Elsevier

Neuroscience

Volume 443, 1 September 2020, Pages 93-109
Neuroscience

Research Article
Long-term Changes in the Central Amygdala Proteome in Rats with a History of Chronic Cocaine Self-administration

https://doi.org/10.1016/j.neuroscience.2020.06.011Get rights and content

Highlights

  • Cocaine self-administration and prolonged abstinence alter global protein expression in the central amygdala.

  • Tyrosine hydroxylase is the most upregulated, while contactin-1 is one of the most downregulated proteins.

  • Protein networks linked to neurobehavioral disorders, energy metabolism, and plasticity are the most significantly altered.

Abstract

The central nucleus of the amygdala (CeA) is a striatum-like structure that contains mainly inhibitory circuits controlling a repertoire of (mal)adaptive behaviors related to pain, anxiety, motivation, and addiction. Neural activity in the CeA is also necessary for the expression of persistent and robust drug seeking, also termed ‘incubation of drug craving.’ However, neuroadaptations within this brain region supporting incubated drug craving have not been characterized. Here, we conducted a comprehensive analysis of protein expression in the CeA of male rats after prolonged (45-day) abstinence from extended-access cocaine self-administration using a quantitative proteomic approach. The proteomic analysis identified 228 unique proteins altered in cocaine rats relative to animals that received saline. Out of the identified proteins, 160 were downregulated, while 68 upregulated. Upregulation of tyrosine hydroxylase and downregulation of neural cell-adhesion protein contactin-1 were validated by immunoblotting. Follow-up analysis by the Ingenuity Pathway Analysis tool revealed alterations in protein networks associated with several neurobehavioral disorders, cellular function and morphology, as well as axogenesis, long-term potentiation, and receptor signaling pathways. This study suggests that chronic cocaine self-administration, followed by a prolonged abstinence results in reorganization of specific protein signaling networks within the CeA that may underlie incubated cocaine craving and identifies potential novel ‘druggable’ targets for the treatment of cocaine use disorder (CUD).

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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      Furthermore, although transcription and protein immunoreactivity of TH is known to increase after extended cocaine exposure in the rodent midbrain (Beitner-Johnson et al., 1991; Logan et al., 2019; Masserano et al., 1996), attempts to measure the effects of cocaine on TH in downstream targets have yielded mixed results. TH immunoreactivity is upregulated in the central amygdala after 45 days of withdrawal from cocaine self-administration in rats; however there appears to be no change in protein levels in the prefrontal or orbitofrontal cortex after cocaine conditioned place preference (Grimm et al., 2002; Hámor et al., 2020). In the dorsal striatum, chronic cocaine injection increases TH protein levels, while the number of TH positive varicosities in the nucleus accumbens has been reported to both increase and decrease after cocaine exposure, depending on tissue sub-sectioning and length of withdrawal (Balda et al., 2009; Schmidt et al., 2001; Todtenkopf et al., 2000).

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