative than VICs in 10% FBS containing osteogenic media40. Future studies will focus on the effect of individual media formulations and FBS concentrations on TPEF metrics and cell proliferation.Digging deeper into our results, we observed that TGFβR1 expression significantly correlated with ORR, which was expected given that TGFβ1 signaling has a major role in inducing disease during valve calcification. RUNX2 expression is known to predict the early osteogenic lineage of the cell41. In our study, OPN and OCN correlated with the ORR, specifically for the early timepoints. While TGFβR1, RUNX2, OPN, and OCN gene expression all were highly correlated with ORR for early timepoints, OPN expression showed the strongest correlation coefficient. OPN did not show any correlations to ORR in late time points, but correlated well with osteogenic cultures under both stiff and compliant cultures at early timepoints. Taken as a whole, our results suggest that the correlation of ORR with OPN can potentially be exploited as a predictor of early metabolic changes during CAVD progression and serve as a biomarker for early disease41.OPN expression has been known to be an early biomarker for CAVD and has been used as a serum marker to study the progression of the disease when it is asymptomatic30. We have previously shown that elongated cells had higher expression of proliferation and αSMA expression19. Dystrophic calcification has been known to be associated with apoptosis and prolonged αSMA activation in VICs30. In our study, ACTA2 did not correlate with ORR but significantly correlated with mitochondrial FD. Stronger correlations were observed between ACTA2 and FD in osteogenic VICs on stiffer substrates, especially at later time points. This suggests that the role played by ACTA2 in stress fiber formation may also affect the regulation of the arrangement of mitochondria in adjusting to the energy demands of the cell under altered stiffness and osteogenesis. Stiffness dependent correlations of TGFβR1 with FD at early timepoints may suggest its role in triggering the aforementioned ACTA2 mediated mitochondrial arrangement32. Additionally, the RHOA gene, which codes for a mechanotransductive protein that regulates the formation of nodules42, also correlated with nodule apoptotic index. RHOA has been known to be activated and expressed at higher levels in stiffer matrices and promote the upregulation of RUNX243. Like ACTA2, RHOA did not correlate with ORR but significantly correlated with mitochondrial FD. RHOA has been shown to increase ACTA2 expression, ultimately increasing stress fiber formation31. This sequence of events is in line with our results that showed that RHOA gene expression correlated with FD at earlier time points, whereas ACTA2 gene expression correlated with FD for later time points.Alpha SMA organizes into stress fibers and affects stiffness, force generation, and cell shape33, the VICs in osteogenic conditions tended to have a lower nuclear aspect ratio, reinforcing the idea that osteogenic cells have reduced mitochondrial clustering. Disorganization of mitochondria has previously been associated with other cardiac ailments44, but the specific functional role of mitochondrial clustering in valve pathophysiology is yet to be elucidated. It is also not fully understood if the increased area and decreased AR of nuclei in osteogenic VICs is a cause or effect of the disorganization of mitochondria.Due to the impact of VIC shape on cellular function, changes in nuclear area and aspect ratio were measured to provide context for the differences we observed in mitochondrial FD. These measures significantly correlated with mitochondrial FD, with nuclear area positively correlated with FD, and aspect ratio negatively correlated with FD. This suggests that mitochondrial clustering decreased with an increase in nuclear area and a decrease in nuclear aspect ratio. We observed an increase in nuclear area and FD in osteogenic VICs, suggesting these cells had decreased mitochondrial clustering. Similar to our previous studyin vitro. Future studies will dig deeper into the exact metabolic changes that occur during CAVD progression and how they relate to observed alteration in measured TPEF metrics, and whether any of these techniques can be translated in vivo.Overall, the correlations we observed between nuclear morphology, gene expression and the TPEF metrics of ORR and FD, suggest that VIC phenotype and shape have an effect on the metabolic state of the cell and its mitochondrial arrangements. This study showed that TPEF microscopy and its derived metrics can be used to assess pathophysiological changes in the structure, function and phenotype of VICs undergoing osteogenic differentiation, suggesting that TPEF imaging markers can serve as a label-free, non-destructive tool to study the cellular changes occurring during CAVD progression 33. Briefly, porcine hearts were acquired from a local abattoir and aortic valves were utilized to aseptically isolate the valve interstitial cells (VICs). Human fibronectin  at a concentration of 100 μg/ml was coated on stiff or compliant PDMS-coated coverslips , 10 ng/ml FGF-2 and 50 ng/ml insulin)36 for 14 days before seeding onto the coverslips. These quiescent VICs from passages three to seven were then seeded at 500,000 cells/coverslip and cultured in either quiescent or pro-osteogenic  conditions33 for 1, 3, 7, 14, 21, and 28 days before analyses were performed as outlined below.Standard cell culture procedures were followed as published earlier45. The samples were then washed in deionized water and brightfield images were acquired using a Nikon Eclipse (Ci-S) microscope in conjunction with NIS Elements software.Three to four fields of view (FOV) from five to six biological replicates per time point per condition were imaged using brightfield phase contrast microscopy before they were utilized for other assays at the 1, 3, 7, 14, 21, and 28-day time points. An image editing software, Fiji Image J  was used to manually count and measure the number and area of nodules per field of view. At the 28-day time-point, the samples were fixed in 4% paraformaldehyde  with 1% Triton X-100 (Electron Microscopy Sciences) and incubated with 40 mM Alizarin Red S  solution for 30 minutes with gentle shaking46. PMT gain and laser power were kept constant and laser power was read after each imaging session19. A custom MATLAB code was used to generate redox images by pixel-wise computation of fluorescence intensities as FAD/(NADH + FAD) after normalization. Optical redox ratios (ORR) were calculated using averaged and normalized field intensities of FAD and NADH images as FAD/(NADH + FAD). NADH fluorescence intensity maps were utilized to assess the spatial arrangement of mitochondria using a custom MATLAB code. Specifically, a modified blanket method was utilized to obtain the mitochondrial fractal dimension, a metric defining the closeness of mitochondrial arrangement, on a pixel-by-pixel basis20. At least two to four fields of view were taken from five to six separate samples per time point, and per treatment group19.At the aforementioned time points, separate samples were transferred to a heated microincubator chamber (37 °C) and media exchanged with 2 ml Tyrode’s salt solution buffer (Sigma-Aldrich). A Bruker Ultima Investigator laser scanning microscope  inverted for cell culture imaging and equipped with an ultrafast Ti:Sapphire tunable laser source  was used to acquire images via a water immersion objective   and non-descanned GaAsP photomultiplier tubes (PMT) . NADH fluorescence was captured with a 460 (±20) nm band pass filter at 755 nm excitation, and FAD fluorescence with a 525 (±25) nm band pass filter at 860 nm excitation. NADH and FAD autofluorescence intensities were normalized by PMT gain and laser power, with PMT gain normalized to fluorescein concentrations (0.1 µM to 20 µM in Tris Buffer of pH 8) as in previous studies33. Briefly, a QIAGEN RNeasy Plus assay kit  was utilized to isolate and purify total mRNA from VIC samples. The mRNA was then reverse transcribed to cDNA using iScript RT supermix . QRT-PCR was performed using the SsoAdvanced Universal SYBR Green supermix (Bio-Rad) in a CFX96 real-time system (BIO-RAD) to quantify the relative expression of genes  was used to quantify gene expression of ACTA2, RHOA, TGFβR1, OPN, OCN, and RUNX2 using previously published methods33. Average nuclear area and aspect ratio per treatment group per condition were then calculated and correlated to the average ORR and FD for that treatment group.ORR maps were additionally used to quantify nuclear aspect ratio and area. Five to eight cells per image field were analyzed using Fiji Image J using previously published methodsSeparate samples were fixed using 4% paraformaldehyde (Electron Microscopy Sciences) with 1% Triton X-100 (Electron Microscopy Sciences), blocked with 10% goat serum , and were immunolabeled with antibodies against either cleaved caspase 3  to assess apoptosis, or Ki67  to assess proliferation. Samples were then fluorescently labelled with appropriate Alexa Fluor-594 conjugated  secondary antibody and 4′,6-diamidino-2-phenylindole  to stain the nuclei. The samples were mounted onto glass coverslips using Prolong Gold antifade agent (Life Technologies) and imaged using a Nikon epifluorescence microscope in conjunction with NIS Elements software. Technical controls were utilized to assess specificity of the secondary antibody . Normally distributed data was analyzed using three-way analysis of variance (ANOVA) and Tukey’s post-hoc tests. For the analysis of ORR, FD, nuclear area and aspect ratio, each FOV was nested within the sample and each sample was nested within the stiffness and media condition. Both sample and FOV were considered a random variable. ANOVA on ranks with Dunn’s multiple comparisons test was used for non-parametric data. A comparison was considered significant if its p-value was less than 0.05. Pearson’s correlation coefficient was used for the correlation analysis. Significant correlations were defined based on a null hypothesis of uncorrelated data (R = 0). If the data was non-normal, Spearman’s rank correlation coefficient was used. All the statistical and correlation analyses was performed using JMP and graphing was performed using SigmaPlot (Systat Software Inc) software.Supplementary Information.


This exploratory analysis of a randomized clinical trial evaluates the association of bone metastasis count and location with survival benefit from prostate radiotherapy in patients with newly diagnosed metastatic prostate cancer. Are bone metastatic burden and site associated with survival benefit from the addition of prostate radiotherapy (RT) to standard-of-care systemic therapy in newly diagnosed metastatic prostate cancer?This exploratory analysis of 1939 participants in a randomized clinical trial shows that survival benefit following prostate RT gradually diminished with increasing bone metastasis number, with survival benefit most pronounced in patients with up to 3 bone metastases. Prostate RT was associated with greater overall and failure-free survival in patients with only nonregional lymph node metastasis (M1a) or 3 or fewer bone metastases without visceral metastasis.In patients with prostate cancer, bone metastatic burden and metastasis location may be useful in predicting survival benefit from prostate RT. Prostate radiotherapy (RT) improves survival in men with low-burden metastatic prostate cancer. However, owing to the dichotomized nature of metastatic burden criteria, it is not clear how this benefit varies with bone metastasis counts and metastatic site.To evaluate the association of bone metastasis count and location with survival benefit from prostate RT.This exploratory analysis of treatment outcomes based on metastatic site and extent as determined by conventional imaging (computed tomography/magnetic resonance imaging and bone scan) evaluated patients with newly diagnosed metastatic prostate cancer randomized within the STAMPEDE trial’s metastasis M1 RT comparison. The association of baseline bone metastasis counts with overall survival (OS) and failure-free survival (FFS) was assessed using a multivariable fractional polynomial interaction procedure. Further analysis was conducted in subgroups.Patients were randomized to receive either standard of care (androgen deprivation therapy with or without docetaxel) or standard of care and prostate RT.The primary outcomes were OS and FFS.P = .003; HR for FFS, 0.87; 95% CI, 0.76-0.99; interaction P = .002).A total of 1939 of 2061 men were included ; 1732 (89%) had bone metastases. Bone metastasis counts were associated with OS and FFS benefit from prostate RT. Survival benefit decreased continuously as the number of bone metastases increased, with benefit most pronounced up to 3 bone metastases. A plot of estimated treatment effect indicated that the upper 95% CI crossed the line of equivalence  above 3 bone metastases without a detectable change point. Further analysis based on subgroups showed that the magnitude of benefit from the addition of prostate RT was greater in patients with low metastatic burden with only nonregional lymph nodes (M1a) or 3 or fewer bone metastases without visceral metastasis  than among patients with 4 or more bone metastases or any visceral/other metastasis (HR for OS, 1.08; 95% CI, 0.91-1.28; interaction In this exploratory analysis of a randomized clinical trial, bone metastasis count and metastasis location based on conventional imaging were associated with OS and FFS benefit from prostate RT in M1 disease.NCT00268476; ISRCTN.com Identifier: ISRCTN78818544ClinicalTrials.gov Identifier:  Also, owing to the historical nature of setting criteria for metastatic burden, the role of prostate RT in treatment for men presenting with only nonregional lymph node (NRLN) or visceral metastases has not been reported. Herein, we use data from the STAMPEDE trial’s M1 RT comparison1 to explore the association of bone metastatic burden and the influence of isolated or concomitant nodal or visceral metastatic sites with treatment outcome following RT.Two randomized clinical trials, HORRAD and STAMPEDE, confirmed that prostate radiotherapy (RT) improves survival in newly diagnosed, low-metastatic-burden prostate cancer.1 Briefly, patients with newly diagnosed mPCa and no contraindication to RT were randomized 1:1 to SOC or SOC plus prostate RT. Patients underwent baseline staging imaging per study protocol prior to randomization. Metastatic sites at baseline were evaluated by conventional imaging (bone scan and computed tomography/magnetic resonance imaging). Pretreatment bone scans were centralized, and metastasis counts were analyzed retrospectively. Reviewers were blinded to treatment allocation and outcomes as previously reported.1 The SOC comprised lifelong androgen deprivation therapy (ADT), with up-front docetaxel permitted in patients randomized after December 17, 2015. Where used, docetaxel was planned as six 3-week cycles of 75 mg/m2 with or without prednisolone, 10 mg, daily. Patients allocated to RT received either 55 Gy in 20 daily fractions over 4 weeks or 36 Gy in 6 weekly fractions over 6 weeks. The schedule was nominated before randomization. All patients provided written informed consent. The trial is registered at ClinicalTrials.gov (NCT00268476) and ISRCTN.com (ISRCTN78818544) and had full regulatory, national ethics committee, and local site approval. Full details of the STAMPEDE trial can be found at http://www.stampedetrial.org, and the trial protocol is in Patients randomly allocated to the STAMPEDE trial’s M1 RT comparison were eligible for study. The first efficacy results from this comparison have been published previously.1 Biochemical failure was defined as a rise in prostate-specific antigen (PSA) level of 50% greater than the lowest reported PSA level within 24 weeks of enrollment and greater than 4 ng/mL ; patients without a decrease of 50% were considered to have biochemical failures at time zero. Patients without the event of interest were censored at the time last known to be event free. Secondary outcomes are described in eMethods in 1The STAMPEDE trial comparison’s primary definitive and intermediate outcome measures were overall survival (OS) and failure-free survival (FFS), and we focus on these outcome measures. Overall survival was defined as the time from randomization to death from any cause, and FFS as the time from randomization to the first of: biochemical failure; progression locally, in lymph nodes or in distant metastases; or death from prostate cancer.P value from a likelihood ratio test of the interaction between treatment group and bone metastasis count is presented. The MFPI model–estimated treatment effect as a function of bone metastasis count was plotted graphically on the HR scale with 95% CI. Further details regarding the MFPI have been published previously.14 Sensitivity analysis was also undertaken using Cox models adjusted for selected clinically relevant baseline variables: age, pre-ADT PSA level, World Health Organization performance status, T stage, Gleason score, N stage, planned docetaxel use, nominated RT schedule, and metastatic sites.All analyses conducted herein are exploratory. To evaluate whether treatment effect varied by bone metastasis count, a multivariable fractional polynomial interaction (MFPI) algorithm using a nested Cox regression model was performed. Cox models were adjusted for minimization factors used at randomization: age (<70 or ≥70 years), N stage (N0/N+/NX), World Health Organization performance status (0 or 1-2), nonsteroidal anti-inflammatory drug or aspirin use (either or no), and planned docetaxel use (yes/no), along with metastatic site . Models with first-degree fractional polynomial, second-degree fractional polynomial, and linear functions of bone metastasis counts were evaluated, and the interaction model with the lowest Bayesian information criterion and Akaike information criterion was chosen , 3 or fewer and 4 or more bone metastases (±NRLN), and any visceral/other metastasis. Balance regarding baseline characteristics between treatment arms was evaluated within each subgroup. Kaplan-Meier (KM) estimates were used to plot survival curves, and relative treatment effects were evaluated using Cox models within the subgroups. Finally, based on information obtained from the previous steps, a metastatic burden classification was devised and evaluated as detailed in eMethods in Following exclusion of patients undergoing nonconventional imaging (n = 60) and where baseline bone scans could not be centralized (n = 62), baseline bone scans from 1939 of 2061 (94%) patients with newly diagnosed mPCa randomized between January 22, 2013, and September 2, 2016, in the STAMPEDE M1 RT comparison were included . BaselinUsing the MFPI procedure, the linear model had the lowest Bayesian information criterion and Akaike information criterion for both OS and FFS outcomes  and 171 patients with any visceral/other metastasis   and FFS . However, as bone metastasis counts in HORRAD were categorized into 1 to 4, 5 to 15 and more than 15, a lower cutoff of 3 bone metastases was not considered,3 highlighting the importance of evaluating such effects on a continuous scale.15 Additionally, some studies published previously have suggested that patients with any number of bone metastases confined to the vertebral column are considered low burden.12 However, in our study of nearly 2000 patients, less than 2% of patients had 4 or more bone metastases solely within the vertebral column. We could not explore treatment effects in such patients given the small numbers.How the treatment effect of prostate RT changes with baseline bone metastasis count is of clinical relevance for patient selection and future trial designs. The first analysis of the STAMPEDE M1 RT comparison16 Also, as NRLN metastatic burden has been shown to be prognostic,17 the role of metastatic NRLN metastasis counts as a predictive factor warrants additional investigation. Primary-site RT should therefore be considered as SOC in these men, who in the present study constituted 9% of the overall primary M1 caseload. By contrast, there was no evidence of benefit on FFS or OS in patients with visceral/other metastasis. Taken together, our study reinforces the predictive role of nonosseous metastatic sites within the metastatic burden criteria.Further exploratory analysis based on metastatic sites indicated survival benefit from prostate RT in patients with only NRLN metastasis (M1a) but not in those with visceral/other metastasis. There are currently no other randomized clinical trial data on the role of prostate RT in patients with M1a disease. We also showed a substantial prostate RT effect on FFS in M1a disease; the absolute improvement was 22% at 3 years. This is consistent with a previously reported nonrandomized analysis from STAMPEDE studying RT in N+M0 patients.18 All such criteria are based on the prognostic relevance of metastatic sites and their extent. In our study, we built upon these prognostic criteria to evaluate systematically the predictive nature of metastatic burden based on conventional imaging using bone scan and computed tomography/magnetic resonance imaging. We show that metastatic burden criteria are not just prognostic; they are predictive of survival benefit when primary-site prostate RT is used. The subgroup of patients with only M1a or 3 or fewer bone metastases (±NRLN) and without any visceral/other metastasis had an 8% estimated absolute survival benefit at 3 years, whereas patients with bone metastasis counts greater than this or with visceral/other metastasis had no such benefit.Currently, the definition of low metastatic burden is not agreed upon internationally; it includes a range of definitions based on metastasis number (<3 to <10), various sites , and different imaging modalities.19 Phylogenetic analysis of metastases in mPCa highlights complex metastatic cascades, wherein both primary-to-metastatic and metastasis-to-metastasis progression is identified.20 Based on this, we can hypothesize that treating the primary in low-burden disease may disrupt metastatic dissemination from the primary and delay metastatic progression. By contrast, with high burden, metastasis-to-metastasis progression could be the dominant mode of dissemination; treating the primary in this setting has minimal benefit. This hypothesis is supported by the observed heterogeneity in metastatic progression-free survival in the current study and the HORRAD trial.2 In our study, an absolute improvement of 7% in 3-year metastatic progression-free survival is observed with addition of prostate RT in patients with low-burden disease. Furthermore, a 2020 study21 using the same high-burden and low-burden criteria as devised herein demonstrated that patients with low-burden disease had a lower fraction of the genome altered, with lower genomic instability and fewer oncogenic alterations in the NOTCH, cell cycle, and epigenetic modifier pathways.Various biologically plausible reasons exist whereby prostate RT could delay metastatic progression and improve survival in patients with low metastatic burden.22 by evaluating bone scans using the automated bone scan index, which yielded similar results. Additionally, around 20% of patients in our study received docetaxel as their SOC. Currently, there is no evidence for combined use of RT and docetaxel nor of the effect of combining them with ADT in M1 disease. The value of prostate RT combined with abiraterone and docetaxel is currently being tested in the PEACE-1 trial (NCT01957436). Other ongoing trials are evaluating local treatment in combination with newer systemic therapies or metastasis-directed treatments. These trials can further validate metastatic burden as a predictor of survival benefit from local treatment. Finally, it is unclear how to translate these data to staging with newer imaging modalities using 68gallium-labeled ligands of the prostate-specific membrane antigen (known as 68Ga-PSMA) or whole-body magnetic resonance imaging, given that these emerging imaging modalities are more sensitive in detecting occult metastases. We emphasize that caution is required in extrapolating these data and cutoffs onto newer sensitive imaging modalities. These will need similar detailed studies to ascertain their true individual clinical relevance relative to their utility in predicting treatment outcome. This will be best evaluated prospectively within well-powered randomized clinical trials.Several caveats to this exploratory analysis require mention, including its retrospective nature. Although the data on quantitative bone metastatic burden were available for most of the patients, some patients had to be excluded because their scans could not be centralized. There was also a lack of information on quantitative lymph node and visceral metastasis, which may also be predictive. However, a sensitivity analysis conducted in patients with only bone metastasis did not alter the predictive nature of the bone metastatic burden. We are also conscious that counting bone metastasis is not an accurate representation of bone metastatic volume. We explored this further in a separate studyBone metastatic burden based on conventional imaging is predictive of OS and FFS benefit when prostate RT is added to SOC in newly diagnosed mPCa. This beneficial effect is most pronounced in patients with up to 3 bone metastases, below which addition of prostate RT to SOC improves survival in patients without visceral or other metastasis. The criteria for low metastatic burden based on conventional imaging, predictive of survival benefit from prostate RT in men with newly diagnosed mPCa, should now also include men with M1a disease.


Twiddler syndrome is an uncommon yet dangerous phenomenon usually resulting in lead displacement with loss of capture of cardiac implantable electronic devices. In this case report, we present an interesting case of Twiddler syndrome without lead dislodgment which was detected by an alert triggered by an increase in impedance on remote monitoring. Twiddler syndrome is a rare complication 0.07-1.1%) following the implantation of cardiac devices and has been observed with different types of cardiac devices .1% follo. Twiddle2. She was not known for a history of dementia or impaired cognition.A 75-year-old woman with a mixed ischemic/nonischemic cardiomyopathy and depressed left ventricular systolic function (ejection fraction of 25%) underwent the implantation of a single-chamber primary prevention ICD (Boston Scientific Autogen) in January 2017. Her past medical history was significant for multiple other cardiovascular comorbidities including stage III obesity with a body mass index of 41 kg/mpectoralis major in a new subcutaneous pocket with a suture in the header of the device to prevent any migration.An active fixation right ventricular (RV) lead (Boston Scientific Reliance 4-Front DF4) was implanted at the RV apex. Six months postimplantation, the RV lead pacing threshold and impedance increased from 0.7 V to 2 V and from 450 ohms to 800 ohms, respectively . It was This case report illustrates a Twiddler syndrome without lead dislodgment detected by an impedance lead alert from the remote monitoring system. Failure of the device is often the clinical manifestation of Twiddler syndrome , and thiIn a cohort of 1074 patients who underwent cardiac implantable electronic devices, lead macrodislodgment occurred in 19 1.8%) patients, and the vast majority (89%) of them were detected by a change in lead parameters on remote monitoring . Hence, % patientOur patient was an elderly woman with an increased BMI who fulfilled three well-known risk factors for Twiddler syndrome: increased BMI, female gender, and elderly patient , 9. ObesThis case report highlights the utility of remote monitoring for early detection of lead abnormalities or arrhythmia, which can be caused by rare device complications such as Twiddler syndrome. Measures to identify risk factors for Twiddler syndrome and fixation of the device at time of implant in high-risk patients can prevent this unusual complication.


Lymnaea neuron selectively releases its classical small molecule and peptide neurotransmitters, acetylcholine and FMRFamide-derived neuropeptides, to differentially influence the activity of distinct postsynaptic targets that coordinate cardiorespiratory behaviour. Using a combination of electrophysiological, molecular, and pharmacological approaches, we found that neuropeptide cotransmitter release was regulated by cross-talk between extrinsic neurotrophic factor signaling and target-specific retrograde arachidonic acid signaling, which converged on modulation of glycogen synthase kinase 3. In this context, we identified a novel role for the Lymnaea synaptophysin homologue as a specific and synapse-delimited inhibitory regulator of peptide neurotransmitter release. This study is among the first to define the cellular and molecular mechanisms underlying the differential release of cotransmitter substances from individual presynaptic terminals, which allow for context-dependent tuning and plasticity of the synaptic networks underlying patterned motor behaviour.Many neurons concurrently and/or differentially release multiple neurotransmitter substances to selectively modulate the activity of distinct postsynaptic targets within a network. However, the molecular mechanisms that produce synaptic heterogeneity by regulating the cotransmitter release characteristics of individual presynaptic terminals remain poorly defined. In particular, we know little about the regulation of neuropeptide corelease, despite the fact that they mediate synaptic transmission, plasticity and neuromodulation. Here, we report that an identified  The use of multiple neurotransmitters enhances a neuron’s capacity to encode information within a circuit, allowing for more complex patterns of synaptic interactions to emerge, and the opportunity to differentially influence the activity of synaptic partners over multiple timescales3. Many neurons form hundreds of presynapses, innervate a variety of postsynaptic targets, and modulate their transmitter output in response to a wide range of intracellular and extracellular signals4. There is accumulating evidence that a neuron’s individual presynaptic terminals can exhibit distinct cotransmitter composition and release characteristics, and that this can be dynamically regulated in order to meet the spatiotemporal functional requirements of each synapse13. However, our understanding of the cellular and molecular mechanisms that allow a neuron to regulate the use of its cotransmitters at distinct synaptic sites is considerably lacking.Most neurons use more than one type of neurotransmitter to mediate synaptic transmission14. Although peptide neurotransmitters are traditionally viewed as slow-acting neuromodulators released from perisynaptic sites in response to Ca2+ diffusion during high-frequency bursting15, their release has been demonstrated to occur over a wide range of firing frequencies18. Peptidergic large dense-core vesicles (LDCVs) have also been shown to cluster directly at presynaptic active zones and be released by single action potentials20. Furthermore, several studies have indicated that postsynaptic target identity influences the selective use of classical and peptide neurotransmitters at the different presynaptic terminals of a given neuron22. Taken together, these observations suggest that the peptidergic characteristics of cotransmitting terminals can be highly variable, although the molecular mechanisms that give rise to such differences are yet to be defined.The concurrent use of classical small molecule and peptide neurotransmitters is common throughout the nervous system and across evolution. Neuropeptides are important modulatory signals that regulate numerous aspects of synaptic development, plasticity and network function, as well as animal physiology and behaviourLymnaea stagnalis to study how the selective use of cotransmitters at synapses with distinct postsynaptic targets influences the assembly and function of the neuronal circuits involved in cardiorespiratory regulation. Reductionist approaches using the simple nervous systems of invertebrate models have enabled fundamental insights into cotransmission because their large identified neurons with known transmitter phenotypes participate in behaviourally-defined synaptic networks that can be studied directly in situ or reconstructed and manipulated in vitro24. Moreover, invertebrate models are especially valuable for functional studies on peptidergic transmission because they are one of the few systems in which electrophysiologically measurable synaptic responses can be directly attributed to the actions of identified peptide neurotransmitters at individual synapses.In this study we sought to identify the cellular and molecular mechanisms underlying presynaptic specificity, with a focus on two unresolved questions: (i) how does a neuron establish functionally distinct presynapses with specific postsynaptic targets, and (ii) how does a presynaptic neuron generate the appropriate patterns of cotransmitter release that are required for the coordinated actions of neurons in behaviourally-relevant networks? To address these outstanding questions, we used cardiorespiratory neurons from the central nervous system (CNS) of the invertebrate mollusc Lymnaea synaptophysin (Syp) homologue in the inhibitory regulation of peptide neurotransmitter release. These findings on the regulation of cotransmitter use at individual synapses uncover a previously undefined mechanism for presynaptic cotransmitter specificity in synaptic networks, with implications for the appropriate expression and plasticity of patterned motor behaviours.Here, we found that presynaptic neuropeptide release competency was regulated in a target- and context-dependent manner. This involved an interplay between extrinsic neurotrophic factors (NTF) and synapse-specific retrograde arachidonic acid (AA) signaling interactions, which converged on glycogen synthase kinase 3 (GSK-3) activity. In this context, we identified a surprising role for the Lymnaea cardiorespiratory interneuron visceral dorsal 4 (VD4) was of interest for this study as it is well known to use the classical small molecule neurotransmitter acetylcholine (ACh) alongside a mixture of neuropeptides derived from the heptapeptide transcript of the FMRFamide gene 28. VD4 is integrated into a three-neuron network via reciprocal inhibitory synapses with the FMRFamidergic input 3 interneuron (IP3I) and the giant dopaminergic neuron right pedal dorsal 1 (RPeD1). These neurons and their reciprocal synapses establish Lymnaea’s respiratory central pattern generator (rCPG) network in vivo, are indispensable for the expression of respiratory behaviour, and recapitulate appropriate synaptic networks and rhythmic activity when reconstructed in vitro29. VD4 is also known to form one-way excitatory synapses with a number of other identified neurons that act to coordinate cardiorespiratory behaviour, including the FMRFamidergic visceral F group cells (VF) and the serotonergic neuron left pedal dorsal 1 (LPeD1)33 36. For FMRF neuropeptides, this is due to cell type-specific coupling of FMRF G protein-coupled receptors (GPCRs) to distinct G protein complexes and metabotropic signaling cascades38. FMRF neuropeptides have also been shown to occlude transmitter release from target neurons by inhibitory regulation of Ca2+ influx and neurosecretory mechanisms39. We therefore wondered whether the release of ACh and FMRF neuropeptides might be selectively tuned at VD4′s presynaptic terminals to establish the target-specific formation of one-way or reciprocal, and inhibitory or excitatory synapses that are required for the appropriate expression and modulation of cardiorespiratory behaviour.To investigate the mechanisms that differentiate cotransmitter use at individual synapses, and the consequences of this for specifying network function and behaviour, we first sought to monitor synaptic transmission by classical and peptide transmitters at functionally-defined synapses. The To investigate this question, we first performed simultaneous intracellular recordings from the isolated intact CNS to determine the classical vs peptidergic nature of synaptic transmission at endogenous synapses between VD4 and the identified cardiorespiratory neurons VF and RPeD1. Synaptic transmission was assessed in response to a ~ 4 s burst induced in VD4, first under control conditions to assay cotransmission, and then in the presence of an AChR antagonist cocktail see “” sectionBecause the identified cardiorespiratory neurons and their synaptic networks cannot be easily resolved or manipulated in the intact brain preparation, we turned to in vitro reconstruction of these synapses to uncover the regulatory mechanisms that specify transmission by classical and peptide neurotransmitters. To this end, we isolated the identified neurons from the CNS and paired VD4 in a soma-soma configuration with RPeD1 or VF in culture. As in the intact preparation, we made simultaneous intracellular recordings from cultured neurons, first under control conditions to study cotransmission, and then in the presence of AChR antagonists to isolate peptidergic transmission.40, we next cultured pairs in NTF-deficient defined media (DM) to ask whether regulated cotransmission is solely target dependent, or whether it might also be influenced by extrinsic factors. Surprisingly, we observed a reversal of peptidergic phenotype, where most VD4-VF pairs exhibited primarily cholinergic transmission, and most VD4-RPeD1 pairs exhibited mixed cotransmission , a NTF-rich media that recapitulates the in vivo environment, VD4-VF exhibited mixed cholinergic-peptidergic cotransmission, whereas most VD4-RPeD1 pairs exhibited primarily cholinergic transmission Fig. A-B. As Nion Fig. A-B. Analion Fig. B, while ion Fig. B, in linThe next question was whether the specificity of classical and peptide cotransmission would be observed if VD4 innervated multiple postsynaptic targets in vitro. We therefore cultured VD4 simultaneously with VF and RPeD1 in a triple-soma configuration in CM, and found that the VD4-VF synapse exhibited mixed cotransmission, while the VD4-RPeD1 synapse exhibited primarily cholinergic transmission Fig. A–B. Simi39, we hypothesized that the degree of FMRF neuropeptide transmission would be inversely correlated with the degree of dopamine (DA) transmission at the reciprocal inhibitory synapse between VD4-RPeD1. Therefore, in parallel with monitoring ACh and FMRF neuropeptide transmission from VD4, we also evaluated DA transmission from RPeD1 in the above experiments. Most RPeD1-VD4 synapses exhibited strong inhibitory DA transmission when cultured in CM, whereas in DM most did not . This suggests that the context- and target-dependent regulation of FMRF neuropeptide transmission observed above may reflect differences in VD4′s FMRF neuropeptide release propensity, such that high-releasing synapses would occlude DA release from RPeD1 and low releasing synapses would facilitate DA release. If so, this NTF-dependent regulation of neuropeptide release holds important implications for the assembly, plasticity, and function of the Lymnaea rCPG network, and how the rhythmic network activity required for breathing behaviour is established and modulated29.As FMRF neuropeptides have been shown to inhibit transmitter releasenot Fig. . When we2 moiety common to FMRF neuropeptides, we found a uniform intensity of somatic neuropeptide immunolabel amongst the culture conditions, suggesting that the translational expression of FMRF neuropeptides does not change  the translational expression of FMRF neuropeptides changes; (ii) FMRF neuropeptides are selectively targeted to specific presynaptic sites; or (iii) FMRF neuropeptides are selectively released from specific presynaptic sites. To explore these possibilities, we cultured VD4 neurons alone or with VF and/or RPeD1 postsynaptic targets in CM or DM, using an axon-axon configuration to facilitate visualization of synaptic sites. Using immunocytochemistry (ICC) and an antibody against the RF-NHnge Fig. A. In painge Fig. B. These 2+ sensor SVPs, considering that different isoforms selectively facilitate or inhibit small synaptic vesicle (SSV) and LDCV release characteristics46. This has also been reported in the invertebrate literature49, indicating that the use of alternative Syt isoforms as selective regulators of transmitter release is an evolutionarily conserved strategy. The second was the vesicle integral membrane protein synaptophysin (Syp), which reversibly complexes with the v-SNARE synaptobrevin (Syb) when phosphorylated, rendering Syb unable to enter the fusogenic SNARE complex52.We next sought to identify the molecular switch responsible for the selective facilitation and inhibition of peptidergic LDCV release. We focused on two synaptic vesicle protein (SVP)-dependent mechanisms that have been reported to selectively inhibit transmitter release. The first was the synaptotagmin (Syt) family of CaLymnaea homologues of Syt and Syp on cotransmitter release, we first identified and cloned them from a Lymnaea CNS cRNA library  activation, which transduce NTF signaling55. This implicates a model for context-dependent inhibitory regulation of neuropeptide release in which (i) GSK-3 activity is high in the absence of NTFs, causing phosphorylation of Syp and enhancing formation of the Syp-Syb complexes that inhibit LDCV fusion, and (ii) GSK-3 activity is low in the presence of NTFs, reducing Syp phosphorylation and promoting Syp-Syb dissociation that promotes neuropeptide release.We next sought to identify the molecular signaling cascades that define FMRF neuropeptide release characteristics, reasoning that it likely involves synapse-delimited post-translational modification (PTM) of Syp. GSK-3 has been shown to promote Syp-Syb complexing via phosphorylation of SypTo test this experimentally, we cultured VD4-VF and VD4-RPeD1 pairs in CM or DM, in the presence of a GSK-3 inhibitor (5 μM SB 216763) or vehicle control (0.1% DMSO). When cultured in CM, VD4-VF peptidergic transmission was not impacted by GSK-3 inhibition, while in DM, GSK-3 inhibition induced peptidergic transmission Fig. A. This s56. In particular, the generation of arachidonic acid (AA) metabolites has been linked to the actions of FMRF neuropeptides58, and was previously implicated in the function of reciprocal VD4-RPeD1 synapses59. Since we observed an atypical GSK-3 signature at VD4-RPeD1 synapses, we wondered whether target-specific retrograde AA might influence neuropeptide release. Therefore, VD4-VF and VD4-RPeD1 pairs were cultured in DM, to avoid NTF/RTK signaling, and supplemented with free arachidonic acid , ETYA , or vehicle control (0.1% DMSO + 0.1% Tocrisolve 100). Relative to control, AA induced peptidergic transmission at VD4-VF pairs, while the inhibitor ETYA had no effect  to determine whether PKC is a molecular target for AA. Relative to vehicle control (0.1% DMSO), PKC inhibition prevented peptidergic transmission at VD4-RPeD1 synapses  family kinasesses Fig. A. We fouses Fig. B.Figure These data reveal that target-specific retrograde AA signaling acts via a PKC family kinase to define the cotransmitter release properties of innervating presynaptic terminals. Taken together, our observations on the regulation of peptide cotransmitter use at individual synapses uncover a previously undefined molecular signaling network that tunes the cotransmitter release characteristics of individual presynaptic terminals Fig. .Figure 1Lymnaea cardiorespiratory neurons, we have identified (i) a new component of NTF-dependent plasticity that selectively defines peptidergic cotransmission, (ii) a novel function for the Lymnaea synaptophysin homologue in the inhibitory regulation of neuropeptide release, and (iii) a role for AA metabolites as a target-specific retrograde signaling mechanism that establishes cotransmitter specificity. While we do not exclude the possibility that there may be concurrent changes in the expression or function of postsynaptic receptors, our observations of presynaptic neuropeptide accumulation and the inhibitory role of synaptophysin identify the presynaptic terminal as an important locus for target- and context-dependent regulation of cotransmission. Taken together, our findings define a molecular signaling network that underlies presynaptic cotransmitter specificity, and can allow for the context-dependent tuning of synaptic network function underlying the plasticity of patterned motor behaviours. Through this pathway, the release characteristics of individual synapses can be tuned to meet the dynamic functional requirements of neuronal networks set by an animal’s demand for behavioural plasticity.In this study we sought to identify the cellular and molecular mechanisms that specify the differential use of neurotransmitters at the individual presynaptic terminals formed by a cotransmitting neuron—a key mechanism for synaptic heterogeneity that underlies the selective processing and relay of information within neuronal circuits. Here, using 63. Here, we show that peptide cotransmitter release at inter-neuronal synapses is regulated by context-dependent inhibition. FMRF neuropeptides accumulated at functionally non-peptidergic presynapses, while functionally peptidergic presynapses showed comparatively little localization, indicating that colocalization does not imply cotransmission63. Consistent with this notion, a previous study on the Drosophila neuromuscular junction showed that neuropeptide stores are low in resting terminals, where they are supplied in a use-dependent manner by the activity-dependent capture of rapidly transiting LDCVs64. This suggests that, in contrast to classical transmission, peptidergic transmission may be sufficiently maintained in the absence of a sizeable reserve of LDCVs in the presynaptic bouton.One particular question in the field of cotransmission that has remained unresolved is whether the synaptic colocalization of multiple neurotransmitter substances necessarily implies their coreleaseDrosophila has demonstrated that neuropeptide autoreceptor signaling via presynaptic convergence adjusts cotransmitter output by enhancing the release of classical transmitters to accelerate the circadian rhythm directing sleep–wake behaviour65. Second, we have previously reported that ACh release competency in VD4 is scaled by presynaptic cAMP/PKA activation during competition for postsynaptic innervation66. Third, in the murine hippocampus, target-specific retrograde AA signaling has been found to induce presynaptic spike broadening by local inhibition of voltage-gated potassium channels, leading to synaptic facilitation67. Taken together, these observations indicate that a variety of mechanisms differentiate the function of individual presynaptic terminals to establish synaptic heterogeneity and enable selective information processing in neuronal circuits.The main focus of our study was to define the molecular mechanisms that selectively regulate neuropeptide cotransmitter release, however, our observation that the release of ACh from VD4 is tuned independently from FMRF neuropeptide release warrants further investigation to advance our understanding of the dynamic regulation of cotransmitter release and presynaptic heterogeneity. We suspect that there are a number of mechanisms, likely involving target-specific signaling interactions and cross-talk amongst signaling networks, at play. First, work in Lymnaea Syp homologue in inhibiting LDCV release, it is important to note that mammalian Syp has been widely regarded to be specifically localized to SSVs, and absent from LDCVs68. If the mechanism described here is conserved, one possibility is that inhibitory regulation of LDCVs in mammals is mediated by another member of the Syp protein family. Another possibility raised by studies in neuroendocrine cells is that Syp is present at varying concentrations in LDCVs, and at lower concentrations than in SSVs70. This is consistent with a study on the protein composition of single SVs reporting that some SVPs, including Syt I, are sorted with high precision, whereas others, including Syp and Syb, show significant intervesicle variability71. In view of our findings on the inhibitory regulation of neuropeptide release by Lymnaea Syp, it seems that synaptic cotransmitter specificity might be achieved by two complementary mechanisms: the differential trafficking of release competent (Syp-) or release incompetent (Syp +) LDCVs to presynaptic terminals; and synapse-delimited PTM of Syp that alters the release competency of Syp + LDCVs.Considering our observations on role of the 72. This suggests that N-glycosylation might act as a selectivity filter to govern the amount of Syp incorporated during LDCV biogenesis, which could regulate the release competency of LDCVs and also account for the controversial and variable presence of Syp on LDCVs. Downstream of biogenic sorting, the synapse-specific targeting of cargo, including LDCVs, is a second highly regulated process that differentiates the composition and function of individual presynaptic terminals during synaptogenesis and synaptic plasticity74. Considering our findings, it may be that NTF/RTK and retrograde AA signaling establish synapse-specific populations of release-competent and -incompetent LDCVs by influencing Syp N-glycosylation and the selective trafficking or capture of Syp + vs Syp- LDCVs at presynaptic terminals.Regarding the differential trafficking of Syp + and Syp− LDCVs, N-glycosylation, a PTM that occurs in the endoplasmic reticulum and affects protein sorting and trafficking, has been shown to be essential for targeting Syp, but not other SVPs such as Syt, out of the cell body and to the synapse53. Here, we found that target-dependent regulation of neuropeptide release involved the regulation of GSK-3 activity by NTF/RTK signaling, and PKC family kinase activity by retrograde AA signaling. The inhibition of GSK-3 by RTKs is known to involve the Par3-Par6-aPKC cell polarity complex61. During CNS development, the asymmetric accumulation of these polarity proteins mediates neurogenic divisions by neural progenitors, and regulates microtubule dynamics underlying axogenesis and directed outgrowth61. An attractive mechanism for the local regulation of neuropeptide release by GSK-3-dependent phosphorylation of Syp would be the differential localization of the Par3-Par6-aPKC cell polarity complex underlying the functional heterogeneity of individual presynaptic terminals.Regarding synapse-delimited PTM of Syp, Syp-Syb affinity has been reported to be modulated by GSK-3 activity, where phosphorylation of Syp by GSK-3 inhibits Syb dissociation and attenuates transmitter releaseLymnaea, NTF-dependent synaptic network plasticity has been proposed to mediate the seasonal regulation of heart rate and breathing behaviour during aestivation75. The regulation of FMRF neuropeptide release described here identifies a new mechanism with significant potential to underlie the plasticity of cardiorespiratory behaviour induced by NTFs. Increases or decreases in NTF/RTK signaling would differentially affect FMRF neuropeptide release at VD4′s excitatory synapse with VF to promote or inhibit cardiac drive, and the reciprocal inhibitory synapse with RPeD1 to facilitate or attenuate rCPG rhythmogenesis and breathing behaviour.In Cancer borealis, a cotransmitting projection neuron (MPN) selectively activates and inhibits the expression of pyloric and gastric mill motor patterns using its neuropeptide cotransmitter at synapses within the STG network and its small molecule cotransmitter at synapses with projection neurons in the commissural ganglia6. Thus, it seems that the selective use of neuropeptide and classical small molecule cotransmitters with distinct postsynaptic targets is a common mechanism underlying the expression of patterned motor behaviour in invertebrates. It would next be pertinent to determine whether an analogous mechanism involving target-specific retrograde AA signaling and Syp-dependent inhibition of LDCV fusion may be involved in expressing gastric mill motor patterns in the Cancer STG, and whether this may be a more general mechanism for the target-specific regulation of cotransmitter release underlying the appropriate expression and context-dependent plasticity of patterned motor behaviours across species.In the stomatogastric ganglion (STG) of the crab 76. Applied to the regulated secretion of neuropeptides, this adjustable reserve function would have interesting implications for our understanding of the dynamic propensity of neurons to modulate the release of their neuropeptides. In Lymnaea, release of the ovulation hormone requires priming by a prolonged period of coordinated network spiking activity77. Activity-dependent dephosphorylation of Syp might switch release-incompetent LDCVs to release-competent ones, perhaps involving activity-dependent NTF/RTK signaling. This mechanism would allow resting secretory terminals to accumulate neurohormones to levels sufficient for sustaining endocrine signaling, and maintain the system in an energetically favorable ‘primed-but-off’ state that would decouple neurohormone release from slower somal-dependent processes.The regulated complexing interactions of Syp and Syb have been suggested to act in establishing a reserve pool of SVs that can be recruited by synaptic activity80. These findings suggest that mammalian Syp is an essential regulator of synaptic plasticity, although previous studies have been unable to elucidate the underlying mechanism. Moreover, Syp is one of the genetic mutations implicated in human X-linked intellectual disability81. A compelling possibility raised by our work is that the synaptic plasticity deficits linked to mammalian Syp family proteins may involve the aberrant release of peptide cotransmitters. Further characterization of this Syp-dependent mechanism for regulated neuropeptide release, as it pertains to synaptic specificity and plasticity, is therefore likely to offer new fundamental insights into neurological disorders.In mice Syp knockout does not result in any readily apparent structural or synaptic deficits, however, changes in short- and long-term plasticity and hippocampus-dependent behaviour have been reportedLymnaea stagnalis were raised under standard conditions in freshwater aquaria at room temperature (~ 22 °C) on a diet of lettuce. Animals (6–8 weeks old) were anesthetized with a 10% Listerine solution (10 min) and sacrificed by dissection of the CNS in normal Lymnaea saline. Identified neurons VD4, VF and RPeD1 were isolated from trypsinized CNS by suction applied through a glass pipette and then cultured on poly-L-lysine coated glass culture dishes, as previously described in detail82. Neurons were maintained overnight (15–20 h) in defined media  or NTF-rich CNS conditioned media , as described elsewhere41.Lymnaea Syp homologue was identified from the Lymnaea EST library83 using a BLAST search  against mouse Syp (Accession number NM_009305), which revealed a single hit containing a complete protein-coding sequence (Accession number ES572211). We screened clones of the Lymnaea Syt I homologue (Accession number AF484090)84, and identified C2B-α (Syt-α) and C2B-β (Syt-β) isoform splice variants, each with a frequency of ~ 50%, suggesting that both transcripts are abundant in the CNS. The coding sequences for Syp, Syt-α, and Syt-β were cloned from Lymnaea CNS cDNA using Platinum Taq DNA polymerase (Invitrogen), and then subcloned into pBlueScript SK- (Clontech), as described elsewhere30. A C-terminal mCherry tag was introduced to Syp with site overlap extension (SOE) PCR using the KAPA HiFi HotStart ReadyMix PCR kit (Kapa Biosystems), as described previously35. See Supplementary Experimental Procedures for primer sequences . Purified synthetic mRNA was dissolved in molecular-grade water (3–6 μg/μL) and microinjected into VD4 neurons using a sterile low resistance glass electrode . Molecular-grade water was used as a vehicle control. Microinjections were performed shortly after neurons were plated, allowing time for the cells to adhere to the poly-L-lysine coated coverslip (~ 2 h). Synapses developed overnight in CM or DM.2 moiety85), or mCherry  were used at 1:500 in IM for 1 h. The secondary antibody  was used at 1:100 in IM for 1 h. Three 15 min washes in 1xPBS were performed between each incubation, and all incubations were performed at room temperature. Cells were mounted with MOWIOL containing DAPI.Neurons were fixed for 30 min with 1% paraformaldehyde and 0.2% picric acid in 1xPBS, then permeabilized for 1 h with incubation media (IM) containing 0.2% Triton, 5% fetal calf serum, and 0.25% fish gelatin in 1xPBS. Primary antibodies against FMRF neuropeptides . Laser excitation wavelengths were 402 and 561, in series, and emission wavelengths were collected through 450/50 and 595/50 filter cubes (Nikon). For quantitative analysis, imaging parameters were kept the same amongst relevant samples. Images were acquired with NIS Elements v4.13.00 (Nikon), and intensity analysis was performed with ImageJ (NIH). To determine the synaptic localization of FMRF neuropeptides and Syp-mCherry, we measured the mean fluorescence intensity at 3 presynaptic varicosities per pair. Presynapses were defined by their distinct morphology coincident with FMRF or mCherry immunoreactivity. We have previously combined this approach with FM1-43 dye uptake to verify that morphologically defined varicosities are functional presynaptic terminals41 and in situ86 preparations. Briefly, for cultured neurons, cells were maintained in CM or DM , and glass microelectrodes (resistance 20–50 MΩ) were filled with a saturated solution of K2SO4. Cells were visualized under an inverted microscope , and impaled using micromanipulators (Narishige). VD4, VF and RPeD1 neurons were identified by size, plating position, and electrophysiological signature. Signals were amplified with a Neuro data dual channel intracellular recording amplifier (Neuron Data Instruments), digitized , and recorded with Axoscope 10.2 (Molecular Devices). For in situ recordings, Lymnaea CNS were dissected as above and briefly treated (5–10 s) with focally applied Protease Type XIV (Sigma-Aldrich) to soften and remove the inner sheath. Isolated CNS were maintained in normal Lymnaea saline , and neurons were visualized under a Wild stereoscope (Wild Leitz AG), identified by size, position and color, then impaled with K2SO4-filled glass microelectrodes (resistance 20–50 MΩ) using micromanipulators (Sutter Instruments). Signals were amplified with a Neuro data amplifier, digitized , and recorded using custom ElectroLite software.Simultaneous intracellular current clamp recordings and Lucifer yellow (LY) dye injections were performed as previously described for in vitro87. To determine FMRF neuropeptide release characteristics from VD4, the excitation of VF at holding potentials of − 60 mV in vitro or − 70 mV in situ (below firing threshold), or inhibition of RPeD1 at − 50 mV in vitro or − 60 mV in situ (at firing threshold), was measured in response to a ~ 4 s burst elicited by depolarizing current injection in VD4. These holding potentials were used for in vitro and in situ recordings because ionic differences between the external recording solutions shift neuronal firing thresholds. Peptidergic transmission was deemed to have occurred if stimulation of VD4 resulted in the induction of action potentials in VF (exhibits an excitatory response to FMRF neuropeptides from VD4) or the cessation of firing and hyperpolarization of RPeD1 (exhibits an inhibitory response to FMRF neuropeptides from VD4). To determine DA release characteristics from RPeD1, the inhibition of VD4 at a holding potential of − 50 mV was measured in response to a ~ 4 s burst elicited by depolarizing current injection in RPeD1. Dopaminergic transmission was deemed to have occurred if stimulation of RPeD1 resulted in the cessation of firing and hyperpolarization of VD4 (exhibits an inhibitory response to DA from RPeD1). In presynaptic neurons neither the duration nor the number of action potentials per burst were significantly different amongst experimental conditions (see Supplementary Information). Data analysis was performed using AxoScope 10.3 (Molecular Devices).To determine ACh release characteristics from VD4, the mean amplitude of 5 consecutive PSPs, recorded in VF or RPeD1 neurons at a holding potential of − 100 mV was measured in response to single action potentials elicited by brief depolarizing current injection in VD4. Peptidergic transmission via FMRF neuropeptides was isolated pharmacologically using an AChR antagonist cocktail containing 5 μM methyllcaconitine (MLA), 10 μM tubocurarine (TC), and 20 μM tetraethylammonium (TEA), incubation time 5–10 min-), 3--4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB 216763) and Tocrisolve 100 were purchased from Tocris. 5,8,11,14-Eicosatetraynoic acid (ETYA) was purchased from Santa Cruz Biotech. Drugs were dissolved in dimethyl sulfoxide (DMSO) or DM according to solubility, and then added to culture media shortly after neurons were plated (~ 1 h). The next day, three washes with DM followed by replacement with fresh DM or CM was performed to remove drugs prior to experiments (~ 1 h). 0.1% DMSO and Tocrisolve 100 vehicle controls were performed for all experiments, as appropriate.TC, MLA, TEA and pertussis toxin (PTX) were purchased from Sigma-Aldrich. AA, Chelerythrine chloride (Ch ClP > 0.05) or non-parametric (P < 0.05) statistical tests were performed as appropriate. Non-parametric data sets were analyzed with Wilcoxon Signed Ranks tests, Mann–Whitney U tests, or Kruskal–Wallis tests. Parametric data sets were analyzed with Student’s Paired or Independent Samples T-tests, or One-way ANOVA with Games-Howell  or Tukey’s HSD  post hoc tests, as appropriate (see Supplementary Information).All reported results are derived from ≥ 2 independent experiments, to ensure reliability and replicability of the data. Sample sizes were limited by uncontrollable factors inherent to the cell culturing techniques used. Data analysis was performed blinded by acquisition file number. Statistical analyses were performed using SPSS Statistics v26 (IBM). Data sets were assessed with Shapiro–Wilk test for normality, and parametric (Supplementary Information.