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Using a combination of patch‐clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. Voltage‐clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low‐threshold dendrotoxin (DTX)‐sensitive current (ILT) and a high‐threshold DTX‐insensitive current (IHT). I HT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mm). The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. An mRNA probe corresponding to the C‐terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1‐ like current is essential for cells to respond to a sustained train of high‐frequency stimuli. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high‐frequency inputs.

Voltage-gated calcium channels are well characterized at neuronal somata but less thoroughly understood at the presynaptic terminal where they trigger transmitter release. In order to elucidate how the intrinsic properties of presynaptic calcium channels influence synaptic function, we have made direct recordings of the presynaptic calcium current (I(pCa)) in a brainstem giant synapse called the calyx of Held. The current was pharmacologically classified as P-type and exhibited marked inactivation. The inactivation was largely dependent upon the inward calcium current magnitude rather than the membrane potential, displayed little selectivity between divalent charge carriers (Ca2+, Ba2+ and Sr+), and exhibited slow recovery. Simultaneous pre- and postsynaptic whole-cell recording revealed that I(pCa) inactivation predominantly contributes to posttetanic depression of EPSCs. Thus, because of its slow recovery, I(pCa) inactivation underlies this short-term synaptic plasticity.

Beyond their role in generating ATP, mitochondria have a high capacity to sequester calcium. The interdependence of these functions and limited access to presynaptic compartments makes it difficult to assess the role of sequestration in synaptic transmission. We addressed this important question using the calyx of Held as a model glutamatergic synapse by combining patch-clamp with a novel mitochondrial imaging method. Presynaptic calcium current, mitochondrial calcium concentration ([Ca(2+)](mito), measured using rhod-2 or rhod-FF), cytoplasmic calcium concentration ([Ca(2+)](cyto), measured using fura-FF), and the postsynaptic current were monitored during synaptic transmission. Presynaptic [Ca(2+)](cyto) rose to 8.5 +/- 1.1 microM and decayed rapidly with a time constant of 45 +/- 3 msec; presynaptic [Ca(2+)](mito) also rose rapidly to >5 microM but decayed slowly with a half-time of 1.5 +/- 0.4 sec. Mitochondrial depolarization with rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished mitochondrial calcium rises and slowed the removal of [Ca(2+)](cyto) by 239 +/- 22%. Using simultaneous presynaptic and postsynaptic patch clamp, combined with presynaptic mitochondrial and cytoplasmic imaging, we investigated the influence of mitochondrial calcium sequestration on transmitter release. Depletion of ATP to maintain mitochondrial membrane potential was blocked with oligomycin, and ATP was provided in the patch pipette. Mitochondrial depolarization raised [Ca(2+)](cyto) and reduced transmitter release after short EPSC trains (100 msec, 200 Hz); this effect was reversed by raising mobile calcium buffering with EGTA. Our results suggest a new role for presynaptic mitochondria in maintaining transmission by accelerating recovery from synaptic depression after periods of moderate activity. Without detectable thapsigargin-sensitive presynaptic calcium stores, we conclude that mitochondria are the major organelle regulating presynaptic calcium at central glutamatergic terminals.

1. An in vitro brainstem slice preparation of the superior olivary complex has been developed permitting patch recording from a presynaptic terminal (calyx of Held) and from its postsynaptic target‐‐the principal neurone of the medial nucleus of the trapezoid body (MNTB). 2. The fluorescent stain DiI (1,1'‐dioctadecyl‐3,3,3',3'‐tetramethylindocarbocyanine perchlorate) was used in fixed tissue and Lucifer Yellow in living slices, to identify calices enclosing single MNTB neuronal somata. 3. Whole‐cell recording from the MNTB neurone shows evoked EPSCs preceded by a prespike, corresponding to the presynaptic action potential (AP). In some cases one patch pipette recorded from both pre‐ and postsynaptic elements, but confirmation of exclusively presynaptic recording was obtained using pipettes containing Lucifer Yellow in a further eleven cases. 4. Under current clamp, the pre‐ and postsynaptic sites could be distinguished by their response to step depolarizations; presynaptic terminals generated a train of APs at frequencies up to 200 Hz, while MNTB neurones gave a single AP. Each presynaptic AP had an after‐hyperpolarization lasting less than 2 ms. 5. Under voltage clamp, step depolarizations of presynaptic terminals generated a tetrodotoxin‐sensitive inward current followed by rapidly activating outward potassium currents at potentials more positive than ‐60 mV. The outward current exhibited little inactivation over the 150 ms steps and 4‐aminopyridine (200 microM) blocked 63.0 +/‐ 14.5% (mean +/‐ S.D., n = 3) of the sustained current at 0 mV. Like the squid giant synapse, mammalian terminals express rapidly activating ‘delayed rectifier’‐type potassium currents.

Metabotropic glutamate receptors (mGluRs) regulate transmitter release at mammalian central synapses. However, because of the difficulty of recording from mammalian presynaptic terminals, the mechanism underlying mGluR-mediated presynaptic inhibition is not known. Here, simultaneous recordings from a giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body were obtained in rat brainstem slices. Agonists of mGluRs suppressed a high voltage-activated P/Q-type calcium conductance in the presynaptic terminal, thereby inhibiting transmitter release at this glutamatergic synapse. Because several forms of presynaptic modulation and plasticity are mediated by mGluRs, this identification of a target ion channel is a first step toward elucidation of their molecular mechanism.

Nitric oxide (NO) is an important signaling molecule that is widely used in the nervous system. With recognition of its roles in synaptic plasticity (long-term potentiation, LTP; long-term depression, LTD) and elucidation of calcium-dependent, NMDAR-mediated activation of neuronal nitric oxide synthase (nNOS), numerous molecular and pharmacological tools have been used to explore the physiology and pathological consequences for nitrergic signaling. In this review, the authors summarize the current understanding of this subtle signaling pathway, discuss the evidence for nitrergic modulation of ion channels and homeostatic modulation of intrinsic excitability, and speculate about the pathological consequences of spillover between different nitrergic compartments in contributing to aberrant signaling in neurodegenerative disorders. Accumulating evidence points to various ion channels and particularly voltage-gated potassium channels as signaling targets, whereby NO mediates activity-dependent control of intrinsic neuronal excitability; such changes could underlie broader mechanisms of synaptic plasticity across neuronal networks. In addition, the inability to constrain NO diffusion suggests that spillover from endothelium (eNOS) and/or immune compartments (iNOS) into the nervous system provides potential pathological sources of NO and where control failure in these other systems could have broader neurological implications. Abnormal NO signaling could therefore contribute to a variety of neurodegenerative pathologies such as stroke/excitotoxicity, Alzheimer's disease, multiple sclerosis, and Parkinson's disease.

Low-threshold voltage-gated potassium currents (I(LT)) activating close to resting membrane potentials play an important role in shaping action potential (AP) firing patterns. In the medial nucleus of the trapezoid body (MNTB), I(LT) ensures generation of single APs during each EPSP, so that the timing and pattern of AP firing is preserved on transmission across this relay synapse (calyx of Held). This temporal information is critical for computation of sound location using interaural timing and level differences. I(LT) currents are generated by dendrotoxin-I-sensitive, Shaker-related K+ channels; our immunohistochemistry confirms that MNTB neurons express Kv1.1, Kv1.2, and Kv1.6 subunits. We used subunit-specific toxins to separate I(LT) into two components, each contributing approximately one-half of the total low-threshold current: (1) I(LTS), a tityustoxin-Kalpha-sensitive current (TsTX) (known to block Kv1.2 containing channels), and (2) I(LTR), an TsTX-resistant current. Both components were sensitive to the Kv1.1-specific toxin dendrotoxin-K and were insensitive to tetraethylammonium (1 mm). This pharmacological profile excludes homomeric Kv1.1 or Kv1.2 channels and is consistent with I(LTS) channels being Kv1.1/Kv1.2 heteromers, whereas I(LTR) channels are probably Kv1.1/Kv1.6 heteromers. Although they have similar kinetic properties, I(LTS) is critical for generating the phenotypic single AP response of MNTB neurons. Immunohistochemistry confirms that Kv1.1 and Kv1.2 (I(LTS) channels), but not Kv1.6, are concentrated in the first 20 microm of MNTB axons. Our results show that heteromeric channels containing Kv1.2 subunits govern AP firing and suggest that their localization at the initial segment of MNTB axons can explain their dominance of AP firing behavior.