Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

doi:10.1016/S0006-8993(97)00632-X

Brain Research

Volume 767, Issue 1, 29 August 1997, Pages 72-80

https://doi.org/10.1016/S0006-8993(97)00632-X Get rights and content

Abstract

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (I_H) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of I_H if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of I_H by Cs⁺ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that I_H, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that I_H does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of I_H were modulated in a circadian manner. However, no substantial day–night differences in I_H were found. We conclude that I_H, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in `rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.

Introduction

It has been repeatedly demonstrated that the suprachiasmatic nucleus (SCN) of the hypothalamus in mammals generates a circadian rhythm 19, 21, 26, 27, 34. Electrophysiological studies have revealed the existence of circadian rhythmicity in spontaneous firing rate of SCN neurons 4, 5, 12, 13, 15. Blocking the neuronal firing of SCN neurons does not prevent the circadian clock from running 28, 29, 35, indicating that these neurons have an intrinsic mechanism for circadian rhythmogenesis. The intracellular mechanism that is responsible for the expression of circadian rhythms in spontaneous firing rate is not yet known.

One prominent feature of many SCN neurons is the presence of a time- and voltage-dependent inward current, I_H1, 16. I_H is a mixed Na⁺/K⁺ current, which is activated by membrane hyperpolarization 7, 8, 9, 20, 24, 31. Upon hyperpolarization of the cell, the net influx of positively charged ions makes a slow excitatory contribution to the membrane potential and will rectify it back towards rest 10, 14, 20, 31. It has been suggested by several authors 14, 20, 22, 31that the physiological role of this current is to counterbalance transient or prolonged membrane hyperpolarizations in order to keep the membrane potential near the firing threshold. Since the reversal potential of I_H is estimated to lie between −50 and −20 mV [24], there is a distinct possibility that I_H contributes positively to the spontaneous firing rate. Results of McCormick and Pape [7]indicate that I_H indeed promotes spontaneous firing in thalamic relay neurons by keeping episodes of hyperpolarization limited in duration.

Akasu and coworkers [1]reported that I_H may contribute to the spontaneous firing mechanism in SCN neurons by shortening the duration of the spike afterhyperpolarization (AHP), an effect which also reduces the interspike interval. This contribution of I_H to the spontaneous firing rate may vary in a circadian manner and thereby generate the circadian rhythm of spontaneous firing rate in SCN neurons. In this study we reassessed the contribution of I_H to the spontaneous firing rate of SCN neurons and also compared I_H across day and night. This reassessment must be considered in the light of the hypothesis that the magnitude and/or kinetics of I_H may follow a circadian rhythm itself, and may thus underlie the rhythm in spontaneous firing rate. Such a rhythm in I_H may result from a circadian modulation by a second messenger or from structural changes in the channel protein. The whole-cell patch-clamp technique allows to do single-electrode voltage-clamp experiments in continuous mode and investigate circadian conductance changes in I_H. However, the whole-cell patch-clamp technique has one important disadvantage, namely that the whole-cell configuration will give rise to intracellular dialysis. Thereby this study does not conclusively address the possibility of second messenger modulation of I_H. Functional consequences of structural channel modifications, other than those at messenger binding sites, are not subject to this problem.

We first investigated the contribution of I_H to the spontaneous firing rate, resting membrane potential (RMP) and spike AHP of SCN neurons in current-clamp mode during the subjective day phase, which is the period expected to reveal a positive contribution of I_H to the spontaneous firing rate if I_H would underlie the circadian rhythm in spontaneous firing rate. Second, we investigated I_H and its activation kinetics in voltage-clamp mode to obtain more insight in the characteristics of this current. Third, we investigated I_H and its activation characteristics at different circadian time points to determine whether the magnitude and/or kinetics of I_H vary in a circadian manner.

Section snippets

Slice preparation

Male Wistar rats, weighing 150–300 g, were housed in a normal (lights-on 07.00 h) or reversed (lights-on 24.00 h) 12:12 h light/dark cycle for at least 5 weeks before they were used. All rats were anesthetized intraperitoneally with Nembutal (60 mg/kg sodium pentobarbital and 9 mg/kg benzylalcohol) followed by a transcardial perfusion of 50 ml ice-cold artificial cerebrospinal fluid (aCSF) with a perfusion pressure of 80–100 mm Hg. The aCSF contained (in mM): 124.0 NaCl, 3.5 KCl, 1.0 NaH₂PO₄,

Basic membrane properties

The data presented in this paper are based on whole-cell patch-clamp recordings from 58 SCN neurons, which were uniformly sampled across the SCN. In 29 neurons, voltage and current clamp recordings were performed between CT 7 and CT 11; the remaining cells were recorded between CT 14 and CT 19. The RMP of both groups were −59.0±1.4 mV and −60.2±1.4 mV, respectively (MW U-test: not significant (n.s.); both n=29). The average spontaneous firing rate was 2.9±0.5 Hz in the period between CT 7–11

Discussion

The results of this study demonstrate that I_H is present in a large majority of SCN neurons and indicate that I_H does not influence the spontaneous firing rate and RMP of the SCN neurons. Furthermore, the magnitude and kinetics of I_H recorded in whole-cell mode are not modulated in a circadian manner.

I_H was detected in 89.7% of our SCN neurons and these neurons were uniformly distributed throughout the SCN. The intracellular recordings of Kim and Dudek [18]and Akasu and Shoji [2]revealed a

Acknowledgements

We would like to thank A. Geurtsen for technical assistance, and R. Buijs and M. Hermes for their helpful comments on the manuscript. This research was supported by Grant 903-52-203 of the Netherlands Organization for Scientific Research.

References (36)

N. Bos et al.
Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasmatic nucleus
Brain Res.
(1990)
M. Gillette
The suprachiasmatic nuclei: circadian phase-shifts induced at the time of hypothalamic slice preparation are preserved in vitro
Brain Res.
(1986)
D. Green et al.
Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice
Brain Res.
(1982)
J. Halliwell et al.
Voltage-clamp analysis of muscarinic excitation in hippocampal neurons
Brain Res.
(1982)
Z. Jiang et al.
Melatonin activates an outward current and inhibits I_H in rat suprachiasmatic nucleus neurons
Brain Res.
(1995)
E. Neher
Correction for liquid junction potential in patch-clamp experiments
Methods Enzymol.
(1992)
S. Shibata et al.
Tetrodotoxin does not affect circadian rhythms in neuronal activity and metabolism in rodent suprachiasmatic nucleus in vitro
Brain Res.
(1993)
D. Welsh et al.
Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms
Neuron
(1995)
T. Akasu et al.
Inward rectifier and low-threshold calcium currents contribute to the spontaneous firing mechanism in neurons of the rat suprachiasmatic nucleus
Eur. J. Physiol.
(1993)
T. Akasu et al.
cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus
Eur. J. Physiol.
(1994)

C. Amstrong et al.

Access resistance and space clamp problems associated with whole-cell patch clamping

Methods Enzymol.

(1992)

Y. Bouskila et al.

Neuronal synchronization without calcium-dependent synaptic transmission in the hypothalamus

Proc. Natl. Acad. Sci. USA

(1993)

T. Budde et al.

Hyperpolarization-activated Na⁺-K⁺ current (I_H) in neocortical neurons is blocked by external proteolysis and internal TEA

J. Neurophysiol.

(1994)

D. McCormick et al.

Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones

J. Physiol.

(1990)

D. DiFranscesco

A new interpretation of the pace-maker current in calf Purkinje fibres

J. Physiol.

(1981)

D. DiFranscesco

A study of the ionic nature of the pace-maker current in calf Purkinje fibres

J. Physiol.

(1981)

D. DiFranscesco

The cardiac hyperpolarizing-activated current, I_f, origins and developments

Prog. Biophys. Mol. Biol.

(1985)

G. Groos et al.

Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro

Neurosci. Lett.

(1982)

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Research reportFunctional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

Abstract

Introduction

Section snippets

Slice preparation

Basic membrane properties

Discussion

Acknowledgements

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Methods Enzymol.

Brain Res.

Neuron

Inward rectifier and low-threshold calcium currents contribute to the spontaneous firing mechanism in neurons of the rat suprachiasmatic nucleus

Eur. J. Physiol.

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Eur. J. Physiol.

Access resistance and space clamp problems associated with whole-cell patch clamping

Methods Enzymol.

Neuronal synchronization without calcium-dependent synaptic transmission in the hypothalamus

Proc. Natl. Acad. Sci. USA

Hyperpolarization-activated Na+-K+ current (IH) in neocortical neurons is blocked by external proteolysis and internal TEA

J. Neurophysiol.

Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones

J. Physiol.

A new interpretation of the pace-maker current in calf Purkinje fibres

J. Physiol.

A study of the ionic nature of the pace-maker current in calf Purkinje fibres

J. Physiol.

The cardiac hyperpolarizing-activated current, If, origins and developments

Prog. Biophys. Mol. Biol.

Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro

Neurosci. Lett.

Research report
Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

Hyperpolarization-activated Na⁺-K⁺ current (I_H) in neocortical neurons is blocked by external proteolysis and internal TEA

The cardiac hyperpolarizing-activated current, I_f, origins and developments