Research reportDay–night variations in zinc sensitivity of GABAA receptor-channels in rat suprachiasmatic nucleus
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 GABAA 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 GABAA receptor itself undergoes circadian variations.
To monitor possible time-dependent changes in GABAA receptor subunit composition, we investigated GABAA receptor sensitivity to zinc during the day and night. In recombinant receptors, zinc inhibitory effect is related to the subunit composition of the receptor; GABAA 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 GABAA receptor in SCN neurons moderately, and that sensitivity to this cation is higher during the day than during the night time.
Section snippets
Animals and brain slices preparation
Brain slices were prepared from 18- to 24-day-old Wistar rats, which were kept from their birth under a controlled 12–12 h light–dark cycle. Animals used for the day experiments had lights on from 6 AM to 6 PM and animals used for the night experiments were kept with lights off from 10 AM to 10 PM. Rats were killed by decapitation and dissected brains were placed in cold oxygenated artificial cerebrospinal fluid (ACSF). Two-hundred-micrometer hypothalamic slices containing SCN were cut with
Bicuculline-sensitive GABAA receptor-mediated currents in SCN neurons
During the whole-cell patch recording from SCN neurons in slices, bicuculline-sensitive spontaneous inhibitory postsynaptic currents (IPSC) were recorded in 167 of 171 tested neurons in VL and DM parts of SCN (Fig. 1A). The addition of 100 μM GABA applied to SCN neurons elicited a large inward current (the maximum peak amplitude ranged from 1 to about 6 nA), which appeared with a delay of 100–200 ms after GABA application, and which was reversibly inhibited by 30 μM bicuculline (Fig. 1B). This
Discussion
The whole-cell patch-clamp recordings from SCN neurons in acutely isolated hypothalamic slices revealed that there is a large heterogeneity and significant day–night variability in the blocking effect of Zn2+ on GABAA receptors. Ligand-induced currents were more sensitive to Zn2+ during the day than during the night. This change was specific for the SCN neurons. These data opened a question what is the mechanism of this variability in zinc sensitivity and whether it has any physiological role
Acknowledgements
This research was supported by the Grant Agency of the Czech Republic (Grant No. 309/02/1519), Internal Grant Agency of Academy of Sciences (Grants No. A5011103 and A5011105) and by the Academy of Sciences of the Czech Republic (Research Project No. AVOZ 501 1922).
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