Spectra analysis and live-neuron imaging of cyclic AMP binding domain fusing circularly permutated GFP: Data for violet light excitable cyclic AMP indicator.

Cyclic adenosine monophosphate (cyclic AMP) is a second messenger, which is involved in the regulation of various cellular processes, including neuronal firing rate, synaptic plasticity, axon formation and axon elongation in brain. Although the main molecules in the cAMP-mediated signaling pathway are well studied, the spatio-temporal dynamics of the cAMP remain to be elucidated. Live imaging is an informative tool to investigate the cell signaling dynamics. It allows continuous monitoring of a specific cell over a period of time. Thus, optical probes for cAMP are important tools for studying the dynamics of cAMP signaling. Multiple genetically encoded cAMP probes are available [1], [2], including Förster resonance energy transfer (FRET) based or circular permutated fluorescent protein (cpFP) based probes. cpFP-based probes have an advantage of easier handling than FRET-based probes caused by monomeric detection and smaller molecular size. However, there is no cAMP probe compatible with violet light excitation. Therefore, we fused violet light excitable cpGFP to cyclic nucleotide binding domain (CBD) in E. coli cAMP receptor protein. This construct successfully responded to cAMP concentration changes. We show here the spectra data and live-cell imaging data of the violet light excitable cAMP probe which can be used for multi-signal fluorescence imaging.

Green fluorescent cAMP indicator of high speed and specificity suitable for neuronal live-cell imaging.

Cyclic adenosine monophosphate (cAMP) is a canonical intracellular messenger playing diverse roles in cell functions. In neurons, cAMP promotes axonal growth during early development, and mediates sensory transduction and synaptic plasticity after maturation. The molecular cascades of cAMP are well documented, but its spatiotemporal profiles associated with neuronal functions remain hidden. Hence, we developed a genetically encoded cAMP indicator based on a bacterial cAMP-binding protein. This indicator "gCarvi" monitors [cAMP]i at 0.2 to 20 µM with a subsecond time resolution and a high specificity over cyclic guanosine monophosphate (cGMP). gCarvi can be converted to a ratiometric probe for [cAMP]i quantification and its expression can be specifically targeted to various subcellular compartments. Monomeric gCarvi also enables simultaneous multisignal monitoring in combination with other indicators. As a proof of concept, simultaneous cAMP/Ca2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca2+ signals. In cerebellar presynaptic boutons, forskolin induced nonuniform cAMP elevations among boutons, which positively correlated with subsequent increases in the size of the recycling pool of synaptic vesicles assayed using FM dye. Thus, the cAMP domain in presynaptic boutons is an important determinant of the synaptic strength.

The influences of a novel anti-adhesion device, thermally cross-linked gelatin film on peritoneal dissemination of tumor cells: The in vitro and in vivo experiments using murine carcinomatous peritonitis models.

To create anti-adhesive materials to be more effective and safer, we developed a thermally cross-linked gelatin film that showed superior anti-adhesive effects with excellent peritoneal regeneration. However, it may act as a convenient scaffold for tumor cell growth, thereby accelerating peritoneal dissemination when used in surgery for abdominal tumors. In this study, we tried to clarify this issue using mouse carcinomatous peritonitis models. First, we examined the in vitro tumor cell growth of mouse B16 melanoma or Colon26 cells on the gelatin film or the conventional hyarulonate/carboxymethylcellulose film. Tumor cell growth on each film was significantly lower than that of the control (no film). Next, we conducted the following in vivo experiments: After the parietal peritoneum was partially removed and covered with each film or without any film, mice were inoculated intraperitoneally with B16 melanoma or Colon26/Nluc cells expressing NanoLuc luciferase gene. At 7 days after the operation, we measured the weight of B16 melanoma tumors or the NanoLuc activity of Colon26/Nluc cells using in vivo imaging at the injured sites. There were no significant differences in the weight of the tumors and the NanoLuc activity among the three groups. We also observed the survival time of mice receiving the same operation and treatments. There was no significant difference in the survival time among the three groups. These results suggest that the gelatin film will likely not accelerate peritoneal dissemination as a convenient scaffold for tumor cell growth when used in surgery for abdominal tumors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2122-2130, 2018.

Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies.

Giant presynaptic terminal brain slice preparations have allowed intracellular recording of electrical signals and molecular loading, elucidating cellular and molecular mechanisms underlying neurotransmission and modulation. However, molecular genetic manipulation or optical imaging in these preparations is hampered by factors, such as tissue longevity and background fluorescence. To overcome these difficulties, we developed a giant presynaptic terminal culture preparation, which allows genetic manipulation and enables optical measurements of synaptic vesicle dynamics, simultaneously with presynaptic electrical signal recordings. This giant synapse reconstructed from dissociated mouse brainstem neurons resembles the development of native calyceal giant synapses in several respects. Thus, this novel preparation constitutes a powerful tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development. SIGNIFICANCE STATEMENT: We have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.

Developmental shift to a mechanism of synaptic vesicle endocytosis requiring nanodomain Ca2+.

Ca(2+) is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca(2+) coordinates these processes remains unclear, particularly at mature synapses. Using membrane capacitance measurements from calyx of Held nerve terminals in rats, we found that vesicle endocytosis is initiated primarily in Ca(2+) nanodomains around Ca(2+) channels, where exocytosis is triggered. Bulk Ca(2+) outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca(2+)-dependent endocytosis required calmodulin and calcineurin activation at immature synapses, but not at more mature synapses. Similarly, GTP-independent endocytosis, which occurred after extensive exocytosis at immature synapses, became negligible after maturation. We propose that nanodomain Ca(2+) simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca(2+)-dependent endocytosis undergo major developmental changes at this fast central synapse.

Involvement of Ca2+ channel synprint site in synaptic vesicle endocytosis.

The synaptic protein interaction (synprint) site of the voltage-gated Ca(2+) channel (VGCC) alpha1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the mu subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2-synprint interaction occurred over a wide range of Ca(2+) concentrations but was inhibited at high Ca(2+) concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca(2+)-dependent interactions with AP-2 and Syt.

Involvement of AMPA receptor desensitization in short-term synaptic depression at the calyx of Held in developing rats.

Paired-pulse facilitation (PPF) and depression (PPD) are forms of short-term plasticity that are generally thought to reflect changes in transmitter release probability. However, desensitization of postsynaptic AMPA receptors (AMPARs) significantly contributes to PPD at many glutamatergic synapses. To clarify the involvement of AMPAR desensitization in synaptic PPD, we compared PPD with AMPAR desensitization, induced by paired-pulse glutamate application in patches excised from postsynaptic cells at the calyx of Held synapse of developing rats. We found that AMPAR desensitization contributed significantly to PPD before the onset of hearing (P10-12), but that its contribution became negligible after hearing onset. During postnatal development (P7-21) the recovery of AMPARs from desensitization became faster. Concomitantly, glutamate sensitivity of AMPAR desensitization declined. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated a developmental decline of GluR1 expression that correlated with speeding of the recovery of AMPARs from desensitization. Transmitter release probability declined during the second postnatal week (P7-14). Manipulation of the extracellular Ca2+/Mg2+ ratio, to match release probability at P7-8 and P13-15 synapses, revealed that the release probability is also an important factor determining the involvement of AMPAR desensitization in PPD. We conclude that the extent of involvement of AMPAR desensitization in short-term synaptic depression is determined by both pre- and postsynaptic mechanisms.

Developmental changes in calcium/calmodulin-dependent inactivation of calcium currents at the rat calyx of Held.

Ca2+-binding to calmodulin (CaM) causes facilitation and/or inactivation of recombinant Ca2+ channels. At the rat calyx of Held, before hearing onset, presynaptic Ca2+ currents (IpCa) undergo Ca2+/CaM-dependent inactivation during repetitive activation at around 1 Hz, implying that this may be a major cause of short-term synaptic depression. However, it remains open whether the Ca2+/CaM-dependent inactivation of IpCa persists in more mature animals. To address this question, we tested the effect of CaM inhibitors on the activity-dependent modulation of IpCa in calyces, before (postnatal day (P) 7-9) and after (P13-15) hearing onset. Our results indicate that the CaM-dependent IpCa inactivation during low-frequency stimulation, and the ensuing synaptic depression, occur only at calyces in the prehearing period. However, CaM immunoreactivity in P8 and P14 calyces was equally strong. Even at P13-15, high frequency stimulation (200-500 Hz) could induce IpCa inactivation, which was attenuated by EGTA (10 mM) or a CaM inhibitor peptide loaded into the terminal. Furthermore, the CaM inhibitor peptide attenuated a transient facilitation of IpCa preceding inactivation observed at 500 Hz stimulation, whereas it had no effect on sustained IpCa facilitations during trains of 50-200 Hz stimulation. These results suggest that the Ca2+/CaM-dependent IpCa modulation requires a high intraterminal Ca2+ concentration, which can be attained at immature calyces during low frequency stimulation, but only during unusually high frequency stimulation at calyceal terminals in the posthearing period.

4-Chloro-m-cresol, an activator of ryanodine receptors, inhibits voltage-gated K(+) channels at the rat calyx of Held.

4-Chloro-m-cresol (4-CmC) is thought to be a specific activator of ryanodine receptors (RyRs). Using this compound, we examined whether the RyR-mediated Ca(2+) release is involved in transmitter release at the rat calyx of Held synapse in brainstem slices. Bath application of 4-CmC caused a dramatic increase in the amplitude of excitatory postsynaptic currents (TIFCs) with the half-maximal effective concentration of 0.12 mm. By making direct patch-clamp whole-cell recordings from presynaptic terminals, we investigated the mechanism by which 4-CmC facilitates transmitter release. 4-CmC markedly prolonged the duration of action potentials, with little effect on their rise time kinetics. In voltage-clamp recordings, 4-CmC inhibited voltage-gated presynaptic K(+) currents (I(pK)) by 53% (at +20 mV) but had no effect on voltage-gated presynaptic Ca(2+) currents (I(pCa)). In simultaneous pre- and postsynaptic recordings, 4-CmC had no effect on the TIFC evoked by I(pCa). Although immunocytochemical study of the calyceal terminals showed immunoreactivity to type 3 RyRs, ryanodine (0.02 mm) had no effect on the 4-CmC-induced TIFC potentiation. We conclude that the facilitatory effect of 4-CmC on nerve-evoked transmitter release is mediated by its inhibitory effect on I(pK).

Mechanisms underlying developmental speeding in AMPA-EPSC decay time at the calyx of Held.

The time course of synaptic conductance is important in temporal precision of information processing in the neuronal network. The AMPA receptor (AMPAR)-mediated EPSCs at the calyx of Held become faster in decay time as animals mature. To clarify how desensitization and deactivation of AMPARs contribute to developmental speeding of EPSCs, we compared the decay time of quantal EPSCs (qEPSCs) with the deactivation and desensitization times of AMPAR currents induced in excised patches by fast glutamate application (AMPA patch currents). Both the deactivation and desensitization times of AMPA patch currents became markedly faster from postnatal day 7 (P7) to P14 and changed little thereafter. In individual neurons, throughout development (P7-P21), the time constants of deactivation and fast desensitization in AMPA patch currents were similar to each other and close to the qEPSC decay time constant. Cyclothiazide (CTZ) abolished the fast desensitization, prolonged deactivation of AMPA patch currents, and slowed the decay time of EPSCs. The effects of CTZ on AMPA patch currents were unchanged throughout development, whereas its effect on EPSCs became weaker as animals matured. In single-cell reverse transcription-PCR analysis, glutamate receptor subunit 4 (GluR4) flop increased from P7 to P14 and changed little thereafter. At P7, the GluR4 flop abundance had an inverse correlation with the qEPSC decay time. These results together suggest that both desensitization and deactivation of AMPARs are involved in the EPSC decay time, but the contribution of desensitization decreases during postnatal development at the calyx of Held.

Distinct roles of Kv1 and Kv3 potassium channels at the calyx of Held presynaptic terminal.

Despite identification of >100 potassium channel subunits, relatively little is known about their roles in synaptic transmission. To address this issue we recorded presynaptic potassium currents (IPK) directly from the calyx of Held terminal in brainstem slices of rats. IPK was composed of a 4-aminopyridine (4-AP)-sensitive component and a smaller 4-AP-insensitive component composed of an iberiotoxin-sensitive current and an unidentified slowly activating potassium current. IPK could also be separated into a tetraethylammonium (TEA; 1 mm)-sensitive high-voltage-activated component and a margatoxin (10 nm)-sensitive low-voltage-activated component, which was also blocked by dendrotoxin-I (200 nm) and tityustoxin-Kalpha (100 nm). In outside-out patches excised from calyceal terminals, TEA (1 mm) consistently and to a large extent attenuated IPK, whereas margatoxin attenuated IPK only in a subset of patches (three of seven). Immunocytochemical examination using Kv subtype-specific antibodies indicated that multiple Kv1 and Kv3 subtypes were present at the calyceal terminal. In paired presynaptic and postsynaptic whole-cell recordings, TEA (1 mm) increased both the duration and peak amplitude of presynaptic action potentials and simultaneously potentiated EPSCs. Margatoxin alone had no such effect but reduced the amount of depolarization required for action potential generation, thereby inducing a burst of spikes when the nerve terminal was depolarized for a prolonged period. Thus, at the calyx of Held terminal, Kv3 channels directly regulate evoked transmitter release, whereas Kv1 channels reduce nerve terminal excitability, thereby preventing aberrant transmitter release. We conclude that both Kv3 and Kv1 channels contribute differentially to maintaining the fidelity of synaptic transmission at the calyx of Held.

Adenosine A(1) receptor-mediated presynaptic inhibition at the calyx of Held of immature rats.

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5-7) rats, adenosine, or the type 1 adenosine (A1) receptor agonist N6-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A1 receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca(2+) currents (IpCa) recorded directly from calyceal terminals, but had no effect on presynaptic K+ currents. When EPSCs were evoked by IpCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of IpCa fully explained that of EPSCs, suggesting that the presynaptic Ca(2+) channel is the main target of A1 receptors. Whereas the N-type Ca(2+) channel blocker omega-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A1 receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A1 receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca(2+) channels.

Novel mutations in C-terminal channel region of the ryanodine receptor in malignant hyperthermia patients.

Malignant hyperthermia (MH) is a pharmacogenetical complication of general anesthesia resulting from abnormal Ca2+-induced Ca2+ release (CICR) via the type 1 ryanodine receptor (RyR1) in skeletal muscles. In this study, we analyzed the genomic DNAs prepared for determination of all the 106 exons of the RyR1 gene from blood samples donated by two MH patients with extremely high CICR rates in their biopsied skeletal muscles and a clear history of MH incidence. Two novel point mutations were found in the exons 96 and 101 with alterations in the coded amino acids within the C-terminal channel region, i.e., Pro4668 to Ser and Leu4838 to Val. The latter mutation was found in both MH patients. Rabbit RyR1 channels carrying corresponding mutations were expressed in CHO cells for functional assay. It was found that the L to V but not the P to S mutation of the RyR1 resulted in enhanced Ca2+ release activity. These results indicate that the L4838V mutation is responsible for the MH incidence. The L4838V mutation is unique because it is the mutation first found within a hydrophobic transmembrane segment of the channel region and should provide further information on the function of the RyR1 as well as for genetic diagnosis of MH.

Neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals.

P/Q-type presynaptic calcium currents (IpCa) undergo activity-dependent facilitation during repetitive activation at the calyx of the Held synapse. We investigated whether neuronal calcium sensor 1 (NCS-1) may underlie this phenomenon. Direct loading of NCS-1 into the nerve terminal mimicked activity-dependent IpCa facilitation by accelerating the activation time of IpCa in a Ca2+-dependent manner. A presynaptically loaded carboxyl-terminal peptide of NCS-1 abolished IpCa facilitation. These results suggest that residual Ca2+ activates endogenous NCS-1, thereby facilitating IpCa. Because both P/Q-type Ca2+ channels and NCS-1 are widely expressed in mammalian nerve terminals, NCS-1 may contribute to the activity-dependent synaptic facilitation at many synapses.