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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/Ca 2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca 2+ 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.

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca 2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca 2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca 2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses. SIGNIFICANCE STATEMENT Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca 2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo , isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia., (Copyright © 2020 Wang et al.)

Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.

At presynaptic terminals vesicular membranes are fused into plasma membrane upon exocytosis and retrieved by endocytosis. During a sustained high-frequency transmission, exoendocytic coupling is critical for the maintenance of synaptic transmission. Here, we show that this homeostatic coupling is supported by cGMP-dependent protein kinase (PKG) at the calyx of Held. This mechanism starts to operate after hearing onset during the second postnatal week, when PKG expression becomes upregulated in the brainstem. Pharmacological tests with capacitance measurements revealed that presynaptic PKG activity is supported by a retrograde signal cascade mediated by NO that is released by activation of postsynaptic NMDA receptors. Activation of PKG also upregulates phosphatidylinositol-4,5-bisphosphate, thereby accelerating endocytosis. Furthermore, presynaptic PKG activity upregulates synaptic fidelity during high-frequency transmission. We conclude that maturation of the PKG-dependent retrograde signal cascade strengthens the homeostatic plasticity for the maintenance of high-frequency synaptic transmission at the fast glutamatergic synapse., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

During early postnatal development, the calyx of Held synapse in the auditory brainstem of rodents undergoes a variety of morphological and functional changes. Among ionic channels expressed in the calyx, voltage-dependent K+ channels regulate transmitter release by repolarizing the nerve terminal. Here we asked whether voltage-dependent K+ channels in calyceal terminals undergo developmental changes, and whether they contribute to functional maturation of this auditory synapse. From postnatal day (P) 7 to P14, K+ currents became larger and faster in activation kinetics, but did not change any further to P21. Likewise, presynaptic action potentials became shorter in duration from P7 to P14 and remained stable thereafter. The density of presynaptic K+ currents, assessed from excised patch recording and whole-cell recordings with reduced [K+]i, increased by 2-3-fold during the second postnatal week. Pharmacological isolation of K+ current subtypes using tetraethylammonium (1 mM) and margatoxin (10 nM) revealed that the density of Kv3 and Kv1 currents underwent a parallel increase, and their activation kinetics became accelerated by 2-3-fold. In contrast, BK currents, isolated using iberiotoxin (100 nM), showed no significant change during the second postnatal week. Pharmacological block of Kv3 or Kv1 channels at P7 and P14 calyceal terminals indicated that the developmental changes of Kv3 channels contribute to the establishment of reliable action potential generation at high frequency, whereas those of Kv1 channels contribute to stabilizing the nerve terminal. We conclude that developmental changes in K+ currents in the nerve terminal contribute to maturation of high-fidelity fast synaptic transmission at this auditory relay synapse.

5-hydroxytryptamine (5-HT) inhibits transmitter release via activating GTP-binding proteins, but the target of 5-HT receptors in the nerve terminal is not determined. We addressed this question at the calyx of Held synapse in the brainstem slice of immature rats. Bath-application of 5-HT attenuated the amplitude of nerve-evoked excitatory postsynaptic currents (EPSCs) associated with an increase in the paired-pulse ratio, whereas it had no effect on the amplitude of spontaneous miniature EPSCs. The 5-HT1B receptor agonist CP93129 mimicked the inhibitory effect of 5-HT, but the 5-HT1A agonist (R)-(+)-8-hydroxy-DPAT (8-OHDPAT) had no effect. The 5-HT1B receptor antagonist NAS-181 blocked the inhibitory effect of 5-HT. These results suggest that 5-HT activated 5-HT1B receptors in calyceal nerve terminals, thereby inhibiting transmitter release. In direct whole-cell recordings from calyceal nerve terminals, 5-HT attenuated voltage-dependent Ca2+ currents, but had no effect on voltage-dependent K+ currents. When EPSCs were evoked by presynaptic Ca2+ currents during simultaneous pre- and postsynaptic recordings, the magnitude of the 5-HT-induced inhibition of Ca2+ currents fully explained that of EPSCs. Upon repetitive applications, 5-HT showed tachyphylaxis, with its effect on both EPSCs and presynaptic Ca2+ currents becoming weaker in the second application. 1,2-bis(o-aminophenoxy)ethane-N-N'-N'-N'-tetraacetic acid (BAPTA; 10 mm) loaded into the nerve terminal abolished this tachyphylaxis. The presynaptic inhibitory effect of 5-HT was prominent at postnatal day 5, but became weaker as animals matured. We conclude that activation of 5-HT1B receptors inhibits voltage-gated Ca2+ channels, thereby inhibiting transmitter release at immature calyceal nerve terminals, and that 5-HT1B receptors undergo Ca2+-dependent tachyphylaxis on repetitive activations.

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ 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 Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-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 Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse. [ABSTRACT FROM AUTHOR]

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 anal)sis, 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. [ABSTRACT FROM AUTHOR]

AbstractThe γ-aminobutyric acid type A (GABAA) receptor agonist muscimol is widely used as a tool for reducing neuronal activities particularly in experiments in vivo. At the synapse formed by the calyx of Held in the rat brainstem slice, the GABAA receptor agonist muscimol (> 10 μm) attenuated the amplitude of excitatory post synaptic currents (EPSCs) accompanied by an increase in the coefficient of variation of EPSCs, suggesting its presynaptic inhibitory effect. This muscimol effect was not affected by bicuculline but occluded the presynaptic inhibitory effect of the GABAB receptor agonist baclofen and was abolished by the type B GABA (GABAB) receptor-specific antagonist (+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911; 20 μm). We conclude that muscimol activates presynaptic GABAB receptors thereby attenuating synaptic transmission. [ABSTRACT FROM AUTHOR]

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ 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 Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-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 Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse. [ABSTRACT FROM AUTHOR]