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.

Short-term facilitation and depression have a profound influence on transmission at many glutamatergic synapses, particularly during trains of stimuli. A major component of these processes is postsynaptic receptor desensitization. Both presynaptic and postsynaptic mechanisms can contribute to synaptic efficacy, but it is often difficult to define their respective contributions. Blockers of desensitization such as cyclothiazide (CTZ) can be used, but many of these drugs have nonspecific effects on transmitter release, complicating attempts to define synaptic effectiveness under physiological conditions. We describe and validate a new method to minimize desensitization during trains of synaptic stimuli that is based on the low-affinity competitive glutamate receptor antagonists gamma-D-glutamylglycine or kynurenic acid. A computational model of AMPA receptor kinetics shows that the mechanism can be accounted for by simple competitive antagonism of AMPA receptors, where the rapid off-rate of the antagonist permits re-equilibration between blocked and unblocked pools during the interstimulus interval. Our results at the calyx of Held show that desensitization makes little contribution to synaptic depression at frequencies below 10 Hz, but at higher frequencies it makes an important contribution, with accumulating desensitization masking short-term facilitation and causing an underestimation of quantal content. This novel method of protection from desensitization is compatible with physiological studies but cannot be used in conjunction with CTZ. Although presynaptic vesicle depletion makes the dominant contribution to short-term depression, our results show that AMPA receptor desensitization contributes to the depression at auditory synapses after hearing onset and in a frequency-dependent manner.

Voltage‐gated K+ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin‐K (DTX‐K) and tityustoxin‐Kα (TsTX‐Kα) respectively. We show that Kv1.2 homomers are responsible for two‐thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K+ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current‐clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after‐potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX‐K did not alter AP firing. However TsTX‐Kα increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre‐ and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after‐potential.

Glutamate uptake into glial cells helps to keep the brain extracellular glutamate concentration, [glu]o, below levels that kill neurons. Uptake is powered by the transmembrane gradients of Na+, K+ and pH. When the extracellular [K+] rises in brain ischaemia, uptake reverses, releasing glutamate into the extracellular space. Here we show, by monitoring glutamate transport electrically and detecting released glutamate with ion channels in neurons placed outside glial cells, that a raised [H+] inhibits both forward and reversed glutamate uptake. No electroneutral reversed uptake was detected, contradicting the idea that forward and reversed uptake differ fundamentally. Suppression of reversed uptake by the low pH occurring in ischaemia will slow the Ca(2+)-independent release of glutamate with can raise [glu]o to a neurotoxic level, and will thus protect the brain during a transient loss of blood supply.

Glutamate uptake is driven by the cotransport of Na+ ions, the countertransport of K+ ions, and either the countertransport of OH- or the cotransport of H+ ions. In addition, activating glutamate uptake carriers has been shown to lead to activation of an anion conductance present in the carrier structure. Here we characterize the ion selectivity and gating of this anion conductance. The conductance is small with Cl- as the permeant anion, but it is large with NO3- or ClO4- present, undermining the earlier use of NO3- and ClO4- to suggest that OH- countertransport rather than H+ cotransport helps drive uptake. Activation of the anion conductance can be evoked by extra- or intracellular glutamate and can occur even when glutamate transport is inhibited. By running the carrier backward and detecting glutamate release with AMPA receptors in neurons placed near the glial cells, we show that anion flux is not coupled thermodynamically to glutamate movement, but OH-/H+ transport is. The possibility that cell excitability is modulated by the anion conductance associated with glutamate uptake suggests a target for therapeutic drugs to reduce glutamate release in conditions like epilepsy.