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The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein-coupled receptors (MT(1) and MT(2) ). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole-cell patch-clamp recordings in brain slices prepared from Per1::d2EGFP-expressing transgenic mice. In current-clamp mode, bath applied melatonin, at near-physiological concentrations (1 nM), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non-Per1::GFP SCN neurones. Melatonin-induced effects were blocked by the MT(1)/MT(2) antagonist, luzindole (1 μM) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nM) or gabazine (20 μM). In voltage-clamp recordings, 1 nM melatonin increased the frequency of GABA-mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones., (© 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.)

Ageing alters fundamental aspects of circadian rhythmicity in mammals; the effects include reduced rhythm amplitude and alterations in period length and in entrainment to the light/dark cycle. Such changes may reflect disruptions in cellular function within the suprachiasmatic nucleus (SCN), the site of the predominant circadian pacemaker. In the SCN, vasoactive intestinal peptide (VIP)-synthesizing neurones receive various inputs, including retinohypothalamic projections containing pituitary adenylate cyclase activating peptide (PACAP). SCN VIP cells establish connections with local neurones and send efferents beyond the nucleus. Considerable evidence implicates VIP and PACAP in circadian rhythm maintenance and/or entrainment to photic Zeitgebers. These actions involve members of a distinct family of receptors; mRNAs for two such receptors, VPAC2 and PAC1, are present in the SCN. This study used isotopic in situ hybridization to examine the effects of ageing on expression of mRNAs for VIP, VPAC2 and PAC1 in the SCN of male rats under a 12 : 12 h light/dark cycle. Analysis of film autoradiographs from young adult (2-3 months) or aged (19-20 months) rats, at eight time points across the light/dark cycle, showed loss of diurnal rhythmicity and reduced levels for VIP mRNA in the aged group. A diurnal rhythm of VPAC2 receptor mRNA was present in both groups, but its levels were reduced in the aged rats. There were no differences between the two groups for PAC1 receptor mRNA expression. The present results indicate that ageing reduces VIP and VPAC2 receptor mRNA and eliminates diurnal expression of VIP mRNA within the SCN of aged male rats.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

The existence of biological clocks was once the subject of considerable debate. However, research has confirmed that a 24 h (circadian) clock in our brain has a pronounced influence on when we sleep, eat, drink, etc. Disruptions in clock function compromise normal daily patterns of hormone release, sleep and body temperature, and can affect mental and physical well-being. The recent identification of clock genes and the neural pathways to the clock will facilitate the development of effective strategies to treat disorders of circadian function.

The suprachiasmatic nuclei (SCN) of the rodent hypothalamus function as a light entrainable circadian pacemaker. The SCN contain moderate to high concentrations of a number of neuropeptides including peptides showing structural homology with the amphibian derived tetradecapeptide, bombesin (BN), called bombesin-like peptides (BNLPs). BNLPs include the 27 amino acid peptide, gastrin releasing peptide (GRP1-27), a smaller decapeptide (GRP18-27) and another decapeptide with less structural homology, neuromedin B (NmB). Immunoreactivity for BN and receptors for BNLPs have been demonstrated in the region of the rat SCN receiving photic input. We studied the effects of local pressure ejections of BNLPs dissolved in saline/1% bovine serum albumin (BSA) vehicle on the extracellularly recorded firing rates of Syrian hamster SCM neurons in a hypothalamic slice preparation. In one study, an ejecting electrode containing BN (10(-8) to 10(-4) M) was positioned 20 to 60 microm from a recording electrode. Of 74 cells tested with BN, 50 (67.6%) showed significant increases in firing rate, while 3 of 29 cells (15.8%) tested with vehicle ejections were activated. In a second study, a single electrode was used for both recordings and pressure ejections. Of 48 cells tested, BN (10(-6) to 10(-4) M) activated 30 (62.5%) and suppressed firing in 4 (8.3%). Of 208 cells tested with GRP1-27 (10(-9) to 10(-4) M), 105 (50.5%) were activated and 2 (1.0%) were suppressed. The percentage of cells responding increased with the concentration of GRP1027 used in the electrode. No circadian variation in responsiveness to GRP1-27 was detected. GRP18-27 (5 x 10(-5) to 10(-4) M) activated 10 out of 18 cells tested (55.6%), while NmB (10(-4) M) activated 2 out of 30 cells tested (6.7%) and vehicle ejections activated 1 out of 36 cells tested (2.8%). GRP1-27, GRP18-27 and BN, the BNLPs showing the greatest degree of structural homology, activate approximately 50% of SCN cells, apparently via the BN/GRP-preferring receptor subtype, and may play a role in photic entrainment.