Dynamic and stable hippocampal representations of social identity and reward expectation support associative social memory in male mice.

Recognizing an individual and retrieving and updating the value information assigned to the individual are fundamental abilities for establishing social relationships. To understand the neural mechanisms underlying the association between social identity and reward value, we developed Go-NoGo social discrimination paradigms that required male subject mice to distinguish between familiar mice based on their individually unique characteristics and associate them with reward availability. We found that mice could discriminate individual conspecifics through a brief nose-to-nose investigation, and this ability depended on the dorsal hippocampus. Two-photon calcium imaging revealed that dorsal CA1 hippocampal neurons represented reward expectation during social, but not non-social tasks, and these activities were maintained over days regardless of the identity of the associated mouse. Furthermore, a dynamically changing subset of hippocampal CA1 neurons discriminated between individual mice with high accuracy. Our findings suggest that the neuronal activities in CA1 provide possible neural substrates for associative social memory.

Noninvasive Screening Tool for Hyperkalemia Using a Single-Lead Electrocardiogram and Deep Learning: Development and Usability Study.

BACKGROUND: Hyperkalemia monitoring is very important in patients with chronic kidney disease (CKD) in emergency medicine. Currently, blood testing is regarded as the standard way to diagnose hyperkalemia (ie, using serum potassium levels). Therefore, an alternative and noninvasive method is required for real-time monitoring of hyperkalemia in the emergency medicine department. OBJECTIVE: This study aimed to propose a novel method for noninvasive screening of hyperkalemia using a single-lead electrocardiogram (ECG) based on a deep learning model. METHODS: For this study, 2958 patients with hyperkalemia events from July 2009 to June 2019 were enrolled at 1 regional emergency center, of which 1790 were diagnosed with chronic renal failure before hyperkalemic events. Patients who did not have biochemical electrolyte tests corresponding to the original 12-lead ECG signal were excluded. We used data from 855 patients (555 patients with CKD, and 300 patients without CKD). The 12-lead ECG signal was collected at the time of the hyperkalemic event, prior to the event, and after the event for each patient. All 12-lead ECG signals were matched with an electrolyte test within 2 hours of each ECG to form a data set. We then analyzed the ECG signals with a duration of 2 seconds and a segment composed of 1400 samples. The data set was randomly divided into the training set, validation set, and test set according to the ratio of 6:2:2 percent. The proposed noninvasive screening tool used a deep learning model that can express the complex and cyclic rhythm of cardiac activity. The deep learning model consists of convolutional and pooling layers for noninvasive screening of the serum potassium level from an ECG signal. To extract an optimal single-lead ECG, we evaluated the performances of the proposed deep learning model for each lead including lead I, II, and V1-V6. RESULTS: The proposed noninvasive screening tool using a single-lead ECG shows high performances with F1 scores of 100%, 96%, and 95% for the training set, validation set, and test set, respectively. The lead II signal was shown to have the highest performance among the ECG leads. CONCLUSIONS: We developed a novel method for noninvasive screening of hyperkalemia using a single-lead ECG signal, and it can be used as a helpful tool in emergency medicine.

Diffusion of a Lifelog-Based Digital Healthcare Platform for Future Precision Medicine: Data Provision and Verification Study.

We propose a method for data provision, validation, and service expansion for the spread of a lifelog-based digital healthcare platform. The platform is an operational cloud-based platform, implemented in 2020, that has launched a tool that can validate and de-identify personal information in a data acquisition system dedicated to a center. The data acquired by the platform can be processed into products of statistical analysis and artificial intelligence (AI)-based deep learning modules. Application programming interfaces (APIs) have been developed to open data and can be linked in a programmatic manner. As a standardized policy, a series of procedures were performed from data collection to external sharing. The proposed platform collected 321.42 GB of data for 146 types of data. The reliability and consistency of the data were evaluated by an information system audit institution, with a defects ratio of approximately 0.03%. We presented definitions and examples of APIs developed in 17 functional units for data opening. In addition, the suitability of the de-identification tool was confirmed by evaluating the reduced risk of re-identification using quasi-identifiers. We presented specific methods for data verification, personal information de-identification, and service provision to ensure the sustainability of future digital healthcare platforms for precision medicine. The platform can contribute to the diffusion of the platform by linking data with external organizations and research environments in safe zones based on data reliability.

Inter-spike mitochondrial Ca2+ release enhances high frequency synaptic transmission.

KEY POINTS: Presynaptic mitochondria not only absorb but also release Ca2+ during high frequency stimulation (HFS) when presynaptic [Ca2+ ] is kept low (<500 nm) by high cytosolic Ca2+ buffer or strong plasma membrane calcium clearance mechanisms under physiological external [Ca2+ ]. Mitochondrial Ca2+ release (MCR) does not alter the global presynaptic Ca2+ transients. MCR during HFS enhances short-term facilitation and steady state excitatory postsynaptic currents by increasing vesicular release probability. The intra-train MCR may provide residual calcium at interspike intervals, and thus support high frequency neurotransmission at central glutamatergic synapses. ABSTRACT: Emerging evidence indicates that mitochondrial Ca2+ buffering contributes to local regulation of synaptic transmission. It is unknown, however, whether mitochondrial Ca2+ release (MCR) occurs during high frequency synaptic transmission. Confirming the previous notion that 2 µm tetraphenylphosphonium (TPP+ ) is a specific inhibitor of the mitochondrial Na+ /Ca2+ exchanger (mNCX), we studied the role of MCR via mNCX in short-term plasticity during high frequency stimulation (HFS) at the calyx of Held synapse of the rat. TPP+ reduced short-term facilitation (STF) and steady state excitatory postsynaptic currents during HFS at mature calyx synapses under physiological extracellular [Ca2+ ] ([Ca2+ ]o  = 1.2 mm), but not at immature calyx or at 2 mm [Ca2+ ]o . The inhibitory effects of TPP+ were stronger at synapses with morphologically complex calyces harbouring many swellings and at 32°C than at simple calyx synapses and at room temperature. These effects of TPP+ on STF were well correlated with those on the presynaptic mitochondrial [Ca2+ ] build-up during HFS. Mitochondrial [Ca2+ ] during HFS was increased by TPP+ at mature calyces under 1.2 mm [Ca2+ ]o , and further enhanced at 32°C, but not under 2 mm [Ca2+ ]o or at immature calyces. The close correlation of the effects of TPP+ on mitochondrial [Ca2+ ] with those on STF suggests that mNCX contributes to STF at the calyx of Held synapses. The intra-train MCR enhanced vesicular release probability without altering global presynaptic [Ca2+ ]. Our results suggest that MCR during HFS elevates local [Ca2+ ] near synaptic sites at interspike intervals to enhance STF and to support stable synaptic transmission under physiological [Ca2+ ]o .

Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergic Synapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells of Postnatal Hippocampus.

Expression of neuregulin-2 (NRG2) is intense in a few regions of the adult brain where neurogenesis persists; however, little is understood about its role in developments of newborn neurons. To study the role of NRG2 in synaptogenesis at different developmental stages, newborn granule cells in rat hippocampal slice cultures were labeled with retrovirus encoding tetracycline-inducible microRNA targeting NRG2 and treated with doxycycline (Dox) at the fourth or seventh postinfection day (dpi). The developmental increase of GABAergic postsynaptic currents (GPSCs) was suppressed by the early Dox treatment (4 dpi), but not by late treatment (7 dpi). The late Dox treatment was used to study the effect of NRG2 depletion specific to excitatory synaptogenesis. The Dox effect on EPSCs emerged 4 d after the impairment in dendritic outgrowth became evident (10 dpi). Notably, Dox treatment abolished the developmental increases of AMPA-receptor mediated EPSCs and the AMPA/NMDA ratio, indicating impaired maturation of glutamatergic synapses. In contrast to GPSCs, Dox effects on EPSCs and dendritic growth were independent of ErbB4 and rescued by concurrent overexpression of NRG2 intracellular domain. These results suggest that forward signaling of NRG2 mediates GABAergic synaptogenesis and its reverse signaling contributes to dendritic outgrowth and maturation of glutamatergic synapses. SIGNIFICANCE STATEMENT: The hippocampal dentate gyrus is one of special brain regions where neurogenesis persists throughout adulthood. Synaptogenesis is a critical step for newborn neurons to be integrated into preexisting neural network. Because neuregulin-2 (NRG2), a growth factor, is intensely expressed in these regions, we investigated whether it plays a role in synaptogenesis and dendritic growth. We found that NRG2 has dual roles in the development of newborn neurons. For GABAergic synaptogenesis, the extracellular domain of NRG2 acts as a ligand for a receptor on GABAergic neurons. In contrast, its intracellular domain was essential for dendritic outgrowth and glutamatergic synapse maturation. These results imply that NRG2 may play a critical role in network integration of newborn neurons.

Ca(2+) clearance by plasmalemmal NCLX, Li(+)-permeable Na(+)/Ca(2+) exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells.

Na(+)/Ca(2+) exchangers are key players for Ca(2+) clearance in pancreatic ß-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li(+)-permeable Na(+)/Ca(2+) exchangers (NCLX), which were known as mitochondrial Na(+)/Ca(2+) exchangers, contributed to the Na(+)-dependent Ca(2+) movement across the plasma membrane in rat INS-1 insulinoma cells. Na(+)/Ca(2+) exchange activity by NCLX was comparable to that by the Na(+)/Ca(2+) exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na(+) replacement for inhibiting Na(+)-dependent Ca(2+) clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na(+) concentration, suggesting a negligible contribution of mitochondrial Na(+)/Ca(2+) exchanger. Taken together, our data indicate that Na(+)/Ca(2+) exchanger-mediated Ca(2+) clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic ß-cells.

Kv1.2 mediates heterosynaptic modulation of direct cortical synaptic inputs in CA3 pyramidal cells.

KEY POINTS: We investigated the cellular mechanisms underlying mossy fibre-induced heterosynaptic long-term potentiation of perforant path (PP) inputs to CA3 pyramidal cells. Here we show that this heterosynaptic potentiation is mediated by downregulation of Kv1.2 channels. The downregulation of Kv1.2 preferentially enhanced PP-evoked EPSPs which occur at distal apical dendrites. Such enhancement of PP-EPSPs required activation of dendritic Na(+) channels, and its threshold was lowered by downregulation of Kv1.2. Our results may provide new insights into the long-standing question of how mossy fibre inputs constrain the CA3 network to sparsely represent direct cortical inputs. ABSTRACT: A short high frequency stimulation of mossy fibres (MFs) induces long-term potentiation (LTP) of direct cortical or perforant path (PP) synaptic inputs in hippocampal CA3 pyramidal cells (CA3-PCs). However, the cellular mechanism underlying this heterosynaptic modulation remains elusive. Previously, we reported that repetitive somatic firing at 10 Hz downregulates Kv1.2 in the CA3-PCs. Here, we show that MF inputs induce similar somatic firing and downregulation of Kv1.2 in the CA3-PCs. The effect of Kv1.2 downregulation was specific to PP synaptic inputs that arrive at distal apical dendrites. We found that the somatodendritic expression of Kv1.2 is polarized to distal apical dendrites. Compartmental simulations based on this finding suggested that passive normalization of synaptic inputs and polarized distributions of dendritic ionic channels may facilitate the activation of dendritic Na(+) channels preferentially at distal apical dendrites. Indeed, partial block of dendritic Na(+) channels using 10 nm tetrodotoxin brought back the enhanced PP-evoked excitatory postsynaptic potentials (PP-EPSPs) to the baseline level. These results indicate that activity-dependent downregulation of Kv1.2 in CA3-PCs mediates MF-induced heterosynaptic LTP of PP-EPSPs by facilitating activation of Na(+) channels at distal apical dendrites.

Costimulation of AMPA and metabotropic glutamate receptors underlies phospholipase C activation by glutamate in hippocampus.

Glutamate, a major neurotransmitter in the brain, activates ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs, respectively). The two types of glutamate receptors interact with each other, as exemplified by the modulation of iGluRs by mGluRs. However, the other way of interaction (i.e., modulation of mGluRs by iGluRs) has not received much attention. In this study, we found that group I mGluR-specific agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) alone is not sufficient to activate phospholipase C (PLC) in rat hippocampus, while glutamate robustly activates PLC. These results suggested that additional mechanisms provided by iGluRs are involved in group I mGluR-mediated PLC activation. A series of experiments demonstrated that glutamate-induced PLC activation is mediated by mGluR5 and is facilitated by local Ca(2+) signals that are induced by AMPA-mediated depolarization and L-type Ca(2+) channel activation. Finally, we found that PLC and L-type Ca(2+) channels are involved in hippocampal mGluR-dependent long-term depression (mGluR-LTD) induced by paired-pulse low-frequency stimulation, but not in DHPG-induced chemical LTD. Together, we propose that AMPA receptors initiate Ca(2+) influx via the L-type Ca(2+) channels that facilitate mGluR5-PLC signaling cascades, which underlie mGluR-LTD in rat hippocampus.

Activity-dependent downregulation of D-type K+ channel subunit Kv1.2 in rat hippocampal CA3 pyramidal neurons.

The intrinsic excitability of neurons plays a critical role in the encoding of memory at Hebbian synapses and in the coupling of synaptic inputs to spike generation. It has not been studied whether somatic firing at a physiologically relevant frequency can induce intrinsic plasticity in hippocampal CA3 pyramidal cells (CA3-PCs). Here, we show that a conditioning train of 20 action potentials (APs) at 10 Hz causes a persistent reduction in the input conductance and an acceleration of the AP onset time in CA3-PCs, but not in CA1-PCs. Induction of such long-term potentiation of intrinsic excitability (LTP-IE) was accompanied by a reduction in the D-type K(+) current, and was abolished by the inhibition of endocytosis or protein tyrosine kinase (PTK). Consistently, the CA3-PCs from Kv1.2 knock-out mice displayed no LTP-IE with the same conditioning. Furthermore, the induction of LTP-IE depended on the back-propagating APs (bAPs) and intact distal apical dendrites. These results indicate that LTP-IE is mediated by the internalization of Kv1.2 channels from the distal regions of apical dendrites, which is triggered by bAP-induced dendritic Ca(2+) signalling and the consequent activation of PTK.

Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic ß-cells.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic ß-cells. In this study, we provide evidence that leptin modulates pancreatic ß-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic ß-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase ß. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic ß-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and ß-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating ß-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating ß-cell excitability.

Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation.

We have previously reported that the surface expression of K(+)-dependent Na(+)/Ca(2+) exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the µ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs ((365)YGKL and (371)YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the (365)YGKL motif is essential for the interaction with the µ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

KIF21A-mediated axonal transport and selective endocytosis underlie the polarized targeting of NCKX2.

We have previously shown that K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) is a major calcium clearance mechanism at the large axon terminals of central neurons, whereas their somata display little NCKX activity. We investigated mechanisms underlying the axonal polarization of NCKX2 in rat hippocampal neurons. We identified NCKX2 as the first neuron-specific cargo molecule of kinesin family member 21A (KIF21A). The intracellular loop of NCKX2 specifically interacted with the WD-40 repeats, a putative cargo-binding domain, of KIF21A. Dominant-negative mutant or depletion of KIF21A inhibited the transport of NCKX2-GFP to axon fibers. Knockdown of KIF21A caused calcium dysregulation at axonal boutons but not at somatodendritic regions. Despite the axonal polarization of the NCKX activity, both somatodendritic and axonal regions were immunoreactive to NCKX2. The surface expression of NCKX2 revealed by live-cell immunocytochemistry, however, displayed highly polarized distribution to the axon. Inhibition of endocytosis increased the somatodendritic surface NCKX2 and thus abolished the axonal polarization of surface NCKX2. These results indicate that KIF21A-mediated axonal transport and selective somatodendritic endocytosis underlie the axonal polarized surface expression of NCKX2.

Target cell-specific involvement of presynaptic mitochondria in post-tetanic potentiation at hippocampal mossy fiber synapses.

Previous studies indicate that boutons from the same axon exhibit distinct Ca2+ dynamics depending on the postsynaptic targets. Mossy fibers of hippocampal granule cells innervate synaptic targets via morphologically distinct boutons. We investigated mitochondrial involvement in the generation of post-tetanic residual Ca2+ (Ca(res)) at large and small en passant mossy fiber boutons (MFBs). Mitochondria limited the [Ca2+]i build-up during high-frequency stimulation (HFS) at large MFBs, but not at small MFBs. The amount of Ca(res), quantified as a time integral of residual [Ca2+]i, was significantly larger at large MFBs than at small MFBs, and that at large MFBs was substantially attenuated by inhibitors of mitochondrial Ca2+ uniporter and mitochondrial Na+/Ca2+ exchanger (mitoNCX). In contrast, blockers of mitoNCX had no effect on the Ca(res) at small MFBs. Post-tetanic Ca(res) has been proposed as a mechanism for post-tetanic potentiation (PTP). We examined mitochondrial involvement in PTP at mossy fiber synapses on hilar mossy cells (MF-->MC synapse) and on hilar interneurons (MF-->HI synapse), which are presumably innervated via large and small MFBs, respectively. Consistent with the differential contribution of mitochondria to Ca(res) at large and small MFBs, mitoNCX blockers significantly reduced the PTP at the MF-->MC synapse, but not at the MF-->HI synapse. In contrast, protein kinase C (PKC) inhibitors significantly reduced the PTP at MF-->HI synapse, but not at the MF-->MC synapse. These results indicate that mitochondria- and PKC-dependent PTP are expressed at distinct hilar mossy fiber synapses depending on postsynaptic targets.

A new cytotoxic prenylated chalcone from Sophora flavescens.

We have isolated a new prenylated chalcone from the roots of Sophora flavescens (Leguminosae). We determined that structure of this compound is 7,9,2',4'-tetrahydroxy-8-isopentenyl-5-methoxychalcone (1) on the basis of spectroscopic analysis (1D and 2D NMR data). Compound 1 exhibited potent cytotoxicity against human acute promyelocytic (HL60), mouse lymphocytic (L1210) and human histiocytic (U937) leukemia cells.

Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.