Adrian Y. C. Wong

Modulation of a presynaptic hyperpolarization‐activated cationic current (Ih) at an excitatory synaptic terminal in the rat auditory brainstem

1 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. 2 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. 3 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. 4 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. 5 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.

Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS

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.

Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses

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.

Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells

1 The single channel properties of P2X ATP receptors were investigated in outside‐out patches from hippocampal granule cells in brain slices from 12‐day‐old rats. The results demonstrate that functional P2X ATP receptors are expressed in hippocampal granule cells and, combined with previously published information on the P2X subunits expressed in the hippocampus, suggest that the receptors may be heteromers of the P2X4 and P2X6 subunits or P2X1, P2X2, P2X4 and P2X6 subunits. 2 Two distinct types of P2X channel openings were observed. A flickery P2X receptor channel was observed in three patches with a mean chord conductance of 32 ± 6 pS, a mean open time of 1.0 ± 0.3 ms and a mean burst length of 11 ± 5 ms at a membrane potential of −60 mV. A large conductance P2X receptor was observed in 19 out of 98 patches with a mean conductance of 56 ± 1.8 pS, a linear current‐voltage relationship between −80 and +60 mV with a reversal potential around 0 mV, a mean open time of 2.6 ± 0.2 ms and a mean burst length of 8.8 ± 1.8 ms at −60 mV. At an ATP concentration of 1 mm, these channels exhibited a low steady‐state open probability (Popen, 0.07 ± 0.008; n= 15), little apparent desensitisation and were also activated by α,β‐methylene ATP (α,β‐meATP, 40 μm; Popen, 0.007 ± 0.0002; conductance, 57 ± 1.1 pS; n= 3). No decrease in the single channel conductance was observed on increasing the free extracellular calcium concentration from 0.3 to 0.85 mm. 3 Channel closed time distributions were fitted with five exponential components with time constants (and relative areas) of 90 μs (20 %), 0.77 ms (32 %), 10 ms (15 %), 90 ms (18 %) and 403 ms (15 %) at 1 mm ATP. Of these, the first two components are suggested to represent gaps within single activations of the receptor based on the lack of agonist concentration dependence of these two shut time components between 1 μm and 1 mm ATP. 4 Suramin (40 μm) significantly increased the single channel conductance (19 ± 7 %; n= 5) and produced a small decrease in Popen (39 ± 9 %; n= 5) by decreasing mean open time, burst length and total open time per burst. These actions of suramin are not consistent with simple competitive antagonism.

External Ions Are Coactivators of Kainate Receptors

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.

Na+/Cl− Dipole Couples Agonist Binding to Kainate Receptor Activation

Kainate-selective ionotropic glutamate receptors (GluRs) require external Na+ and Cl− 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+ 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+ and Cl− 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.

Detecting synaptic connections in the medial nucleus of the trapezoid body using calcium imaging

Abstract. The study of synaptic transmission in brain slices generally entails the patch-clamping of postsynaptic neurones and stimulation of identified presynaptic axons using a remote electrical stimulating electrode. Although patch recording from postsynaptic neurones is routine, many presynaptic axons take tortuous turns and are severed in the slicing procedure, blocking propagation of the action potential to the synaptic terminal and preventing synaptic stimulation. Here we demonstrate a method of using calcium imaging to select postsynaptic cells with functional synaptic inputs prior to patch-clamp recording. We have used this method for exploring transmission in the auditory brainstem at the medial nucleus of the trapezoid body neurones, which are innervated by axons from the contralateral cochlear nucleus. Brainstem slices were briefly loaded with the calcium indicator fura-2 AM and stimulated with an electrode placed on the midline. Electrical stimulation caused a rise in intracellular calcium concentration in those postsynaptic neurones with active synaptic connections. Since <10% of the medial nucleus of the trapezoid body neurones retain viable synaptic inputs following the slicing procedure, preselecting those cells with active synapses dramatically increased our recording success. This detection method will greatly ease the study of synaptic responses in brain areas where suprathreshold synaptic inputs occur but connectivity is sparse.

A computational model of synaptic transmission at the calyx of Held

The calyx of Held is a giant glutamatergic synapse in the mammalian auditory pathway designed to ensure faithful transmission of high frequency action potential trains. Pre- and postsynaptic recordings from this synapse reveal several forms of facilitation and depression. A computational model of synaptic transmission has been developed to investigate the mechanisms underlying modulation at the calyx of Held. The model includes paired-pulse facilitation and depression due to vesicle depletion and postsynaptic AMPA receptor desensitization. Accurately matching the experimental time course and magnitude of the depression requires both slow and fast replenishment of vesicles at the presynaptic release sites. The model demonstrates that the temporal accuracy of postsynaptic spike generation depends on the amplitude of the EPSC and so accuracy decreases as the EPSCs depress. 2001 Elsevier Science B.V. All rights reserved.

A multi-component model of depression at the calyx of Held

An average-response model of depression at the calyx of Held, a giant glutamatergic synapse in the mammalian auditory pathway, is presented. The model is 2t to experimentally recorded EPSC amplitudes resulting from 10 to 100 Hz presynaptic stimulation for 1 s. This data exhibits a strong depression of the EPSC amplitude. The model best 2ts the time course and magnitude of depression when signi2cant postsynaptic receptor desensitisation and vesicle replenishment from a small, depleting reserve pool were included. c

Cations But Not Anions Regulate the Responsiveness of Kainate Receptors

Kainate-selective ionotropic glutamate receptors are unique among ligand-gated ion channels in their obligate requirement of external anions and cations for activation. Although it is established that the degree of kainate receptor (KAR) activation is shaped by the chemical nature of the agonist molecule, the possible complementary role of external ions has yet to be examined. Here we show that external cations but not anions regulate the responsiveness to a range of full and partial agonists acting on rat GluK2 receptors. This observation is unexpected as previous work has assumed anions and cations affect KARs in an identical manner through functionally coupled binding sites. However, our data demonstrate that anion- and cation-binding pockets behave discretely. We suggest cations uniquely regulate a pregating or flipping step that impacts the closed-cleft stability of the agonist-binding domain (ABD). This model departs from a previous proposal that KAR agonist efficacy is governed by the degree of closure elicited in the ABD by ligand binding. Our findings are, however, in line with recent studies on Cys-loop ligand-gated ion channels suggesting that the “flipping” mechanism has been conserved by structurally diverse ligand-gated ion channel families as a common means of regulating neurotransmitter behavior.