Elsevier

Neuropharmacology

Volume 102, March 2016, Pages 92-102
Neuropharmacology

Endocannabinoid regulation of nausea is mediated by 2-arachidonoylglycerol (2-AG) in the rat visceral insular cortex

https://doi.org/10.1016/j.neuropharm.2015.10.039Get rights and content

Highlights

  • Visceral insular cortex (VIC) 2-AG is increased during LiCl-induced nausea in rats.

  • Intra-VIC MAGL inhibition supresses nausea-induced conditioned gaping.

  • The anti-nausea effects of MAGL inhibition are attenuated following CB1 antagonism.

  • MAGL inhibition reduces LiCl-induced Fos immunoreactivity within the VIC.

  • The endocannabinoid system regulates nausea via 2-AG and CB1 signaling in the VIC.

Abstract

Cannabinoid (CB) agonists suppress nausea in humans and animal models; yet, their underlying neural substrates remain largely unknown. Evidence suggests that the visceral insular cortex (VIC) plays a critical role in nausea. Given the expression of CB1 receptors and the presence of endocannabinoids in this brain region, we hypothesized that the VIC endocannabinoid system regulates nausea. In the present study, we assessed whether inhibiting the primary endocannabinoid hydrolytic enzymes in the VIC reduces acute lithium chloride (LiCl)-induced conditioned gaping, a rat model of nausea. We also quantified endocannabinoid levels during an episode of nausea, and assessed VIC neuronal activation using the marker, c-Fos. Local inhibition of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of 2-arachidonylglycerol (2-AG), reduced acute nausea through a CB1 receptor mechanism, whereas inhibition of fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of anandamide (AEA), was without effect. Levels of 2-AG were also selectively elevated in the VIC during an episode of nausea. Inhibition of MAGL robustly increased 2-AG in the VIC, while FAAH inhibition had no effect on AEA. Finally, we demonstrated that inhibition of MAGL reduced VIC Fos immunoreactivity in response to LiCl treatment. Taken together, these findings provide compelling evidence that acute nausea selectively increases 2-AG in the VIC, and suggests that 2-AG signaling within the VIC regulates nausea by reducing neuronal activity in this forebrain region.

Introduction

Although the central neural circuitry underlying the vomiting reflex is well established (Hornby, 2001, Andrews and Horn, 2006, Horn, 2008), the precise brain mechanisms underlying nausea are less clear. Two recent functional neuroimaging studies in humans explored the brain circuitry involved in nausea (Napadow et al., 2013, Sclocco et al., 2014), and revealed an important role for the insular cortex; specifically, a strong sensation of nausea resulted in sustained activation of the interoceptive insula, as well as limbic and sub-cortical regions. Importantly, autonomic activation, which is important for nausea perception (LaCount et al., 2011), appears to be modulated by the insular cortex (Sclocco et al., 2014). This work suggests that the insula is part of a broad network of brain areas mediating the experience of nausea in humans (Napadow et al., 2013), and may play a particularly critical role in regulating autonomic control in response to nausea-inducing stimuli (Sclocco et al., 2014).

An intact forebrain is also necessary for the establishment of conditioned gaping responses in rats (Grill and Norgren, 1978a), a well-established rodent model of nausea (Parker, 2003, Parker et al., 2015). The insular cortex (IC) is particularly critical in this response, as its ablation was found to selectively disrupt conditioned gaping behavior (Kiefer and Orr, 1992). The IC (Cechetto and Saper, 1987, Allen et al., 1991) is comprised of gustatory neurons occupying the dysgranular (and agranular) layers (gustatory insular cortex [GIC]; Kosar et al., 1986), and visceral neurons located in the posterior granular layer (visceral insular cortex [VIC]; Cechetto and Saper, 1987). We have previously shown that localized administration of the anti-emetic drug, ondansetron (a 5-hydroxytryptamine 3 [5-HT3] antagonist), selectively blocked conditioned gaping upon intra-VIC - but not GIC – administration in rats (Tuerke et al., 2012a). Conversely, intra-VIC administration of a 5-HT3 agonist, potentiated conditioned gaping caused by the emetic agent lithium chloride (LiCl), and even produced conditioned gaping on its own (Tuerke et al., 2012a). Moreover, Contreras et al. (2007) reported that administration of LiCl produced enhanced Fos expression in the VIC, and inactivation of this region attenuated LiCl-induced lying-on-belly behavior (lying with flattened belly on cage [LOB]), a measure of unconditioned nausea in rats (Parker et al., 1984, Tuerke et al., 2012b). Collectively, these studies demonstrate the importance of the IC, and particularly the VIC, in mediating behaviors reflective of the sensation of nausea in rats.

It is well known that exogenous cannabinoids exert robust anti-nausea and anti-emetic effects (Sharkey et al., 2014). We have shown that systemically administered cannabinoid (CB) agonists (Limebeer and Parker, 1999, Parker and Mechoulam, 2003, Parker et al., 2003) or intra-cerebral administration of the synthetic cannabinoid agonist HU-210 into the VIC (Limebeer et al., 2012) reduced nausea-induced conditioned gaping in rats. Furthermore, exogenous administration of the endocannabinoid 2-arachidonoylglycerol (2-AG; Mechoulam et al., 1995, Sugiura et al., 1995), but not anandamide (AEA; Devane et al., 1992) within the VIC suppressed nausea-induced conditioned gaping, as well (Sticht et al., 2015). Surprisingly, this effect of 2-AG was not mediated via CB1 receptors, but rather dependent on endocannabinoid metabolic products (Sticht et al., 2015). The physiologic role of endogenously released endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the VIC during nausea is still unknown, however; thus, the role of the endocannabinoid system in regulating this sensation remains to be determined.

We hypothesize that the VIC endocannabinoid system is recruited during an episode of nausea, and as such, regulates this sensation in rats. The rapid hydrolysis of endocannabinoids in vivo represents a significant challenge in investigating the physiological functions of these lipids; therefore, we investigated the role of endocannabinoids in the VIC via localized inhibition of their catabolic enzymes. This approach has been effectively used to elucidate the physiological role of the endocannabinoid system in several pre-clinical models, including pain (Davis, 2014), anxiety and depression (Gaetani et al., 2009), addiction and withdrawal (Sidhpura and Parsons, 2011, Muldoon et al., 2013), and nausea (Parker et al., 2014). Specifically, drugs that block their respective catabolic enzymes, fatty acid amide hydrolase (FAAH; Cravatt et al., 1996) and monoacylglycerol lipase (MAGL; Dinh et al., 2002) produce elevated brain levels of 2-AG and AEA. Additionally, we quantified endocannabinoid levels in the VIC during an episode of nausea to infer whether this system plays a tonic role in the regulation of nausea, an effect that is achieved through endocannabinoid (and CB1)-mediated inhibition of neuronal signaling in this region. To this end, given the role for VIC activation during an episode of nausea (Contreras et al., 2007), we assessed whether the parameters of LiCl administration in the current study leads to elevated c-Fos within the VIC, and whether the anti-nausea effects following MAGL inhibition are associated with a subsequent decrease in neuronal activity in this region.

Section snippets

Animals

Sprague–Dawley rats (300–350 g; Charles River Lab, St Constant, Quebec) were single-housed in opaque plastic cages (10.25″W × 18.75″D × 8″H; 22 °C) with Teklad corncob bedding (Harlan). Shredded paper and opaque tube/container were placed in each cage for environmental enrichment. Rats were maintained on a reverse light/dark cycle (7:00 am lights off; 7:00 pm lights on) with free access to food (Iams rodent chow, 18% protein). Behavioral testing occurred during the dark cycle; thus,

Intra-VIC administration of the dual FAAH/MAGL JZL195 reduces nausea-induced conditioned gaping

The dual FAAH/MAGL inhibitor JZL195 suppressed LiCl-induced conditioned gaping reactions. Fig. 1A shows that rats receiving intra-VIC infusions of JZL195 prior to LiCl treatment displayed fewer gapes to LiCl-paired saccharin (t(12) = 2.48, p < 0.05) during the TR test. To assess whether JZL195 selectively interfered with nausea – as opposed to a general interference with learning (conditioning) – we assessed whether this compound modified CTA of LiCl-paired saccharin in a two-bottle consumption

Discussion

The insular cortex is increasingly recognized as a critical forebrain region involved in the sensation of nausea (e.g., Contreras et al., 2007, Napadow et al., 2013, Sclocco et al., 2014). Here, we demonstrate that the endocannabinoid 2-AG serves as an endogenous regulator of nausea in the rat VIC, an effect mediated through CB1 receptors. Indeed, 2-AG was found to be selectively elevated in the VIC during an episode of LiCl-induced nausea. Furthermore, LiCl-induced neuronal activation in this

Funding and disclosures

The authors declare that there are no financial interests or conflicts of interest. The research was funded by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC:92057) and Canadian Institutes of Health Research (CIHR: 334086) to LAP and KAS, and National Institutes of Health (P30DA033934, RO1DA032933 and P01DA009789) to AHL and BFC, as well as an NSERC doctoral Canada Graduate Scholarship award to MAS. KAS is the Crohn's and Colitis Foundation of Canada

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