Multiple co-localizations in arcuate GHRH-eGFP neurons in the mouse hypothalamus Karine Bouyer a , Catherine Loudes a , Iain C.A.F. Robinson b , Jacques Epelbaum a, * , Annie Faivre-Bauman a a UMR 549 INSERM, Faculte ´ de Me ´decine, Universite ´ Paris-Rene ´ Descartes, IFR Broca Sainte Anne, 2ter rue d’Ale ´sia, 75014 Paris, France b Molecular Neuroendocrinology, NIMR, London NW7 1AA, United Kingdom Received 9 June 2006; received in revised form 12 October 2006; accepted 12 October 2006 Available online 29 November 2006 Abstract In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month- old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40–44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25–22% in 8-month-old mice. TH immunoreactivity occurred in 36–35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co- localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se, may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse. # 2006 Published by Elsevier B.V. Keywords: Galanin; Neurotensin; Tyrosine hydroxylase; Sexual dimorphism; Ageing 1. Introduction The arcuate nucleus is located in the periventricular region of the tuberal zone of the hypothalamus and contains two distinct cytoarchitectural subdivisions: a dorsomedial part which contains small neurons and a larger ventro-lateral part in which neurons are medium-sized. Many arcuate neurons exhibit specific neuroendocrine phenotypes (Everitt et al., 1986) amongst which hypophysiotropic neurons expressing growth-hormone-releasing-hormone (GHRH) which stimulates GH secretion. In the rat, these neurons are located in the ventrolateral portion of the arcuate nucleus. Like many neuropeptidergic neurons, subpopulations of GHRH-contain- ing cells are known to synthetize other peptides, such as galanin (GAL) (Niimi et al., 1990), neurotensin (NT) (Sawchenko et al., 1985; Niimi et al., 1991) and tyrosine hydroxylase (TH) (Meister et al., 1986), the rate limiting enzyme in the synthesis of dopamine. However, TH-immunoreactive neurons in the ventrolateral arcuate nucleaus do not contain L-amino acid decarboxylase (AADC) which indicates they can produce only L-dopa (Meister et al., 1988; Komori et al., 1991). In mammals, the pulsatile pattern of GH secretion is markedly sexually dimorphic (Tannenbaum and Martin, 1976; Clark and Robinson, 1985; Tannenbaum et al., 2003), and it is possible that co-expression of hypophysiotrophic peptides is differentially affected by the gonadal steroid milieu. For example, GAL expression in GHRH neurons is sex-dependent, being higher in male than in female rats (Delemarre-Van De Waal et al., 1994), and in ovariectomized rats, up to 30% of GHRH-containing neurons are able to concentrate [ 3 H]estra- diol, in a subpopulation of GHRH neurons that do not contain TH immunoreactivity (Shirasu et al., 1990). In contrast, www.elsevier.com/locate/jchemneu Journal of Chemical Neuroanatomy 33 (2007) 1–8 * Corresponding author. Tel.: +33 1 40 78 92 82; fax: +33 1 45 80 72 93. E-mail address: epelbaum@broca.inserm.fr (J. Epelbaum). 0891-0618/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.jchemneu.2006.10.002