Margaret Barnes-Davies

Presynaptic Calcium Current Modulation by a Metabotropic Glutamate Receptor

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.

INACTIVATION OF PRESYNAPTIC CALCIUM CURRENT CONTRIBUTES TO SYNAPTIC DEPRESSION AT A FAST CENTRAL SYNAPSE

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.

Pre- and postsynaptic glutamate receptors at a giant excitatory synapse in rat auditory brainstem slices.

1. Whole‐cell patch recordings were used to examine the EPSC generated by the calyx of Held in neurones of the medial nucleus of the trapezoid body (MNTB). Each neurone receives a somatic input from a single calyx (giant synapse). 2. A slow NMDA receptor‐mediated EPSC peaked in 10 ms and decayed as a double exponential with time constants of 44 and 147 ms. A fast EPSC had a mean rise time of 356 microseconds (at 25 degrees C), while the decay was described by a double exponential with time constants of 0.70 and 3.43 ms. 3. Cyclothiazide slowed the decay of the fast EPSC, indicating that it is mediated by AMPA receptors. The slower time constant was slowed to a greater extent than the faster time constant. Cyclothiazide potentiated EPSC amplitude, partly by a presynaptic mechanism. 4. The metabotropic glutamate receptor (mGluR) agonists, 1S,3S‐ACPD, 1S,3R‐ACPD and L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) reversibly depressed EPSC amplitude. A dose‐response curve for 1S,3S‐ACPD gave an EC50 of 7 microM and a Hill coefficient of 1.2. 5. Analysis of the coefficient of variation ratio showed that the above mGluR agonists acted presynaptically to reduce the probability of transmitter release. Adenosine and baclofen also depressed transmission by a presynaptic mechanism. 6. alpha‐Methyl‐4‐carboxyphenylglycine (MCPG; 0.5‐1 mM) did not antagonize the effects of 1S,3S‐ACPD, while high concentrations of L‐2‐amino‐3‐phosphonopropionic acid (L‐AP3; 1 mM) and 4‐carboxy‐3‐hydroxyphenyglycine (4C3HPG; 500 microM) depressed transmission. 7. There was a power relationship between [Ca2+]o and EPSC amplitude with co‐operativity values ranging from 1.5 to 3.4. 8. The mechanism by which mGluRs modulate transmitter release appeared to be independent of presynaptic Ca2+ or K+ currents, since ACPD caused no change in the level of paired‐pulse facilitation or the duration of the presynaptic action potential (observed by direct recording from the terminal), indicating that the presynaptic mGluR transduction mechanism may be coupled to part of the exocytotic machinery. 9. Our data are not consistent with the presence at the calyx of Held of any one known mGluR subtype. Comparison of the time course and pharmacology of the fast EPSC with data from cloned AMPA receptors is consistent with the idea that GluR‐Do subunits dominate the postsynaptic channels.

The binaural auditory pathway: excitatory amino acid receptors mediate dual timecourse excitatory postsynaptic currents in the rat medial nucleus of the trapezoid body.

We show here that synaptic transmission to the medial nucleus of the trapezoid body (MNTB) is mediated principally by excitatory amino acid receptors and has two components. A fast excitatory postsynaptic current (EPSC) is mediated by non-NMDA receptors and a slow EPSC is mediated by NMDA receptors. Each neuron receives a large synaptic input (calyx of Held) which produces an EPSC with a mean peak conductance of 37 nS. The somatic location of this synapse gives good resolution of the EPSC timecourse with the fast EPSC decaying with a time constant of 1.1 ms (at 25 °C). The slow EPSC exhibits a double exponential decay with time constants of 41 ms and 106 ms and is voltage dependent in the presence of extracellular magnesium. Other smaller EPSCS mediated by NMDA and non-NMDA receptors, and a strychnine-sensitive synaptic current, are also present. Although the intrinsic membrane properties of MNTB neurons (Forsythe & Barnes-Davies (Proc. R. Soc. Lond. B 251, 143 (1993)), preceding paper) promote high-fidelity transmission, we show that voltage-dependent modulation of synaptic transmission can occur. Given the specialization of the calyx of Held, it seems that the NMDA—receptor ion channel complex is not primarily serving to potentiate a subthreshold input, but may be involved in the development and maintenance of this exuberant somatic synapse.

Kv1 currents mediate a gradient of principal neuron excitability across the tonotopic axis in the rat lateral superior olive.

Principal neurons of the lateral superior olive (LSO) detect interaural intensity differences by integration of excitatory projections from ipsilateral bushy cells and inhibitory inputs from the medial nucleus of the trapezoid body. The intrinsic membrane currents active around firing threshold will form an important component of this binaural computation. Whole cell patch recording in an in vitro brain slice preparation was employed to study conductances regulating action potential (AP) firing in principal neurons. Current‐clamp recordings from different neurons showed two types of firing pattern on depolarization, one group fired only a single initial AP and had low input resistance while the second group fired multiple APs and had a high input resistance. Under voltage‐clamp, single‐spiking neurons showed significantly higher levels of a dendrotoxin‐sensitive, low threshold potassium current (ILT). Block of ILT by dendrotoxin‐I allowed single‐spiking cells to fire multiple APs and indicated that this current was mediated by Kv1 channels. Both neuronal types were morphologically similar and possessed similar amounts of the hyperpolarization‐activated nonspecific cation conductance (Ih). However, single‐spiking cells predominated in the lateral limb of the LSO (receiving low frequency sound inputs) while multiple‐firing cells dominated the medial limb. This functional gradient was mirrored by a medio‐lateral distribution of Kv1.1 immunolabelling. We conclude that Kv1 channels underlie the gradient of LSO principal neuron firing properties. The properties of single‐spiking neurons would render them particularly suited to preserving timing information.

The Binaural Auditory Pathway: Membrane Currents Limiting Multiple Action Potential Generation in the Rat Medial Nucleus of the Trapezoid Body

In this paper we describe the membrane currents of neurons in the medial nucleus of the trapezoid body (MNTB), which serves as an inverting relay in the binaural auditory pathway. In the following paper (Forsythe & Barnes-Davies (Proc. R. Soc. Lond. B 251, 151 (1993))) we describe the synaptic inputs to the MNTB and discuss the significance of these results for transmission through this nucleus, where the fidelity of information transfer will depend on the integration of synaptic responses with the intrinsic postsynaptic membrane properties. Whole-cell patch clamp recordings were made from MNTB neurons using a thin-slice preparation of the rat brain stem. Resting potentials were —70 mV with a neuronal input resistance of 250 MΩ and a membrane time constant of 14 ms. Voltage-clamp studies showed that MNTB neurons possess an inward sodium current, an outward current similar to a delayed rectifier and an inward rectifier. In addition, a novel transient outward current exhibiting rapid kinetics and a sustained current are present, which are both blocked by micromolar concentrations of 4-aminopyridine (4AP). Current-clamp recording showed that MNTB neurons respond to depolarization with a single overshooting action potential (AP) ; 4AP blocked a fast after-hyperpolarization, increased AP duration, and converted the single AP response on depolarization to a train of action potentials.

Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones

1 Whole‐cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). 2 Both high voltage‐activated (HVA) and low voltage‐activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. 3 The transient LVA calcium current activated rapidly on depolarization (half‐activation, −59 mV) and inactivated during maintained depolarization (half‐inactivation, −89 mV). This T‐type current was observed in somatic recordings but was absent from presynaptic terminals. 4 On the basis of their pharmacological sensitivity, P/Q‐type Ca2+ channels accounted for only 6 % of the somatic HVA, while L‐, N‐ and R‐type Ca2+ channels each accounted for around one‐third of the somatic calcium current. 5 The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. 6 We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P‐type contribute little to somatic currents but predominate at the synaptic terminal; L‐, N‐ and R‐types dominate at the soma, but contribute negligibly to calcium currents in the terminal.

Lateral olivocochlear (LOC) neurons of the mouse LSO receive excitatory and inhibitory synaptic inputs with slower kinetics than LSO principal neurons

We examined membrane properties and synaptic responses of neurons in the mouse lateral superior olivary nucleus (LSO). Two clear populations were identified consistent with: principal neurons which are involved in detecting interaural intensity differences (IIDs) and efferent neurons of the lateral olivocochlear (LOC) system which project to the cochlea. Principal neurons fired a short latency action potential (AP) often followed by an AP train during maintained depolarization. They possessed sustained outward K(+) currents, with little or no transient K(+) current (I(A)) and a prominent hyperpolarization-activated non-specific cation conductance, I(H). On depolarization, LOC neurons exhibited a characteristic delay to the first AP. These neurons possessed a prominent transient outward current I(A), but had no I(H). Both LOC and principal neurons received glutamatergic and glycinergic synaptic inputs. LOC synaptic responses decayed more slowly than those of principal neurons; the mean decay time constant of AMPA receptor-mediated EPSCs was around 1 ms in principal neurons and 4 ms in LOC neurons. Decay time constants for glycinergic IPSCs were around 5 ms in principal neurons and 10 ms in LOC neurons. We conclude that principal cells receive fast synaptic responses appropriate for integration of IID inputs, while the LOC cells possess excitatory and inhibitory receptors with much slower kinetics.

Protection from Noise-Induced Hearing Loss by Kv2.2 Potassium Currents in the Central Medial Olivocochlear System

The central auditory brainstem provides an efferent projection known as the medial olivocochlear (MOC) system, which regulates the cochlear amplifier and mediates protection on exposure to loud sound. It arises from neurons of the ventral nucleus of the trapezoid body (VNTB), so control of neuronal excitability in this pathway has profound effects on hearing. The VNTB and the medial nucleus of the trapezoid body are the only sites of expression for the Kv2.2 voltage-gated potassium channel in the auditory brainstem, consistent with a specialized function of these channels. In the absence of unambiguous antagonists, we used recombinant and transgenic methods to examine how Kv2.2 contributes to MOC efferent function. Viral gene transfer of dominant-negative Kv2.2 in wild-type mice suppressed outward K+ currents, increasing action potential (AP) half-width and reducing repetitive firing. Similarly, VNTB neurons from Kv2.2 knock-out mice (Kv2.2KO) also showed increased AP duration. Control experiments established that Kv2.2 was not expressed in the cochlea, so any changes in auditory function in the Kv2.2KO mouse must be of central origin. Further, in vivo recordings of auditory brainstem responses revealed that these Kv2.2KO mice were more susceptible to noise-induced hearing loss. We conclude that Kv2.2 regulates neuronal excitability in these brainstem nuclei by maintaining short APs and enhancing high-frequency firing. This safeguards efferent MOC firing during high-intensity sounds and is crucial in the mediation of protection after auditory overexposure.

Calcium channels triggering transmitter release in the rat medial superior olive

We used whole cell voltage clamp recordings from neurones in rat auditory brainstem slices to study the Ca(2+) channel types involved in triggering synaptic glutamate and glycine release in the medial superior olivary nucleus. Glutamate release from the anterior ventral cochlear (aVCN) bushy neurone synapse did not involve L-type Ca(2+) channels (alpha(1C-D); Ca(V)1.2-1.3), but was mediated with similar efficacies by both N-type (alpha(1B); Ca(V)2.2) and the P/Q-type Ca(2+) channels (alpha(1A); Ca(V)2.1). Glycine release from the medial nucleus of the trapezoid body (MNTB) synapse was mediated predominantly by P/Q-type Ca(2+) channels, but with a significant contribution from N-type Ca(2+) channels. Combined application of the P/Q- and N-type Ca(2+) channel toxins, omega-agatoxin IVA and omega-conotoxin GVIA, left a very small remnant of both the inhibitory and excitatory postsynaptic currents, probably reflecting a minimal contribution of R-type Ca(2+) channels (alpha(1E); Ca(V)2.3) to transmitter release. In contrast with aVCN bushy neurones, MNTB somata lacked both T- (alpha(1G-I); Ca(V)3.1-3.3) and L-type channels, but expressed a higher proportion of P/Q-type Ca(2+) channels.