Tenpei Akita

Bradykinin-induced astrocyte–neuron signalling: glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels

Glial cells release gliotransmitters which signal to adjacent neurons and glial cells. Previous studies showed that in response to stimulation with bradykinin, glutamate is released from rat astrocytes and causes NMDA receptor‐mediated elevation of intracellular Ca2+ in adjacent neurons. Here, we investigate how bradykinin‐induced glutamate release from mouse astrocytes signals to neighbouring neurons in co‐cultures. Astrocyte‐to‐neuron signalling and bradykinin‐induced glutamate release from mouse astrocytes were both inhibited by the anion channel blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS) and phloretin. Glutamate release was also sensitive to 4‐(2‐Butyl‐6,7‐dichlor‐2‐cyclopentylindan‐1‐on‐5‐yl) oxybutyric acid (DCPIB), a specific blocker of the volume‐sensitive outwardly rectifying anion channel (VSOR). Astrocytes, but not neurons, responded to bradykinin with activation of whole‐cell Cl− currents. Although astrocytes stimulated with bradykinin did not undergo cell swelling, the bradykinin‐activated current exhibited properties typical of VSOR: outward rectification, inhibition by osmotic shrinkage, sensitivity to DIDS, phloretin and DCPIB, dependence on intracellular ATP, and permeability to glutamate. Bradykinin increased intracellular reactive oxygen species (ROS) in mouse astrocytes. Pretreatment of mouse astrocytes with either a ROS scavenger or an NAD(P)H oxidase inhibitor blocked bradykinin‐induced activation of VSOR, glutamate release and astrocyte‐to‐neuron signalling. By contrast, pretreatment with BAPTA‐AM or tetanus neurotoxin A failed to suppress bradykinin‐induced glutamate release. Thus, VSOR activated by ROS in mouse astrocytes in response to stimulation with bradykinin, serves as the pathway for glutamate release to mediate astrocyte‐to‐neuron signalling. Since bradykinin is an initial mediator of inflammation, VSOR might play a role in glia–neuron communication in the brain during inflammation.

Regulation of bradykinin‐induced activation of volume‐sensitive outwardly rectifying anion channels by Ca2+ nanodomains in mouse astrocytes

Non‐technical summary  Cell volume regulation is an essential function involved not only in homeostasis, but also in cell migration, fission and programmed cell death. The volume‐sensitive outwardly rectifying (VSOR) anion channel provides the main pathway for anion transport across the cell membrane during the regulation. We previously demonstrated that an inflammatory mediator, bradykinin, activates the VSOR channels in the major glial cells, astrocytes, in the brain derived from mice and the channels send signals to adjacent neurons through the release of glutamate. Here we demonstrate that this activation is controlled in the immediate vicinity of Ca2+‐permeable channel proteins in the astrocytes via high concentrations of intracellular Ca2+, so‐called ‘Ca2+ nanodomains’. This mechanism would provide a basis for responding quickly and certainly to even a minute amount of bradykinin released from surrounding tissues (e.g. slightly damaged blood vessel walls) with local control of cell shape changes and signal transmission by astrocytes.

Characteristics and roles of the volume-sensitive outwardly rectifying (VSOR) anion channel in the central nervous system

Cell volume regulation (CVR) is essential for all types of cells in the central nervous system (CNS) to counteract cell volume changes that may be associated with neuronal activities or diseases and with osmosensing in the hypothalamus, to facilitate morphological changes during cell proliferation, differentiation and migration, and to execute apoptosis of cells. The regulation is attained by regulating the net influx or efflux of solutes and water across the plasma membrane. The volume-sensitive outwardly rectifying (VSOR) anion channel plays a major role in providing a pathway for anion flux during the regulation. The VSOR anion channel is permeable not only to Cl(-) ions but also to amino acids like glutamate and taurine. This property confers a means of intercellular communications through the opening of the channel in the CNS. Thus exploring the roles of VSOR anion channels is crucial to understand the basic principles of cellular functions in the CNS. Here we review biophysical and pharmacological characteristics of the VSOR anion channel in the CNS, discuss its activation mechanisms and roles in the CNS reported so far, and give some perspectives on the next issues to be examined in the near future.

Ca2+ nanodomain-mediated component of swelling-induced volume-sensitive outwardly rectifying anion current triggered by autocrine action of ATP in mouse astrocytes.

The volume-sensitive outwardly rectifying (VSOR) anion channel provides a major pathway for anion transport during cell volume regulation. It is typically activated in response to cell swelling, but how the channel senses the swelling remains unclear. Meanwhile, we recently found that in mouse astrocytes the channel is activated by an inflammatory chemical mediator, bradykinin, without cell swelling and that the activation is regulated via high concentration regions of intracellular Ca2+ ([Ca2+]i) in the immediate vicinity of open Ca2+-permeable channels, so-called Ca2+ nanodomains. Here we investigated whether a similar mechanism is involved in the swelling-induced VSOR channel activation in the astrocytes. A hypotonic stimulus (25% reduction in osmolality) caused the [Ca2+]i rises in the astrocytes, and the rises were abolished in the presence of an ATP-degrading enzyme, apyrase (10 U/ml). Application of ATP (100 µM) under isotonic conditions generated the current through VSOR channels via Ca2+ nanodomains, as bradykinin does. The current induced by the hypotonic stimulus was suppressed by ∼40% in the Ca2+-depleted condition where the ATP-induced VSOR current was totally prevented. Thus the swelling-induced VSOR channel activation in mouse astrocytes is partly regulated via Ca2+ nanodomains, whose generation is triggered by an autocrine action of ATP.

De Novo KCNB1 Mutations in Infantile Epilepsy Inhibit Repetitive Neuronal Firing.

The voltage-gated Kv2.1 potassium channel encoded by KCNB1 produces the major delayed rectifier potassium current in pyramidal neurons. Recently, de novo heterozygous missense KCNB1 mutations have been identified in three patients with epileptic encephalopathy and a patient with neurodevelopmental disorder. However, the frequency of KCNB1 mutations in infantile epileptic patients and their effects on neuronal activity are yet unknown. We searched whole exome sequencing data of a total of 437 patients with infantile epilepsy, and found novel de novo heterozygous missense KCNB1 mutations in two patients showing psychomotor developmental delay and severe infantile generalized seizures with high-amplitude spike-and-wave electroencephalogram discharges. The mutation located in the channel voltage sensor (p.R306C) disrupted sensitivity and cooperativity of the sensor, while the mutation in the channel pore domain (p.G401R) selectively abolished endogenous Kv2 currents in transfected pyramidal neurons, indicating a dominant-negative effect. Both mutants inhibited repetitive neuronal firing through preventing production of deep interspike voltages. Thus KCNB1 mutations can be a rare genetic cause of infantile epilepsy, and insufficient firing of pyramidal neurons would disturb both development and stability of neuronal circuits, leading to the disease phenotypes.

Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay.

Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compound heterozygous SLC12A5 (encoding the neuronal K+-Cl− co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compound heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp analysis demonstrated strongly suppressed Cl− extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl− level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl− extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS.

Activity-dependent endogenous taurine release facilitates excitatory neurotransmission in the neocortical marginal zone of neonatal rats

In the developing cerebral cortex, the marginal zone (MZ), consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA) in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/β subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl−]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na+, K+-2Cl− cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na+ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of glycine receptors in the MZ.

A Newly Cloned ClC-3 Isoform, ClC-3d, as well as ClC-3a Mediates Cd2+-Sensitive Outwardly Rectifying Anion Currents

Background: ClC-3, a member of the ClC family, is predicted to have six isoforms, ClC-3a to -3f, with distinct N- and C-terminal amino acid sequences. There have been conflicting reports on the properties of ClC-3a (also known as the N-terminal short form of ClC-3) and ClC-3b (the N-terminal long form of ClC-3) as plasmalemmal Cl- channels. Meanwhile, little is known about other isoforms. The amino acid sequence of ClC-3d (a C-terminal variant of the short form) listed in the NCBI database was derived from the genomic sequence, but there has been no experimental evidence for the mRNA. Methods: PCR-cloning was made to obtain the full coding region of ClC-3d from mouse liver. Its molecular expression on the plasma membrane was microscopically examined in HEK293T cells transfected with GFP-tagged ClC-3d. Its functional plasmalemmal expression and the properties of currents were studies by whole-cell recordings in the cells transfected with ClC-3d. Results: The cloned ClC-3d was found to be the only isoform which has an N-terminal amino acid sequence identical to ClC-3a. When introduced into HEK293T cells, a minor fraction of exogenous ClC-3d proteins was detected at the plasma membrane, and activation of anion currents was observed at neutral pH under normotonic conditions. The properties of ClC-3d currents were found to be shared by ClC-3a-mediated currents. Also, both ClC-3d and -3a currents were found to be sensitive to Cd2+. ClC-3d overexpression never affected the endogenous activity of acid- or swelling-activated anion channels. Conclusion: We thus conclude that plasmalemmal ClC-3d, like ClC-3a, mediates Cd2+-sensitive outwardly rectifying anion currents and that ClC-3d is distinct from the molecular entities of acid- and volume-sensitive anion channels.

Adenosine depresses a Ca2+-independent step in transmitter exocytosis at frog motor nerve terminals

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca2+‐imaging techniques. Adenosine (200 µm) quickly and reversibly decreased the amplitude and quantal content of end‐plate potentials (EPPs) with no change in quantal size in a low‐Ca2+, high‐Mg2+ solution, and EPP amplitude in normal Ringer containing d‐tubocurarine. Likewise, adenosine (200 µm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high‐K+ (6 mm) solution. Adenosine (40–200 µm), however, did not affect single or repetitive impulse(s)‐induced rises in Ca2+ in the nerve terminals or its basal level. Adenosine (100–200 µm) reduced the Ca2+‐independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d‐tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 µm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca2+‐independent step of transmitter exocytosis at frog motor nerve terminals.

Ion channels, guidance molecules, intracellular signaling and transcription factors regulating nervous and vascular system development

Our sophisticated thoughts and behaviors are based on the miraculous development of our complex nervous network system, in which many different types of proteins and signaling cascades are regulated in a temporally and spatially ordered manner. Here we review our recent attempts to grasp the principles of nervous system development in terms of general cellular phenomena and molecules, such as volume-regulated anion channels, intracellular Ca2+ and cyclic nucleotide signaling, the Npas4 transcription factor and the FLRT family of axon guidance molecules. We also present an example illustrating that the same FLRT family may regulate the development of vascular networks as well. The aim of this review is to open up new vistas for understanding the intricacy of nervous and vascular system development.