The Role of Mesolimbic Reward Neurocircuitry in Prevention and Rescue of the Activity-Based Anorexia (ABA) Phenotype in Rats
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Neuropsychopharmacology (2017) 42, 2292–2300 © 2017 American College of Neuropsychopharmacology. All rights reserved 0893-133X/17 www.neuropsychopharmacology.org The Role of Mesolimbic Reward Neurocircuitry in Prevention and Rescue of the Activity-Based Anorexia (ABA) Phenotype in Rats 1 1 ,1 Claire J Foldi , Laura K Milton and Brian J Oldfield* 1 Department of Physiology, Monash University, Clayton, VIC, Australia Patients suffering from anorexia nervosa (AN) become anhedonic; unable or unwilling to derive normal pleasures and avoid rewarding outcomes, most profoundly in food intake. The activity-based anorexia (ABA) model recapitulates many of the characteristics of the human condition, including anhedonia, and allows investigation of the underlying neurobiology of AN. The potential for increased neuronal activity in reward/hedonic circuits to prevent and rescue weight loss is investigated in this model. The mesolimbic pathway extending from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) was activated using a dual viral strategy, involving retrograde transport of Cre (CAV-2-Cre) to the VTA and coincident injection of DREADD receptors (AAV-hSyn-DIO-hM3D(Gq)-mCherry). Systemic clozapine-n-oxide (CNO; 0.3 mg/kg) successfully recruited a large proportion of the VTA-NAc dopaminergic projections, with activity evidenced by colocalization with elevated levels of Fos protein. The effects of reward circuit activation on energy balance and predicted survival was investigated in female Sprague-Dawley rats, where free access to running wheels was paired with time-limited (90 min) access to food, a paradigm (ABA) which will cause anorexia and death if unchecked. Excitation of the reward pathway substantially increased food intake and food anticipatory activity (FAA) to prevent ABA-associated weight loss, while overall locomotor activity was unchanged. Similar activation of reward circuitry, delayed until establishment of the ABA phenotype, rescued rats from their precipitous weight loss. Although these data are consistent with shifts primarily in food intake, the contribution of mechanisms including energy expenditure to survival remains to be determined. These results will inform the neurobiological underpinnings of AN, and provide insight into the mechanisms of reward circuitry relevant to feeding and weight loss. Neuropsychopharmacology (2017) 42, 2292–2300; doi:10.1038/npp.2017.63; published online 19 April 2017 INTRODUCTION Although genetic, sociocultural and psychosocial factors undoubtedly contribute to the pathogenesis of AN, it is now Anorexia nervosa (AN) is a disorder with complex etiology and the highest mortality rate of any psychiatric disease clear that there are underlying neurobiological drivers (Harris and Barraclough, 1998). AN is characterized by a (Gorwood et al, 2016; Kaye et al, 2013; van Kuyck et al, dramatic reduction in body weight caused by excessive caloric 2009). Indeed, mounting evidence from neuroimaging restriction, which is accompanied by physiological, biochem- studies supports a role for altered central reward processing ical and behavioral disturbances including the diminished in the development and maintenance of AN (Frank et al, capacity to experience pleasure or reward (Klein and Walsh, 2012; Frank et al, 2016b; Kaye et al, 2009; O'Hara et al, 2015). 2004). These patients have been described as anhedonic or For example, patients with AN have reduced neural activity ascetic (Davis and Woodside, 2002). A lack of understanding in the striatum in response to sucrose (Wagner et al, 2008) of the pathophysiology of AN has hindered the development and increased dopamine (DA) receptor binding in the of effective treatments, with no clear evidence to support the anterior ventral striatum (indicating decreased endogenous efficacy of existing pharmacotherapeutic interventions beyond DA concentration (Frank et al, 2005)) compared to healthy improving the depressive symptoms (SSRIs) or delusional controls. In addition, a disrupted dopaminergic system may dysmorphic features (olanzapine and haloperidol) that often play a role in susceptibility to AN, with multiple DA complicate the course of the disorder (Miniati et al, 2016). receptor polymorphisms associated with the condition (Bergen et al, 2005). The link between anorexia and *Correspondence: Professor BJ Oldfield, Department of Physiology, anhedonia is highlighted by studies showing that patients Monash University, 26 Innovation Walk, Clayton, VIC 3800, Australia, with AN dislike highly palatable foods (Fernstrom et al, Tel: +61 3 990 52507, Fax: +61 3 990 20707, E-mail: [email protected] 1994), rate food as negative when hungry (Santel et al, 2006) Received 24 January 2017; revised 23 February 2017; accepted 1 March and report dysphoric mood following food consumption 2017; accepted article preview online 21 March 2017 (Kaye, 2008). Reward neurocircuitry and activity-based anorexia CJ Foldi et al 2293 Anhedonia has its biological underpinnings in the studies (approved by the Monash Animal Resource Platform mesolimbic DA system (Davis and Woodside, 2002) and it ethics committee; MARP/2015/046). Animals were allowed is therefore plausible that disrupted DA signaling in reward- to lose a total of 20–25% of baseline body weight before being based neural circuits gives rise to anhedonia in AN (Der- removed from the experiment and euthanized with 300 mg/ Avakian and Markou, 2012). The microstructure of neural kg sodium pentobarbitone (Lethabarb; Virbac, Australia). circuits and transmitters coding for reward can be simply Rats were housed individually in transparent activity wheel subdivided into (1) the activation of μ opioid, endocanna- and living chambers (Lafayette Instruments, model no. binoid or GABA receptors in the nucleus accumbens (NAc), 80859) in a temperature (22.5–25 °C) and humidity which enhance the affective value or hedonic ‘liking’ (60 ± 10%) controlled room, under a 12 h light/dark cycle characteristics of reward; and (2) dopaminergic projections (lights off at 15:00 hours). A singly housed male rat was originating in the ventral tegmental area (VTA) and present in the experimental room to synchronize the estrous extending to the NAc which encode for motivated behaviors cycles of the female rats. (Berridge and Kringelbach, 2008; Der-Avakian and Markou, 2012). Viral Activation Strategy Activity-based anorexia (ABA) is a robust and widely recognized rodent model, which mimics many of the A dual viral strategy was employed, involving microinjection important characteristics of human AN (Dixon et al, 2003), into two brain regions in the same rat. CAV-2-CreEGFP including hyperactivity and reduced food consumption and, (IGMM; Montpellier, France) was injected into the NAc with of particular relevance to the present study, anhedonia subsequent retrograde transport to the VTA, amongst other (Brown et al, 2008; Verty et al, 2011). In the ABA paradigm, well defined sources of afferent input to the NAc. AAV-hSyn- unrestricted access to running wheels (RW) and time-limited DIO-hM3D(Gq)-mCherry (abbreviated +M3-mCherry; UNC access to food leads to a profound reduction in body weight Vector Core, USA) was injected into the VTA and contains that, without intervention, results in death by starvation the floxed inverted sequence of hM3D(Gq)-mCherry, which (Epling et al, 1983; Routtenberg and Kuznesof, 1967). Several reorients in the presence of Cre to allow DREADD receptor lines of evidence implicate disrupted DA signaling and expression and ligand binding. This ensures that +M3- anhedonia as central to the ABA phenotype. First, DA mCherry is not expressed in all AAV-infected cells, but antagonism with cis-flupenthixol reduces body weight loss exclusively in those that are also infected with CAV-2- associated with ABA by increasing food intake and reducing CreEGFP, thus defining the VTA to NAc neuronal pathway. hyperactivity (Verhagen et al, 2009a). Furthermore, +M3-mCherry is activated by clozapine-n-oxide (CNO; Δ9-tetrahydrocannabinol (THC) increases DA levels in the Tocris Bioscience), delivered via daily intraperitoneal injec- striatum (Malone and Taylor, 1999), and in ABA rats THC tion (0.3 mg/kg), causing infected cells to depolarize. administration increases highly palatable food (high-fat diet; HFD) consumption leading to attenuation of body weight Surgical and Viral Injection Procedures loss (Verty et al, 2011). Moreover, access to highly palatable food alone (sweet HFD), that is known to activate All rats were injected bilaterally with 250 nl of neurochemistry associated with reward, can both prevent CAV-2-CreEGFP (5.0 × 108 genomic copies/μl) into NAc and rescue ABA-associated body weight loss (Brown et al, (from bregma: AP: +1.6 mm, ML: ± 0.8 mm, and DV: 2008). This suggests that increasing the hedonic value of food − 6.6 mm) using a stereotaxic apparatus (Kopf Instruments, is pertinent to prevention of and recovery from the ABA CA, USA). Rats then received bilateral injections of 250 nl of 12 μ phenotype in rats. +M3-mCherry (4.5 × 10 genomic copies/ l) into VTA The combination of designer receptor exclusively activated (from bregma; AP: − 5.3 mm, ML: ± 0.6 mm, DV: by designer drugs (DREADD) technologies with Cre- − 7.7 mm). Viruses were infused over 5 min through glass mediated homologous recombination enables specific target- micropipettes with a tip diameters ~ 40 μm. Both viruses ing and manipulation of neural pathways (Gore et al, 2013). were injected under the same anesthetic (2.5% isoflurane in Activation of