Day–night variations in zinc sensitivity of GABA_A receptor-channels in rat suprachiasmatic nucleus

Abstract

In the suprachiasmatic nucleus (SCN), electrical activity, secretion, and other cellular functions undergo profound rhythm during day–night cycle due to oscillatory expression of clock gene constituents. Although SCN is enriched with γ-aminobutyric acid (GABA)-ergic neurons, it is unknown whether there are circadian changes in the GABA_A receptor expression and/or function. Here we investigated the possible daily variations in zinc sensitivity of GABA_A channels in rat SCN neurons maintained in brain slices. Extracellular zinc inhibited GABA-induced currents in all ventrolateral (VL) and dorsomedial (DM) SCN neurons studied, as well as in neurons of non-SCN regions. In SCN neurons, the currents evoked by 30 μM GABA were inhibited by Zn²⁺ with an IC₅₀ of 50.3±3.2 μM, whereas currents evoked by 100 μM GABA were inhibited with an IC₅₀ of 181.6±32.0 μM. The antagonist action of zinc saturated at 97.4±0.7% for 30 μM GABA and 91.6±2.7% for 100 μM GABA. These observations indicate that Zn²⁺ inhibits SCN GABA_A receptor competitively and in part non-competitively. In SCN neurons, but not in other neurons, the zinc sensitivity varied with daily time. During the day, the calculated IC₅₀ for zinc was significantly lower than during the night (43.9±4.7 μM vs. 58.6±3.8, respectively). These results indicate that native GABA_A receptors in SCN neurons display pharmacological properties of receptors having and not having γ subunit and that the proportionality of these receptors could change during the day and night.

Introduction

In mammals, the circadian pacemaker is situated in the suprachiasmatic nucleus (SCN) [25], [37]. SCN neurons exhibit circadian rhythm in spontaneous electrical activity, with the frequency of action potentials firing higher during the day [5], [19], [32], in both day-active and night-active species [22]. Investigation of the molecular clock mechanism by which SCN neurons generate spontaneous activity rhythms with a period of 24 h is already advanced [2], [10], but the underlying mechanism(s) for rhythmic changes in electrical activities of the membrane is still unknown [32], [33].

The SCN has two regions that differ in neuronal inputs and contents of neuropeptides: the ventrolateral (VL) part receives glutamatergic optical inputs from retina and produces vasoactive intestinal polypeptide (VIP), and the dorsomedial (DM) part is enriched with arginine–vasopressin (AVP)-containing neurons and does not receive direct visual inputs [24], [40]. Synaptic contacts within the both parts of SCN are mediated by γ-aminobutyric acid (GABA) [5], [26], [39], [42] and autoradiographic studies have shown GABA_A receptor binding in the SCN [11]. The GABA content [1] and the expression of genes coding for glutamic acid decarboxylase [7], [12], [29], the GABA-forming enzyme, undergo circadian changes in the SCN. However, it is not yet clear whether the GABA_A receptor itself undergoes circadian variations.

To monitor possible time-dependent changes in GABA_A receptor subunit composition, we investigated GABA_A receptor sensitivity to zinc during the day and night. In recombinant receptors, zinc inhibitory effect is related to the subunit composition of the receptor; GABA_A receptors having αβ subunit composition are highly sensitive to Zn²⁺, whereas the receptors containing γ subunit in combination with α and β subunits are much less sensitive to Zn²⁺ [9], [17], [36]. The results of our investigations indicate that zinc inhibited GABA_A receptor in SCN neurons moderately, and that sensitivity to this cation is higher during the day than during the night time.