Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid

Highlights

• Cerebrospinal fluid ghrelin reaches most brain areas expressing ghrelin receptors.

• Ependymal cells are able to uptake ghrelin present in the cerebrospinal fluid.

• Ghrelin uptake in ependymal cells occurs in a ghrelin receptor-independent manner.

Abstract

Ghrelin is a stomach-derived peptide hormone that acts in the brain to regulate many important physiological functions. Ghrelin receptor, named the growth hormone secretagogue receptor (GHSR), is present in many brain areas with or without obvious direct access to ghrelin circulating in the bloodstream. Ghrelin is also present in the cerebrospinal fluid (CSF) but the brain targets of CSF ghrelin are unclear. Here, we studied which brain areas are accessible to ghrelin present in the CSF. For this purpose, we centrally injected mice with fluorescein-labeled ghrelin (F-ghrelin) peptide tracer and then systematically mapped the distribution of F-ghrelin signal through the brain. Our results indicated that centrally injected F-ghrelin labels neurons in most of the brain areas where GHSR is present. Also, we detected F-ghrelin uptake in the ependymal cells of both wild-type and GHSR-null mice. We conclude that CSF ghrelin is able to reach most of brain areas expressing GHSR. Also, we propose that the accessibility of CSF ghrelin to the brain parenchyma occurs through the ependymal cells in a GHSR-independent manner.

Introduction

Ghrelin is an octanoylated peptide hormone, synthesized mainly by endocrine cells located within the gastric oxyntic mucosa (Kojima et al., 1999). Ghrelin acts in the brain to regulate growth hormone secretion, food intake, energy expenditure, glucose homeostasis, anxiety/depression-related behaviors and stress responses (Kojima and Kangawa, 2005). Two subtypes of ghrelin receptors have been described, the growth hormone secretagogue receptor 1a (GHSR-1a) isoform, which is activated by ghrelin, and the splice variant isoform GHSR-1b, which is a functionally inactive truncated form of GHSR (Howard et al., 1996). Analyses of the distribution of GHSR-1a within the rodent brain using many different techniques have shown that GHSR-1a is present in several brain sites (Guan et al., 1997, Mitchell et al., 2001, Zigman et al., 2006, Perello et al., 2012). For instance, GHSR-1a mRNA is highly expressed in brain areas that presumably have direct access to circulating ghrelin, such as the hypothalamic arcuate nucleus (ARC) and the dorsal vagal complex (DVC) of the medulla (Guan et al., 1997, Willesen et al., 1999, Zigman et al., 2006). However, GHSR-1a is also present in some brain areas distantly situated from circumventricular organs and without immediate access to ghrelin circulating in the bloodstream (Guan et al., 1997, Mitchell et al., 2001, Zigman et al., 2006, Perello et al., 2012, Scott et al., 2012). The physiological relevance of GHSR in many of these brain areas is unclear. Since ghrelin is also present in the cerebrospinal fluid (CSF) (Tritos et al., 2003, Grouselle et al., 2008), we decided to study which brain areas have ghrelin targets accessible to CSF ghrelin. For this purpose, we injected mice with fluorescein-labeled ghrelin (F-ghrelin) peptide tracer via an intra-cerebro-ventricular (ICV) cannula and then systematically mapped the distribution of F-ghrelin signal through the whole brain. Our results indicated that CSF ghrelin is able to access most of the areas where GHSR-1a is present. Interestingly, we also detected F-ghrelin uptake in the ependymal cells of both wild-type and GHSR-null mice, suggesting that ghrelin can interact with these specialized cells in a GHSR-independent manner.