Research ArticleLong-term Changes in the Central Amygdala Proteome in Rats with a History of Chronic Cocaine Self-administration
Introduction
Recent estimates suggest that up to 5.5 million people in the U.S. (Substance Abuse and Mental Health Services Administration, 2019), and 18 million people worldwide have used cocaine in the past year (UNODC, 2019). Out of the population of U.S. cocaine users, about 1 million are diagnosed with cocaine use disorder (CUD; Substance Abuse and Mental Health Services Administration, 2019). However, despite years of preclinical and clinical research efforts, there are no FDA-approved medications for the treatment of CUD. Persistent drug craving represents a key neurobehavioral feature in CUD and is responsible for high rates of relapse even long after the discontinuation of cocaine use (O’Brien, 1997). Drug craving (and drug seeking) in CUD is most frequently triggered by cocaine-associated environmental cues that recruit specific limbic and basal ganglia circuits – with the striatum, frontal cortex, and amygdala identified as ‘hot-spots’ of craving-related neural activity (Grant et al., 1996, Childress et al., 1999, Garavan et al., 2000). Recent animal models successfully recapitulated both the persistent character of cue-elicited cocaine seeking, as well as its time-dependent increase (also termed ‘incubation of cocaine craving’; for reviews see: Pickens et al., 2011, Li et al., 2015a, Li et al., 2015b) that were previously documented in abstinent cocaine users (Gawin and Kleber, 1986). Animal studies have also shown that one of the critical brain regions recruited to support incubated cocaine seeking is the central nucleus of the amygdala (CeA; Lu et al., 2007, Lu et al., 2005b, Roura-Martínez et al., 2020, Xi et al., 2013).
The CeA is a part of the amygdaloid complex of 13 nuclei collectively implicated in initiating and regulating specific behaviors such as fear and anxiety, chronic pain, learning and attention, decision making, as well as reward and motivation (Davis, 1992, Gallagher and Holland, 1994, Gallagher and Chiba, 1996, Maren and Fanselow, 1996, Baxter and Murray, 2002, Sah et al., 2003, Neugebauer et al., 2004, Gupta et al., 2011). Importantly, the CeA does not ‘only’ orchestrate innate behavioral responses, but also plays an important role during the acquisition, consolidation, and expression of conditioned behaviors (Fadok et al., 2018). As such, the CeA, together with the basolateral amygdala (BLA), are known to regulate drug-related associative learning and drug relapse (for review see: Buffalari and See, 2010). Interestingly, under certain conditions, inactivation of the CeA, but not BLA, reduce drug seeking (Lu et al., 2005b, Li et al., 2015b). Studies show that CeA is recruited to control drug seeking following extensive drug self-administration experience and/or during protracted drug withdrawal. In particular, neural activity within the CeA is necessary for the expression of incubated drug craving after chronic exposure to cocaine, methamphetamine, and morphine (Lu et al., 2007, Lu et al., 2005b, Uejima et al., 2007, Li et al., 2015b, Li et al., 2008). Initial studies also revealed the identity of a few neural substrates in the CeA involved in the regulation of persistent (incubated) cocaine seeking. A recent study by Xi et al. (2013) found that inhibition of D3 dopamine receptors in the CeA reduced cocaine relapse after both brief and prolonged abstinence, suggesting a role of mid-brain dopaminergic input in the development and expression of incubated cocaine seeking. Other evidence suggests that the activity of glutamatergic inputs is also necessary for the expression of incubated cocaine seeking (Lu et al., 2007). Finally, increased activity of the MAPK/ERK pathway in the CeA has been identified as a cellular signature permitting the expression of incubated cocaine seeking (Lu et al., 2005b). While these findings collectively support the importance of this brain structure in persistent cocaine seeking, neuroadaptations that may be responsible for aberrant neural activity within the CeA have not been comprehensively studied. A single study limited to evaluation of ionotropic glutamate receptor expression has found a time-dependent increase in glutamate receptor GluN1 subunit expression in the CeA after 30-day withdrawal from extended-access cocaine self-administration (Lu et al., 2005a).
In this study, a comprehensive proteomic approach was used to identify differentially expressed proteins in the rat CeA 45 days after discontinuation of extended-access cocaine self-administration. First, we identified CeA proteins differentially expressed in cocaine vs. saline rats through LC–MS/MS approach and ranked them by the magnitude of their change. Next, we analyzed selected significantly altered proteins by immunoblotting to validate proteomic findings. And finally, we explored and identified altered protein interaction networks and canonical pathways using the Ingenuity Pathway Analysis (IPA). As both the brain region and post-cocaine abstinence period analyzed in this study have been associated with heightened cocaine seeking (Grimm et al., 2001, Pickens et al., 2011), we hypothesize that our approach is suitable for identification of candidate neurobiological substrates related to relapse vulnerability in CUD.
Section snippets
Subjects
Adult male Sprague-Dawley rats (Charles River Laboratories; 275 g on arrival; N = 16) were acclimated to the animal facility for one week, housed individually in 12-h reverse light/dark cycle and had ad libitum access to food (standard rat chow) and water, except for the period of self-administration and abstinence (see below). All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida in accordance with the Guide for the Care and Use of
Self-administration
Animals underwent 6 days of 1 h/session, and 12 days of 6 h/session cocaine self-administration, followed by 45 days of abstinence (Fig. 1A). Rats assigned to the cocaine self-administration group successfully learned to discriminate between the active and inactive nose poke ports (Fig. 1B). Using repeated measures ANOVA, we found an interaction between nose port and time (F(17, 238) = 9.385, p < 0.0001), with animals significantly discriminating between the nose ports during all days of LgA,
Discussion
Utilizing an unbiased shotgun proteomic approach, this study shows for the first time wide-spread changes in protein expression and dysregulation of specific protein networks in the rat CeA, as detected 45 days after discontinuation of extended access cocaine self-administration. Specifically, out of 228 proteins with significantly altered expression in the cocaine group (when compared to saline), we identified TH as the most upregulated protein, and a cluster of proteins involved in the
Acknowledgments
UF McKnight Brain Research Institute pilot award (MS). Mass Spectrometry Research and Education Center at the University of Florida - NIH S10 OD021758-01A1. We thank Dr. Kari Basso (University of Florida, Department of Chemistry) for the help of analyzing tissue samples.
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