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