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© Copyright 2004 The British Pharmacological Society

025P University of Buckingham
3th Focused Meeting April 2004

Lateral hypothalamic neurones which express
NO-donor induced cGMP but do not contain orexin, NOS or MCH
Ornsiri Cheunsuang* & Richard Morris. Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool, UK. *Department
of Veterinary Anatomy, Chulalongkorn University, Bangkok, Thailand.

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Cheunsuang O
Morris R

The induction of cGMP by exogenous application of NO-donor compound to brain slices was investigated in order to identify the targets for NO released in the lateral hypothalamus.

Wistar rats, either sex, 15-30 days old were deeply anaesthetised (ketamine HCl 60mg/kg) and decapitated. The brains were quickly removed and 200µm-thick transverse slices from the hypothalamic region were cut on a vibrating microtome in ice-cold, low sodium-artificial cerebral spinal fluid (aCSF), which was continuously gassed with 95%O2,5%CO2. The brain slices were then transferred to flasks containing normal aCSF and slowly warmed up to 37°C in a rocking bath for 15 minutes before incubation in NO donor, NOC-12, (50 mM) for a further 15 minutes. They were then fixed and immunostained with anti-cGMP antibody (gift from Dr J. de Vente). Double immunofluorescent labelling for either orexin-A, melanin concentrating hormone (MCH), neuronal nitric oxide synthase (nNOS), glia fibrillary acid protein (GFAP) or the neuronal marker, NeuN, was carried out.

Brain slices treated with NOC-12 showed cGMP immunoreactivities in GFAP-positive glia as well as NeuN-positive neurones. The latter were predominant in the perifonical area, the lateral hypothalamus, and less extensively in the paraventricular nucleus. Numerous nerve fibres in these areas were also cGMP-positive. Double immunofluorescent staining for nNOS, orexin or MCH revealed that none of cGMP-containing neurones expressed these molecules. In the perifonical area, cGMP-containing neurones appeared smaller when compared to orexin A-, NOS- and MCH-expressing neurones.

Fig.1 cGMP immunoreactivities in hypothalamus

Fig.2. cGMP-expressing neurones in PVN

This study has revealed a new population of hyopothalamic neurones, which are neither orexin-, nNOS- nor MCH-containing neurones, and are a potential target for NO released in this region. The cGMP-NO system may play a part in the feeding-sleep regulation (Fetissov et al., 2003: Mariano and Cudeiro, 2003). Further investigation is required for a more comprehensive knowledge of its circuitry and interactions between neurones in this region.

Fetissov et al.(2003). J. Neuroendoclinol. 15,754-760.
Marino & Cudeiro (2003). J. Neurosci. 23,4299-4307.