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.

The activation of ligand-gated ion channels is thought to depend solely on the binding of chemical neurotransmitters. In this study, we demonstrate that kainate (KA) ionotropic glutamate receptors (iGluRs) require not only the neurotransmitter L-glutamate (L-Glu) but also external sodium and chloride ions for activation. Removal of external ions traps KA receptors (KARs) in a novel inactive state that binds L-Glu with picomolar affinity. Moreover, occupancy of KARs by L-Glu precludes external ion binding, demonstrating crosstalk between ligand-and ion-binding sites. AMPA iGluRs function normally in the absence of external ions, revealing that even closely related iGluR subfamilies operate by distinct gating mechanisms. This behavior is interchangeable via a single amino acid residue that operates as a molecular switch to confer AMPA receptor behavior onto KARs. Our findings identify a novel allosteric site that singles out KARs from all other ligand-gated ion channels.

A hyperpolarization‐activated non‐specific cation current, Ih, was examined in bushy cell bodies and their giant presynaptic terminals (calyx of Held). Whole‐cell patch clamp recordings were made using an in vitro brain slice preparation of the cochlear nucleus and the superior olivary complex. The aim was to characterise Ih in identified cell bodies and synaptic terminals, to examine modulation by presynaptic cAMP and to test for modulatory effects of Ih activation on synaptic transmission. Presynaptic Ih was activated by hyperpolarizing voltage‐steps, with half‐activation (V1/2) at –94 mV. Activation time constants were voltage dependent, showing an e‐fold acceleration for hyperpolarizations of –32 mV (time constant of 78 ms at –130 mV). The reversal potential of Ih was –29 mV. It was blocked by external perfusion of 1 mm CsCl but was unaffected by BaCl2. Application of internal cAMP shifted the activation curve to more positive potentials, giving a V1/2 of –74 mV; hence around half of the current was activated at resting membrane potentials. This shift in half‐activation was mimicked by external perfusion of a membrane‐permeant analogue, 8‐bromo‐cAMP. The bushy cell body Ih showed similar properties to those of the synaptic terminal; V1/2 was –94 mV and the reversal potential was –33 mV. Somatic Ih was blocked by CsCl (1 mm) and was partially sensitive to BaCl2. Somatic Ih current density increased with postnatal age from 5 to 16 days old, suggesting that Ih is functionally relevant during maturation of the auditory pathway. The function of Ih in regulating presynaptic excitability is subtle. Ih had little influence on EPSC amplitude at the calyx of Held, but may be associated with propagation of the action potential at branch points. Presynaptic Ih shares properties with both HCN1 and HCN2 recombinant channel subunits, in that it gates relatively rapidly and is modulated by internal cAMP.

Sigma-1 receptors (-1Rs) are endoplasmic reticulum resident chaperone proteins implicated in many physiological and pathological processes in the CNS. A striking feature of -1Rs is their ability to interact and modulate a large number of voltage-and ligand-gated ion channels at the plasma membrane. We have reported previously that agonists for -1Rs potentiate NMDA receptor (NMDAR) currents, although the mechanism by which this occurs is still unclear. In this study, we show that in vivo administration of the selective -1R agonists (ϩ)-SKF 10,047 [2S-(2␣,6␣,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride (N-allylnormetazocine) hydrochloride], PRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate hydrochloride), and (ϩ)-pentazocine increases the expression of GluN2A and GluN2B subunits, as well as postsynaptic density protein 95 in the rat hippocampus. We also demonstrate that -1R activation leads to an increased interaction between GluN2 subunits and -1Rs and mediates trafficking of NMDARs to the cell surface. These results suggest that -1R may play an important role in NMDAR-mediated functions, such as learning and memory. It also opens new avenues for additional studies into a multitude of pathological conditions in which NMDARs are involved, including schizophrenia, dementia, and stroke.

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca2+]i) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca2+-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca2+]i increase in OV2008 cells was not effected by the elimination of extracellular Ca2+, but this was attenuated by the depletion of internal Ca2+ store, indicating that mobilization of intracellular Ca2+] stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca2+-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca2+/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.

Presynaptic group III metabotropic glutamate receptor (mGluR) activation by exogenous agonists (such as L-2-amino-4-phosphonobutyrate (L-AP4)) potently inhibit transmitter release, but their autoreceptor function has been questioned because endogenous activation during high-frequency stimulation appears to have little impact on synaptic amplitude. We resolve this ambiguity by studying endogenous activation of mGluRs during trains of high-frequency synaptic stimuli at the calyx of Held. In vitro whole-cell patch recordings were made from medial nucleus of the trapezoid body (MNTB) neurones during 1 s excitatory postsynaptic current (EPSC) trains delivered at 200 Hz and at 37• C. The group III mGluR antagonist (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG, 300 µM) had no effect on EPSC short-term depression, but accelerated subsequent recovery time course (τ : 4.6 ± 0.8 s to 2.4 ± 0.4 s, P = 0.02), and decreased paired pulse ratio from 1.18 ± 0.06 to 0.97 ± 0.03 (P = 0.01), indicating that mGluR activation reduced release probability (P). Modelling autoreceptor activation during repetitive stimulation revealed that as P declines, the readily releasable pool size (N ) increases so that the net EPSC (NP) is unchanged and short-term depression proceeds with the same overall time course as in the absence of autoreceptor activation. Thus, autoreceptor action on the synaptic response is masked but the synapse is now in a different state (lower P, higher N ). While vesicle replenishment clearly underlies much of the recovery from short-term depression, our results show that the recovery time course of P also contributes to the reduced response amplitude for 1-2 s. The results show that passive equilibration between N and P masks autoreceptor modulation of the EPSC and suggests that mGluR autoreceptors function to change the synaptic state and distribute metabolic demand, rather than to depress synaptic amplitude.

Kainate-selective ionotropic glutamate receptors (GluRs) require external Na ϩ and Cl Ϫ as well as the neurotransmitter L-glutamate for activation. Although, external anions and cations apparently coactivate kainate receptors (KARs) in an identical manner, it has yet to be established how ions of opposite charge achieve this. An additional complication is that KARs are subject to other forms of cation modulation via extracellular acidification (i.e., protons) and divalent ions. Consequently, other cation species may compete with Na ϩ to regulate the time KARs remain in the open state. Here we designed experiments to unravel how external ions regulate GluR6 KARs. We show that GluR6 kinetics are unaffected by alterations in physiological pH but that divalent and alkali metal ions compete to determine the time course of KAR channel activity. Additionally, Na ϩ and Cl Ϫ ions coactivate GluR6 receptors by establishing a dipole, accounting for their common effect on KARs. Using charged amino acids as tethered ions, we further demonstrate that the docking order is fixed with cations binding first, followed by anions. Together, our findings identify the dipole as a novel gating feature that couples neurotransmitter binding to KAR activation.

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