The impact of synaptic conductance on action potential waveform: Evoking realistic action potentials with a simulated synaptic conductance

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Abstract

Most current clamp studies trigger action potentials (APs) by step current injection through the recording electrode and assume that the resulting APs are essentially identical to those triggered by orthodromic synaptic inputs. However this assumption is not always valid, particularly when the synaptic conductance is of large magnitude and of close proximity to the axon initial segment. We addressed this question of similarity using the Calyx of Held/MNTB synapse; we compared APs evoked by long duration step current injections, short step current injections and orthodromic synaptic stimuli. Neither injected current protocol evoked APs that matched the evoked orthodromic AP waveform, showing differences in AP height, half-width and after-hyperpolarization. We postulated that this ‘error’ could arise from changes in the instantaneous conductance during the combined synaptic and AP waveforms, since the driving forces for the respective ionic currents are integrating and continually evolving over this time-course. We demonstrate that a simple Ohm's law manipulation of the EPSC waveform, which accounts for the evolving driving force on the synaptic conductance during the AP, produces waveforms that closely mimic those generated by physiological synaptic stimulation. This stimulation paradigm allows supra-threshold physiological stimulation (single stimuli or trains) without the variability caused by quantal fluctuation in transmitter release, and can be implemented without a specialised dynamic clamp system. Combined with pharmacological tools this method provides a reliable means to assess the physiological roles of postsynaptic ion channels without confounding affects from the presynaptic input.

Introduction

The intrinsic properties of a neuron are crucial in determining how it integrates, computes and transmits information to other cells. Although influenced by morphology and passive cable structure (Mainen and Sejnowski, 1996), the suite of ion channels expressed and their location on the cell membrane are a major determinant of a neuron's processing abilities (Dodson et al., 2003, Ogawa and Rasband, 2008). There are two common methods of stimulation used to approximate physiological inputs under current clamp: injection of step depolarizing current through the recording electrode or electrical stimulation of presynaptic axons (orthodromic synaptic stimulation).

For cells that require excitatory synaptic input to generate action potentials (APs), orthodromic synaptic activation is an ideal physiological stimulus. However, some disadvantages arise when studying the physiology of the postsynaptic cell: first, pharmacological tools may act at pre- and postsynaptic sites (e.g. tetraethylammonium TEA, blocks Kv3 channels and broadens the AP at both sites, causing increased transmitter release, Ishikawa et al., 2003). Second, there are many presynaptic changes which may confound interpretation (e.g. quantal fluctuation, short-term depression, facilitation, presynaptic modulation) particularly during repetitive stimulation.

Direct somatic current injection avoids these problems, but the resultant APs are not accompanied by the synaptic conductance and associated changes in resistance. A more physiological stimulus should take account of the ionic driving forces caused by the voltage changes during the AP.

This study compares different methods of AP generation and provides some simple insights into designing a current injection paradigm that more closely mimics a supra-threshold orthodromic excitatory input. We have used the calyx of Held preparation, since this is a secure synapse with a large safety factor. Our approach has been to make this method broadly applicable, avoiding use of dynamic clamp methods by using a simple computation of the AP and synaptic current. We show that the synaptic conductance has a profound influence on the AP waveform, and assert that this should be considered when studying physiological roles of channels. This method can be simply applied to evoke more physiological AP waveforms without the expense or detailed knowledge of the existing conductances required to implement dynamic clamp methods.

Section snippets

In vitro preparation

Brain slices were prepared as described previously (Postlethwaite et al., 2007, Johnston et al., 2008a). Briefly, CBA/Ca mice or Lister Hooded rats aged P10–P19 were decapitated in accordance with the UK animals (Scientific Procedures) Act 1986 and the brain was removed in a slush of iced artificial CSF (aCSF) of composition (in mM) 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 0.5 ascorbic acid, 26 NaHCO3, 4 MgCl2, 0.1 CaCl2, gassed with 95% O2/5% CO2 (pH 7.4). The brain was placed ventral

Results

Comparison of postsynaptic action potentials generated by three standard types of depolarizing stimulus clearly illustrates that they are not equivalent (Fig. 2) with each producing a different waveform AP in the same neuron. A long current step is often employed in excitable cells to study intrinsic properties and evoke APs, while a brief pulse has the advantage of generating a short latency response and is commonly used to trigger trains of APs. We compared APs evoked by long duration current

Discussion

This paper addresses the simple yet fundamental observation that evoked AP waveforms differ depending on the nature of the triggering stimulus. Step current injections evoked APs with waveforms which were distinct from those elicited by orthodromic synaptic stimulation. By definition, the orthodromic synaptic input is the most physiological method of AP generation, but current injection is often assumed to be equivalent; we show here that it is not. There are several situations where synaptic

Acknowledgements

Funded by the MRC.

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1

Present address: Department of Biology, University of Victoria, BC, Canada.

2

Present address: GU Biology, Pfizer Global R & D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK.

3

Both these authors contributed equally to this work.

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