Elsevier

Brain Research

Volume 750, Issues 1–2, 7 March 1997, Pages 67-80
Brain Research

Research report
Neuromedin C decreases potassium conductance and increases a non-specific conductance in rat suprachiasmatic neurones in brain slices in vitro

https://doi.org/10.1016/S0006-8993(96)01332-7Get rights and content

Abstract

Whole-cell recordings were made, in both current and voltage clamp, from suprachiasmatic neurones maintained in coronal rat brain slices. In current clamp doses of 10 and 100 nM neuromedin C (NMC) were shown to increase basal firing rate in 9 out of 32 neurones. The excitatory responses to 100 nM NMC were accompanied by small increases in neuronal input resistance (25.0±9.9% in 4 out of 7 neurones tested) and depolarisations of membrane potential (9.8±3.4 mV in 4 out of 7 neurones tested). However, 10 nM NMC caused no changes in either neuronal input resistance or membrane potential despite the clear increases in neuronal firing rate. When voltage-clamped at −60 mV, 100 nM NMC induced an inward current of 14.8±1.2 pA in 46 of 210 neurones. The NMC-induced inward current was shown to be unaffected by perfusion with 1 μM tetrodotoxin (TTX). The inward current recorded at −60 mV was typically associated with a decrease in membrane conductance. Construction of current–voltage relationships in the absence and presence of 100 nM NMC showed that with the majority of the NMC-sensitive neurones the inward current either reversed polarity close to the potassium reversal potential or decreased at hyperpolarised potentials. This reversal potential was shifted to more depolarised potentials when the extracellular concentration of potassium was increased. The NMC-induced inward current was unaffected by reduction of the extracellular concentration of sodium or by addition of 0.2 mM cadmium. In potassium-free conditions, in both the dialysing pipette solution and perfusing saline, NMC was still able to induce an inward current. The additional reduction of the extracellular concentration of sodium, whilst recording in potassium-free conditions, was also unable to abolish the inward current. Recordings made with an electrode containing the non-hydrolysable guanosine triphosphate analogue, guanosine 5′-thio-triphosphate, resulted in NMC-induced inward currents which failed to recover to baseline. It is concluded that NMC excites a subpopulation of suprachiasmatic neurones by decreasing a resting potassium conductance and increasing a non-specific conductance, via a G-protein link.

Introduction

Neuromedin C (NMC) is one of three mammalian counterparts to the bombesin-like peptide family, which includes peptides derived from amphibian and mammalian tissues. All three mammalian peptides, which includes gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been isolated from porcine spinal cord and non-antral gastric tissue 18, 19, 24, 25, 26. The peptide NMC is the carboxy terminal decapeptide fragment of the mammalian counterpart gastrin-releasing peptide (GRP). Bombesin and a number of related amphibian peptides were themselves isolated from the skin of a number of species of European and American frogs 2, 10, 11. Both amphibian and mammalian members of the bombesin-like peptide family have been shown to evoke a range of biological responses in vivo which includes effects on feeding, thermoregulation, blood pressure, locomotor activity and circadian rhythms 1, 3, 8, 10, 12, 21. To date, two mammalian bombesin receptor subtypes have been cloned, termed the GRP and NMB receptor subtypes 6, 41. In situ hybridisation studies have shown that each receptor subtype displays a distinct distribution throughout the rat CNS, with overlap in some regions [5].

Evidence supports the view that the biological clock or circadian pacemaker is located within the suprachiasmatic nucleus (SCN), a structure located in the rostral hypothalamus 20, 24, 27, 38. Albers et al. (1991) have recently demonstrated that GRP is able to induce phase shifts in circadian rhythms, noted as delays in the onset of wheel-running activity in hamsters [1]. This evidence supports a role for the mammalian form of bombesin in the re-entrainment of circadian rhythms. In a previous study, we reported the use of the three mammalian counterparts and the selective GRP-receptor antagonist [d-Phe6]bombesin(6–13)ethylamide to examine the pharmacology of the bombesin receptors expressed within the SCN [32]. The data obtained were supportive of the predominant expression of the GRP-preferring bombesin receptor subtype by a subpopulation of suprachiasmatic neurones. In this present study using NMC we have examined the ionic mechanism which underlies the acute excitatory responses to the bombesin-like peptides. The peptide GRP, and the decapeptide fragment NMC, have been shown to be expressed by neurones which appear to be intrinsic to the nucleus 24, 40. This aim has been achieved by application of the whole-cell recording technique to acutely prepared rat brain slices.

Section snippets

Preparation of brain slices

Coronal brain slices containing the suprachiasmatic nuclei were prepared from rats as previously described [30]. Prior to sacrifice, male Wistar rats were housed in a 12-h light–dark cycle. During the subjective day only, rats (50–150 g) lightly anaesthetized with ether were killed by cervical dislocation and the brain was rapidly removed. Coronal slices (350–400 μm) were cut at the level of the rostral hypothalamus in a vibratome (Oxford instruments) filled with artificial cerebrospinal fluid

Results

All whole-cell recordings were made in the dorsal region of the nucleus, on the border of the SCN and periventricular nucleus of the hypothalamus. In our previous report [30]this region was referred to as the PVN/SCN region.

Discussion

Using the whole-cell recording technique we have demonstrated that nanomolar concentrations of NMC can evoke excitatory responses from a subpopulation of suprachiasmatic neurones. A comparison of the present current clamp recordings of the potency, magnitude and duration of the responses to the bombesin related peptides, with our previously reported study [30], suggests that the responses to NMC were little affected by the invasive nature of the whole-cell recording technique. The most notable

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    Present address: Merck Sharpe and Dohme Neuroscience Research Center, Terlings Park, Harlow, Essex, UK

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