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.

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.

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.

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.

METHODS Preparation and solutionsAll experimental procedures were performed in accordance with the animal welfare guidelines of the Physiological Society of Japan. Wistar rats from P5 to P21 were decapitated under halothane anaesthesia (Forsythe & Barnes-Davies, 1993). Brainstem slices (200-400 mm thick) were cut using a tissue slicer (Leica, VT1000S). The solution for cutting tissue contained (mM); 250 sucrose, 2.5 KCl, 26 NaHCO 3 , 10 glucose, 1.25 NaH 2 PO 4 , 1 CaCl 2 , 4 MgCl 2 , 0.3 myo-inositol, 2 sodium pyruvate and 0.5 ascorbic acid (pH 7.4 when bubbled with 5 % CO 2 and 95 % O 2 ). Slices were incubated for 30 min at 35-37°C and maintained thereafter at room temperature in artificial cerebrospinal fluid (aCSF) bubbled with 95 % O 2 and 5 % CO 2 . The standard aCSF for superfusion contained (mM): 120 NaCl, 2.5 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2, 10 D-glucose, 3 myo-inositol, 2 sodium pyruvate, and 0.5 ascorbate; pH was adjusted to 7.4 when saturated with 5 % CO 2 and 95 % O 2 . The superfusate routinely contained bicuculline methiodide (10 mM, Sigma) and strychnine hydrochloride (0.5 mM, Sigma) to block inhibitory synaptic responses. The postsynaptic pipette solution contained (mM): 110 CsF, 30 CsCl, 10 Hepes, 5 EGTA and 1 MgCl 2 (pH 7.4, adjusted with CsOH), and also N-(2,6-diethylphenylcarbamoylmethyl)-triethyl-ammonium chloride (QX-314; 5 mM) to block action potential generation. For recording presynaptic Ca 2+ currents, TTX (1 mM) and tetraethylammonium (TEA) chloride (10 mM) were added to the aCSF, and the pipette solution contained (mM): CsCl 110, Hepes 40, TEA-Cl 10, EGTA 0.5, MgCl 2 1, sodium phosphocreatinine 12, ATP-Mg 2 and GTP 0.5._For recording presynaptic K + currents, TTX (1 mM) was added to the aCSF, and the pipette solution contained (mM): potassium gluconate 97.5, KCl 32.5, Hepes 10, EGTA 5, MgCl 2 1, sodium phosphocreatinine 12, ATP-Mg 2 and GTP 0.5.

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 Ca 2+ /Mg 2+ 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.

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic ␥-aminobutyric acid type B receptors (GABAB receptors) or metabotropic glutamate receptors inhibits presynaptic P͞Q-type Ca 2؉ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein ␤␥ subunits (G␤␥) directly into the calyceal nerve terminal through whole-cell patch pipettes. G␤␥ slowed the activation of presynaptic Ca 2؉ currents (IpCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABAB receptor agonist. The effects of both G␤␥ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, G␤␥ partially occluded the inhibitory effect of baclofen on IpCa. In contrast, guanosine 5 -O-(3-thiotriphosphate)-bound Go␣ loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins Go, but not the Gi, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of Go ␤␥ subunits with presynaptic Ca 2؉ channels.I n the central nervous system, synaptic transmission is regulated by presynaptic autoreceptors or heteroreceptors. These receptors are coupled by G proteins to various targets such as Ca 2ϩ channels, K ϩ channels, or the exocytotic machinery downstream of Ca 2ϩ influx (1). At a rat brainstem auditory synapse formed by a giant nerve terminal, the calyx of Held, metabotropic glutamate receptors (mGluRs), or ␥-aminobutyric acid type B receptors (GABA B receptors) primarily inhibit P͞Q-type Ca 2ϩ channels (2-4). The inhibitory effect of the GABA B receptor agonist baclofen on presynaptic Ca 2ϩ currents can be blocked by the GDP analog guanosine 5Ј-O-(2-thiodiphosphate) (GDP [␤S]) and occluded by the nonhydrolyzable GTP analog guanosine 5Ј-O-(3-thiotriphosphate) (GTP[␥S]), both loaded directly into the calyx, indicating that G proteins mediate the presynaptic inhibition (3). At the cell soma, heterotrimeric G proteins attenuate Ca 2ϩ currents acting either directly via a membrane-delimited pathway involving G protein ␤␥ subunits (G␤␥) or indirectly via second messengers (5, 6). At the nerve terminal, however, it is not known which mechanism underlies this presynaptic inhibition. At the calyx of Held, which can be visually identified in slice, it is possible to load various molecules into the nerve terminal through whole-cell recording pipette (3,7,8). Using this technique, we examined the effect of G␤␥ on I pCa . Also, because of its large structure, it was possible to use immunocytochemistry to determine the subtype of G proteins expressed in the calyceal nerve terminal. Our results...

Activation of protein kinase C (PKC) by phorbol ester facilitates hormonal secretion and transmitter release, and phorbol esterinduced synaptic potentiation (PESP) is a model for presynaptic facilitation. A variety of PKC isoforms are expressed in the central nervous system, but the isoform involved in the PESP has not been identified. To address this question, we have applied immunocytochemical and electrophysiological techniques to the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of rat auditory brainstem. Western blot analysis indicated that both the Ca 2؉ -dependent ''conventional'' PKC and Ca 2؉ -independent ''novel'' PKC isoforms are expressed in the MNTB. Denervation of afferent fibers followed by organotypic culture, however, selectively decreased ''novel'' PKC isoform expressed in this region. The afferent calyx terminal was clearly labeled with the PKC immunofluorescence. On stimulation with phorbol ester, presynaptic PKC underwent autophosphorylation and unidirectional translocation toward the synaptic side. Chelating presynaptic Ca 2؉ , by using membrane permeable EGTA analogue or high concentration of EGTA directly loaded into calyceal terminals, had only a minor attenuating effect on the PESP. We conclude that the Ca 2؉ -independent PKC isoform mediates the PESP at this mammalian central nervous system synapse.