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The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses. In the presynaptic terminal, Munc13-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin-1, a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein essential for synaptic vesicle exocytosis. The multiplicity of these Munc13-1 assemblies affords multiple stable states conferring presynaptic weight, potentially encoding several bits of information at individual synapses. Supramolecular assembling enables a stable synaptic weight, which confers robustness of synaptic computation on neuronal circuits and may be a general mechanism by which biological processes operate despite the presence of molecular noise.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca 2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca 2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca 2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca 2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca 2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca 2+ channel abundance and transmitter release at mossy fiber synapses.

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF 1 -ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo. Biologists often refer to a small molecule called adenosine triphosphate – or ATP for short – as ‘the currency of life’. This molecule carries energy all through the body, and most cells and proteins require ATP to perform their various roles. Nerve cells (also known as neurons) in the brain release ATP when activated, and use this molecule to send signals to other active neurons or other cells in the brain. But ATP can also signal danger in the brain. A molecule derived from ATP is involved in transmitting the pain signals of migraines and severe headaches; and ATP levels can become imbalanced after strokes, when parts of the brain are deprived of blood. Despite its importance, ATP remains difficult to visualize in the body, and monitoring the molecule in the active brain in real time is challenging. To address this issue, Kitajima et al. designed an optical sensor that could monitor ATP in the healthy brain, and was sensitive enough to detect when and where it was released. First, Kitajima et al. made several potential sensors by attaching various fluorescent tags to different locations on a protein that binds ATP. Next each sensor was tested to determine whether it could bind ATP tightly and get bright upon binding. This is important because previous sensors could not detect ATP release in the brains of living animals. To illustrate the new sensors’ potential, Kitajima et al. used the sensor to image ATP in the brains of live mice. A ‘wave’ of ATP was seen spreading through the brain after neurons were stimulated with a small electric pulse, mimicking a sudden migraine or stroke. The results confirm that this new sensor is suitable for imaging how ATP signals in the brain, and it may help resolve the underlying mechanisms of migraines and strokes. This sensor could also be used to understand other cellular process which rely on ATP to carry out their role.

Background Hepatic cyst disease is often asymptomatic, but treatment is warranted if patients experience symptoms. We describe our management approach to these patients and review the technical nuances of the laparoscopic approach. Methods Medical records were reviewed for operative management of hepatic cysts from 2012 to 2019 at a single, tertiary academic medical center. Results Fifty-three patients (39 female) met the inclusion criteria with median age at presentation of 65 years. Fifty cases (94.3%) were performed laparoscopically. Fourteen patients carried diagnosis of polycystic liver disease. Dominant cyst diameter was median 129 mm and located within the right lobe (30), left lobe (17), caudate (2), or was bilobar (4). Pre-operative concern for biliary cystadenoma/cystadenocarcinoma existed for 7 patients. Operative techniques included fenestration (40), fenestration with decapitation (7), decapitation alone (3), and excision (2). Partial hepatectomy was performed in conjunction with fenestration/decapitation for 15 cases: right sided (7), left sided (7), and central (1). One formal left hepatectomy was performed in a polycystic liver disease patient. Final pathology yielded simple cyst (52) and one biliary cystadenoma. Post-operative complications included bile leak (2), perihepatic fluid collection (1), pleural effusion (1), and ascites (1). At median 7.1-month follow-up, complete resolution of symptoms occurred for 34/49 patients (69.4%) who had symptoms preoperatively. Reintervention for cyst recurrence occurred for 5 cases (9.4%). Conclusions Outcomes for hepatic cyst disease are described with predominantly laparoscopic approach, approach with minimal morbidity, and excellent clinical results.

Brain injury induces phenotypic changes in astrocytes, known as reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP ₃)-dependent Ca ²⁺ signaling in astrocytes, and that the Ca ²⁺ signaling is required for astrogliosis. We found that type 2 IP ₃ receptor knockout (IP ₃R2KO) mice deficient in astrocytic Ca ²⁺ signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified N-cadherin and pumilio 2 (Pum2) as downstream signaling molecules, and found that brain injury induces up-regulation of N-cadherin around the injured site. This effect is mediated by Ca ²⁺-dependent down-regulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca ²⁺ signaling and the downstream function of N-cadherin play indispensable roles in the cellular responses to brain injury. These findings define a previously unreported signaling axis required for reactive astrogliosis and neuroprotection following brain injury.

To understand how neurons and neural circuits function during behaviors, it is essential to record neuronal activity in the brain in vivo . Among the various technologies developed for recording neuronal activity, molecular tools that induce gene expression in an activity-dependent manner have attracted particular attention for their ability to clarify the causal relationships between neuronal activity and behavior. In this review, we summarize recently developed activity-dependent gene expression tools and their potential contributions to the study of neural circuits.

Introduction Assessing patient-specific risk factors for long-term mortality following resection of pancreatic adenocarcinoma can be difficult. Sarcopenia—the measurement of muscle wasting—may be a more objective and comprehensive patient-specific factor associated with long-term survival. Methods Total psoas area (TPA) was measured on preoperative cross-sectional imaging in 557 patients undergoing resection of pancreatic adenocarcinoma between 1996 and 2010. Sarcopenia was defined as the presence of a TPA in the lowest sex-specific quartile. The impact of sarcopenia on 90-day, 1-year, and 3-year mortality was assessed relative to other clinicopathological factors. Results Mean patient age was 65.7 years and 53.1 % was male. Mean TPA among men (611 mm 2 /m 2 ) was greater than among women (454 mm 2 /m 2 ). Surgery involved pancreaticoduodenectomy (86.0 %) or distal pancreatectomy (14.0 %). Mean tumor size was 3.4 cm; 49.9 % and 88.5 % of patients had vascular and perineural invasion, respectively. Margin status was R0 (59.0 %) and 77.7 % patients had lymph node metastasis. Overall 90-day mortality was 3.1 % and overall 1- and 3-year survival was 67.9 % and 35.7 %, respectively. Sarcopenia was associated with increased risk of 3-year mortality (HR = 1.68; P  < 0.001). Tumor-specific factors such as poor differentiation on histology (HR = 1.75), margin status (HR = 1.66), and lymph node metastasis (HR = 2.06) were associated with risk of death at 3-years (all P  < 0.001). After controlling for these factors, sarcopenia remained independently associated with an increased risk of death at 3 years (HR = 1.63; P  < 0.001). Conclusions Sarcopenia was a predictor of survival following pancreatic surgery, with sarcopenic patients having a 63 % increased risk of death at 3 years. Sarcopenia was an objective measure of patient frailty that was strongly associated with long-term outcome independent of tumor-specific factors.

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia’s morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell–cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

BackgroundGuidelines recommend limiting minimally invasive pancreaticoduodenectomy (MIPD) to high-volume centers. However, the definition of high-volume care remains unclear. We aimed to objectively define a minimum number of MIPD performed annually per hospital associated with improved outcomes in a contemporary patient cohort.Patients and MethodsResectable pancreatic adenocarcinoma patients undergoing MIPD were included from the National Cancer Database (2010–2017). Multivariable modeling with restricted cubic splines was employed to identify an MIPD annual hospital volume threshold associated with lower 90-day mortality. Outcomes were compared between patients treated at low-volume (≤ model-identified cutoff) and high-volume (> cutoff) centers.ResultsAmong 3079 patients, 141 (5%) died within 90 days. Median hospital volume was 6 (range 1–73) cases/year. After adjustment, increasing hospital volume was associated with decreasing 90-day mortality for up to 19 (95% CI 16–25) cases/year, indicating a threshold of 20 cases/year. Most cases (82%) were done at low-volume (< 20 cases/year) centers. With adjustment, MIPD at low-volume centers was associated with increased 90-day mortality (OR 2.7; p = 0.002). Length of stay, positive surgical margins, 30-day readmission, and overall survival were similar. On analysis of the most recent two years (n = 1031), patients at low-volume centers (78.2%) were younger and had less advanced tumors but had longer length of stay (8 versus 7 days; p < 0.001) and increased 90-day mortality (7% versus 2%; p = 0.009).ConclusionsThe cutpoint analysis identified a threshold of at least 20 MIPD cases/year associated with lower postoperative mortality. This threshold should inform national guidelines and institution-level protocols aimed at facilitating the safe implementation of this complex procedure.

Disc1 is a susceptibility gene for psychiatric disorders including schizophrenia. It has been suggested that excess transmission through dopamine type 2 receptors (D2Rs) in the striatum is an underlying mechanism of pathogenesis. In this study, we used super-resolution microscopy to study the distribution of D2Rs at the nanoscale in mice lacking exons 2 and 3 of Disc1 ( Disc1 -deficient mice). We found that D2Rs in the nucleus accumbens (NAc) of wild-type mice form nanoclusters (~ 20,000 nm 2 ), and that Disc1 -deficient mice have larger and more D2R nanoclusters than wild-type mice. Interestingly, administration of clozapine reduced the size and spatial distribution of the nanoclusters only in Disc1 -deficient mice. Moreover, we observed that medium spiny neurons in the NAc of Disc1 -deficient mice had reduced spine density on their dendrites than did wild-type mice, and this was also reversed by clozapine administration. The altered D2R nanoclusters might be morphological representations of the altered dopaminergic transmission in disease states such as schizophrenia.