Preproglucagon (PPG) neurons innervate neurochemicallyidentified autonomic neurons in the mouse brainstem
Highlights
► GLP-1 neurons directly innervate brainstem cholinergic and monoaminergic neurons. ► GLP-1 may influence satiety via heavy GLP-1 innervation of brainstem 5-HT neurons. ► Innervation of dorsal vagal neurons underpins GLP-1’s role in control of digestion. ► Central GLP-1 may affect cardiovascular control via GLP-1 inputs to C1 neurons.
Introduction
Glucagon-like peptide 1 (GLP-1) is an incretin that is released from enteroendocrine cells and facilitates absorption of nutrients (Holst, 2007). Like other gut peptides, such as cholecystokinin, GLP-1 is also synthesised by neurons within the central nervous system. GLP-1 is produced by post-translational processing of preproglucagon (PPG) and immunoreactivity for the products of PPG processing is found in many brain regions, with highest levels occurring in the dorsomedial (DMH) and paraventricular nucleus (PVN) of the hypothalamus and lowest levels in the cortex and hindbrain (Jin et al., 1988, Vrang et al., 2007, Tauchi et al., 2008). Somata capable of synthesising GLP-1, however, are restricted to the lower brainstem. The largest population of GLP-1-containing somata occurs in the caudal nucleus of the solitary tract (NTS) and there are also some cell bodies in the dorsomedial part of the medullary reticular nucleus (Jin et al., 1988, Larsen et al., 1997). Similarly, in situ hybridisation has revealed PPG mRNA only in the caudal NTS, the intermediate reticular nucleus (IRT) and the olfactory bulb (Merchenthaler et al., 1999). Retrograde tracing has confirmed that the hypothalamic axons containing GLP-1 arise from the cell bodies in the NTS and IRT (Larsen et al., 1997, Vrang et al., 2007).
Microinjection of GLP-1 or GLP-1 agonists into the brain has a multitude of effects, including suppression of food intake, control of blood glucose levels, nausea, changes in blood pressure and heart rate, as well as neuroprotection and effects on learning and memory (Tang-Christensen et al., 1996, Turton et al., 1996, Van Dijk et al., 1996, Thiele et al., 1998, Kinzig et al., 2002, Yamamoto et al., 2002, During et al., 2003, Cabou et al., 2008, Sandoval et al., 2008). Because of the functional importance of GLP-1 in the brain, we recently reinvestigated the distribution of central neurons capable of synthesising GLP-1 using a transgenic mouse in which GLP-1 neurons express yellow fluorescent protein (YFP) throughout their cytoplasm under the control of the PPG promoter (Reimann et al., 2008). This approach allowed us to visualise PPG neurons and their processes with an unprecedented level of detail. Our study revealed the full distribution of PPG cell bodies and dendrites within the medulla and demonstrated that axons were widespread throughout the brain with the notable exception of the cerebellum, hippocampus and cerebral cortex (Llewellyn-Smith et al., 2011). These observations matched well with the distribution of GLP-1 receptors within the brain (Shughrue et al., 1996, Merchenthaler et al., 1999). Notably, we found varicose axons in many central sites that are involved in regulating autonomic functions. GLP-1 released in these areas could contribute to the global and coordinated control of food intake, energy balance and maintenance of cardiovascular homoeostasis.
Recent functional studies have highlighted the importance of brainstem circuitry and brainstem GLP-1 receptors for physiological function (Yamamoto et al., 2003, Wan et al., 2007a, Wan et al., 2007b, Hayes et al., 2008, Hayes et al., 2009, Holmes et al., 2009, Williams et al., 2009, Barrera et al., 2011; see also (Trapp and Hisadome, 2011 for review). These findings, together with the fact that our immunohistochemical study revealed many more varicose immunoreactive axons in the brainstem than previously reported, prompted us to define the innervation targets of PPG neurons within the brainstem in more detail. Catecholamine neurons in the area postrema (AP) have been shown to express GLP-1 receptors (Yamamoto et al., 2003) and a subpopulation of cholinergic dorsal vagal motor neurons responds electrically to GLP-1 (Wan et al., 2007a, Wan et al., 2007b, Holmes et al., 2009), consistent with these cell types receiving inputs from GLP-1 neurons.
In this study, we used transgenic YFP–PPG mice (Reimann et al., 2008) in order to take advantage of the strong YFP expression that occurs throughout the cytoplasm of PPG neurons, including their terminals (Hisadome et al., 2010, Llewellyn-Smith et al., 2011). To reveal GLP-1 innervation of cholinergic, catecholamine and serotonin neurons in the brainstem, we detected YFP-immunoreactivity in combination with immunoreactivity for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) or 5-hydroxytryptamine (5-HT), using two-colour or three-colour immunoperoxidase staining.
Section snippets
Experimental procedures
These studies were performed on 11 adult male and 11 adult female mGLU-124 Venus YFP mice (Reimann et al., 2008), which will be referred to here as YFP–PPG mice. Mice were perfused at 12–16 weeks of age and weighed between 25 and 35 g with males consistently being heavier than females of the same age. Mice were bred at Imperial College, kept on a 12-h light:dark cycle and had unlimited access to food and water. All experiments were carried out in accordance with the UK Animals (Scientific
Results
YFP-immunoreactive innervation of catecholamine neurons (Fig. 1, Fig. 2, Fig. 3, Fig. 4), cholinergic neurons (Fig. 1, Fig. 2), and serotonin neurons (Fig. 5, Fig. 6) in the brainstem was assessed in 11 adult male and 11 adult female YFP–PPG mice. After immunoperoxidase staining, YFP–PPG neurons in the NTS and IRT showed intense YFP-immunoreactivity throughout their cell bodies and dendrites (Fig. 1A–C, I and J). In the brainstem, varicose axons arising from the YFP–PPG neurons were also very
Discussion
In this study, we used two- and three-colour immunoperoxidase labelling to assess the GLP-1 innervation of neurochemically identified brainstem neurons in YFP–PPG mice (Reimann et al., 2008, Llewellyn-Smith et al., 2011). YFP expression in these mice is restricted to cells in which the PPG promoter is active (Reimann et al., 2008, Hisadome et al., 2010). Consequently, in these mice, YFP-immunoreactivity occurs exclusively in cells that synthesise PPG, the precursor for glucagon, as well as
Conclusions
In summary, this study provides the first detailed description of the GLP-1 innervation of neurochemically identified monoaminergic and cholinergic neurons in the brainstem. Our observations create an anatomical scaffold for understanding the diverse array of autonomic functions affected by central GLP-1. We expect that the information about the distribution of GLP-1 inputs to brainstem neurons presented here will provide a valuable guide for functional studies probing the roles of central
Acknowledgments
This research was supported by grants from the Medical Research Council, UK (G0600928) to S.T and the National Health & Medical Research Council of Australia (Project Grants 480414 and 1025031) to I.L.S, and Wellcome Trust Senior Research Fellowships (WT088357 and WT084210) to F.M.G and F.R, respectively. Lee Travis provided expert technical assistance.
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