Elsevier

Neuroscience

Volume 218, 30 August 2012, Pages 12-19
Neuroscience

Ghrelin-deficient mice have fewer orexin cells and reduced cFOS expression in the mesolimbic dopamine pathway under a restricted feeding paradigm

https://doi.org/10.1016/j.neuroscience.2012.05.046Get rights and content

Abstract

Ghrelin is an orexigenic stomach peptide previously found to be important for the full display of anticipatory locomotor activity and hypothalamic neuronal activation that precedes a daily scheduled meal in mice. Ghrelin is also important for food-related motivation and seems to have direct effects in the mesocorticolimbic dopamine reward system. Here we hypothesized that neuronal activation in reward-related areas in anticipation of a scheduled meal could be mediated by elevated ghrelin induced by scheduled feeding, and therefore this would be attenuated in ghrelin receptor knock-out (GHSR KO) animals. We found that this was indeed the case for the ventral tegmental area and the shell, but not the core, of the nucleus accumbens. In addition, our results show a reduction in the proportion of activated orexin-immunoreactive (IR) neurons in GHSR KO animals in anticipation of the scheduled meal in comparison to the proportion of activated orexin neurons in wild type (WT) mice. Interestingly we observed that both GHSR and ghrelin KO mice had fewer orexin-IR cells than their WT littermates suggesting that lack of ghrelin or sensitivity to ghrelin may play a role in the development of the orexin system. Our data also suggest that ghrelin may mediate food anticipation, in part, by stimulating both the orexin system and the mesolimbic reward system.

Highlights

► cFOS induced by scheduled feeding is reduced in the VTA and accumbens of GHSR knock-out mice. ► Mice lacking ghrelin or its receptor have reduced orexin neurons in the lateral hypothalamus. ► Ghrelin may be important for the development of the orexin system.

Introduction

The mammalian circadian system controls the daily temporal organization of most physiological, emotional, and behavioral functions, by synchronizing the daily rhythms of rest and activity, hormonal levels, mood, and motivated behaviors such as feeding. Light is the primary synchronizer of the master circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus (Reppert and Weaver, 2002). However, feeding can also synchronize circadian rhythms (Stephan, 2002). Rodents given access to food for only a few hours during the day, a time when these nocturnal animals do not typically feed, will show an increase in activity several hours prior to food availability, a phenomenon known as food anticipatory activity (Stephan, 2002). This type of feeding schedule can synchronize circadian oscillators in the limbic forebrain and hypothalamus, including the bed nucleus of the stria terminalis (BNST-OV), the central and the basolateral nuclei of the amygdala, and the hippocampus (Lamont et al., 2005, Waddington Lamont et al., 2007), and can induce a diurnal rhythm in the dorsomedial hypothalamus (Verwey et al., 2007).

Ghrelin is an orexigenic stomach peptide important for energy balance (Cowley et al., 2003, Wertz-Lutz et al., 2006, Szentirmai et al., 2007). Ghrelin neurons show synapses on hypothalamic neurons known to regulate energy balance and also project to limbic forebrain regions synchronized by scheduled daily feeding including the BNST and amygdala (Cowley et al., 2003). When administered either peripherally or directly into the brain, ghrelin causes feeding (Wertz-Lutz et al., 2006, Szentirmai et al., 2007), and it is secreted in response to fasting and hypoglycemia (Toshinai et al., 2001).

Importantly, ghrelin seems to be related to the motivation to feed, as plasma ghrelin levels increase prior to meals in humans (Cummings et al., 2001), rodents (Sanchez et al., 2004), and cattle (Wertz-Lutz et al., 2006), and are rapidly reduced following a meal (Toshinai et al., 2001). The relationship between the timing of the meal and the ghrelin peak is particularly evident when animals are placed on a restricted feeding (RF) schedule (Bodosi et al., 2004, Wertz-Lutz et al., 2006). This precise timing suggests a role for ghrelin in the circadian regulation of feeding and, indeed, ghrelin seems to play an important, although non-essential role (Blum et al., 2009, LeSauter et al., 2009). Timed ghrelin injections (at zeitgeber time (ZT) 3) and scheduled feeding limited to several hours during the day can cause increased neuronal activation (as measured by cFOS immunoreactivity (IR)) in numerous brain regions implicated in energy balance and circadian regulation of feeding, including the BNST-OV, paraventricular nucleus of the thalamus (PVT), and the hypothalamic dorsomedial (DMH), ventromedial (VMH), arcuate (ARC), and lateral (LH) nuclei (LH; (Blum et al., 2009)). However, ghrelin receptor (GHSR)-deficient mice (Blum et al., 2009, LeSauter et al., 2009), show attenuation, and/or decrease in the speed of acquisition of food anticipatory activity, and less neuronal activation in anticipation of the scheduled meal.

Ghrelin also modulates food-related motivation at the level of the midbrain dopamine (DA) reward system (reviewed in (Abizaid, 2009). GHSR are present on ventral tegmental area (VTA) DA neurons (Guan et al., 1997, Zigman et al., 2005) and ghrelin rapidly lowers the threshold of excitability in the VTA (Abizaid et al., 2006). Ghrelin also causes an increase in DA turnover in the ventral striatum, and the release of DA from the nucleus accumbens (NAcc) when administered intra peritoneally (Abizaid et al., 2011), in the third ventricle (Jerlhag et al., 2006), or directly into the VTA (Jerlhag et al., 2007). Finally, ghrelin administration was able to affect synaptic plasticity in VTA neurons, resulting in a greater number of synapses overall, more excitatory synapses, inputs, and excitatory post-synaptic currents, and fewer inhibitory inputs, synapses, and inhibitory post-synaptic currents (Abizaid et al., 2006). Together, these findings suggest that ghrelin plays a role in food rewards by modulating the release of DA from the VTA (Abizaid, 2009).

In addition to the VTA, GHSR are also found on orexin neurons in the lateral hypothalamus (LH). Orexin is a peptide transmitter that plays a role in both feeding and arousal (see (Tsujino and Sakurai, 2009) for review). Like ghrelin, orexin plays a role in circadian feeding mechanisms. The loss of orexin neurons, either by targeted genetic ablation of orexin neurons (Akiyama et al., 2004, Mieda et al., 2004), or by knock out of the orexin gene (Kaur et al., 2008, Gunapala et al., 2011), causes an attenuation, or delay in acquisition of locomotor activity in anticipation of a scheduled meal. Furthermore, orexin also modulates the midbrain DA reward system (Borgland et al., 2006) via receptors on the VTA (Fadel and Deutch, 2002), and, notably ghrelin’s effects on the reward value of a high-fat diet may be mediated in part via action on the orexin receptor (Perello et al., 2010). In this study we hypothesized that neuronal activation in reward-related areas could be mediated by elevated ghrelin induced by scheduled feeding, and therefore this would be attenuated in GHSR knock-out (GHSR KO) animals. We also hypothesized that the orexin system of GHSR KO mice would be less active in anticipation of the scheduled meal than that of their wild type (WT) littermates.

Section snippets

Animals

Male mice with targeted mutations to the ghrelin (ghrelin KO) and ghrelin-receptor genes (GHSR KO) and their WT littermates were bred at the Carleton University Department of Neuroscience animal facilities. Animals were approximately 3–4 months of age at the beginning of the experiment. Both types of knock-out mice originated from heterozygous breeding pairs obtained from Regeneron Pharmaceuticals (Tarrytown, NY, USA). These mice were generated using a mixed C57BL/6J and DBA strain as background

GHSR KO animals show attenuated cFOS expression in major regions of the mesolimbic DA pathway in anticipation of a scheduled meal

As can be seen in Fig. 2, the expression of cFOS in the VTA was significantly lower in anticipation of a scheduled meal in GHSR KO (n = 3) mice relative to WT (n = 4) mice (T[5] = 2.7, p < 0.05). In the VTA, GHSR KO mice had only 21% (±3.6%) of the number of cFOS-IR cells observed in WT animals (100% ± 24.4%). An examination of some of the major projection areas of the VTA DA neurons, the dorsal and ventral striatum, shown in Fig. 3, reveals that the expression of cFOS in the NAcc shell was also lower in

Discussion

In the present study, we examined the role of ghrelin in activating areas of the brain related to reward and arousal including the VTA, NAcc core and shell, dorsal striatum and LH, in anticipation of a scheduled meal. Our findings confirm that cFOS expression is elevated in anticipation of a scheduled meal in some regions of the mesolimbic reward pathway in WT mice, and this effect is attenuated in GHSR KO mice. These effects were observed in the VTA and the shell portion of the NAcc, a

Acknowledgements

This project was funded by Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI) awarded to A.A. We would like to thank Dr. Mark Sleeman and Dr. Tamas Horvath for the GHSR and ghrelin knock-out mice. We also thank Stuart Davidson for his excellent technical expertise and Darin Kinsey for his assistance.

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