Elsevier

Brain Research

Volume 1221, 24 July 2008, Pages 24-29
Brain Research

Research Report
Tetraethylammonium (TEA) increases the inactivation time constant of the transient K+ current in suprachiasmatic nucleus neurons

https://doi.org/10.1016/j.brainres.2008.05.025Get rights and content

Abstract

Identifying the mechanisms that drive suprachiasmatic nucleus (SCN) neurons to fire action potentials with a higher frequency during the day than during the night is an important goal of circadian neurobiology. Selective chemical tools with defined mechanisms of action on specific ion channels are required for successful completion of these studies. The transient K+ current (IA) plays an active role in neuronal action potential firing and may contribute to modulating the circadian firing frequency. Tetraethylammonium (TEA) is frequently used to inhibit delayed rectifier K+ currents (IDR) during electrophysiological recordings of IA. Depolarizing voltage-clamped hamster SCN neurons from a hyperpolarized holding potential activated both IA and IDR. Holding the membrane potential at depolarized values inactivated IA and only the IDR was activated during a voltage step. The identity of IA was confirmed by applying 4-aminopyridine (5 mM), which significantly inhibited IA. Reducing the TEA concentration from 40 mM to 1 mM significantly decreased the IA inactivation time constant (τinact) from 9.8 ± 3.0 ms to 4.9 ± 1.2 ms. The changes in IAτinact were unlikely to be due to a surface charge effect. The IA amplitude was not affected by TEA at any concentration or membrane potential. The isosmotic replacement of NaCl with choline chloride had no effect in IA kinetics demonstrating that the TEA effects were not due to a reduction of extracellular NaCl. We conclude that TEA modulates, in a concentration dependent manner, τinact but not IA amplitude in hamster SCN neurons.

Introduction

Neurons in the hypothalamic suprachiasmatic nucleus (SCN) contain a molecular clock that drives circadian rhythms. The circadian clock determines many facets of SCN neuronal activity including the frequency of action potential firing and also the strength of afferent excitatory and inhibitory synapses (Gompf and Allen, 2004, Itri et al., 2005, Lundkvist et al., 2002, Pennartz et al., 2001). The mechanisms underlying the circadian regulation of cellular electrical activity remain largely unknown. Several ionic currents including a persistent Na+ current, delayed rectifier K+ channels (IDR), large conductance Ca2+-activated K+ channels (BK), transient K+ current (A-type, IA), and voltage-dependent L- and T-type Ca2+ channels regulate action potential firing or show significant day–night differences in activity or both (Itri et al., 2004, Jackson et al., 2004, Kim et al., 2005, Kononenko et al., 2004, Kuhlman and McMahon, 2004, Meredith et al., 2006, Pennartz et al., 2002, Pitts et al., 2006). Identification of the contribution that each ion channel makes to the firing of SCN neurons requires selective chemical tools with defined mechanisms of action.

IA is a rapidly activating, rapidly inactivating K+ current that contributes to setting the timing between action potentials and the postsynaptic responses to synaptic inputs (Connor and Stevens, 1971). IA is observed in the majority of SCN neurons and may play a role in setting the action potential firing frequency in these cells (Bouskila and Dudek, 1995, Huang, 1993, Huang et al., 1993). IA is carried by ion channels composed of α subunits of the Kv4 family (Shal, Jerng et al., 2004). In neurons, the observed IA activation and inactivation kinetics require the presence of Kv Channel Interacting Proteins (KChIP). KChIP are EF-hand Ca2+-binding proteins that associate with the cytoplasmic tail of the Kv α subunits and alter the expression of the α subunits and IA inactivation kinetics (An et al., 2000, Rhodes et al., 2004).

Activation of IA increases the interspike interval and slows the action potential firing frequency by reducing the rate of membrane depolarization (Rudy et al., 1999). IA is largely inactivated in the range of resting membrane potentials (− 40 mV to − 55 mV) recorded from SCN neurons during the daytime (Kuhlman et al., 2003, Schaap et al., 1999, Teshima et al., 2003). IA rapidly recovers from inactivation during the afterhyperpolarization that follows the upstroke an action potential. The membrane hyperpolarization moves the Kv4 channels to closed states from which IA can be activated during a subsequent subthreshold depolarization (Bardoni and Belluzzi, 1993, Campbell et al., 1993). Therefore, a careful characterization of IA properties is required to understand the physiological properties of SCN neurons and for the development of accurate computational models of neuronal function and circuitry in the SCN.

In addition to IA, SCN neurons have both fast and slow delayed rectifier K+ currents (IDR) and depolarization of an SCN neuron will activate both IA and IDR (Bouskila and Dudek, 1995, Itri et al., 2005, Kuhlman and McMahon, 2004). Two experimental strategies are usually used individually or together to isolate IA from the IDR. The first takes advantage of the fact that IA and IDR have different rates of inactivation at depolarizing voltages. Holding the membrane potential at relatively depolarized levels inactivates IA and a subsequent membrane depolarization only activates IDR. Alternately, tetraethylammonium (TEA) is used to separate IA from the IDR because IA is much less sensitive to TEA block than IDR (Andreasen and Hablitz, 1992). To produce a significant reduction in the IDR requires a TEA concentration of 20 mM to 60 mM (Bardoni and Belluzzi, 1993, Zhou and Hablitz, 1996). Together, the low TEA sensitivity of IA and the voltage dependence of IDR activation and IA inactivation, allow accurate recording of IA. However, some authors have found that TEA alters IA amplitude but not the kinetics (Bardoni and Belluzzi, 1993, Sanchez et al., 1998). While studying the kinetics of IA in hamster SCN neurons we observed that the TEA concentrations required to block IDR significantly increased IA inactivation time constant.

Section snippets

Results

The first experiments were designed to determine IA inactivation time constant (τinact) in hamster SCN neurons. In the presence of TEA (40 mM), SCN neurons were voltage-clamped at − 100 mV then sequentially stepped in 10 mV increments to membrane potentials ranging from − 30 mV and + 60 mV (Fig. 1A). The IDR recorded under these conditions was only a small fraction of the total IDR due to TEA inhibition (Fig. 1B). The membrane holding potential was then set at − 40 mV to inactivate IA and a second

Discussion

Identification of the mechanisms that drive SCN neurons to fire action potentials with a higher frequency during the day than during the night is an important topic in circadian neurobiology. IA plays an active role in neuronal action potential repolarization and may contribute to setting the circadian firing frequency (Bouskila and Dudek, 1995, Huang, 1993, Huang et al., 1993). IA may also modulate the response of SCN neurons to retinohypothalamic tract excitatory input. Thus, the accurate

Experimental procedures

Golden hamsters (Mesocricetus auratus) were housed under a 14:10 h light–dark cycle for at least a week. The hamsters were deeply anesthetized with halothane at least 1 h before the beginning of the dark period, decapitated and the brain removed (Gillette, 1986). The Oregon Health & Science University Institutional Animal Care and Use Committee approved in advance all procedures involving animals. Thin slices containing the SCN (250 µm) were cut with a vibrating blade microtome (Leica, VT1000S,

Acknowledgments

We would like to thank Dr. Mykhaylo Moldavan and Dr. David W. Robinson for critically reading the manuscript. The experimental work was supported by grant NS36607 (CNA) from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health.

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