Phenotyping CCL2 Containing Central Amygdala Neurons Controlling Alcohol Withdrawal-Induced Anxiety.

Kathryn M Harper,George R Breese,Laure Aurelian,Irina Balan,Darin J Knapp,Caroline A Todd,Hugh E Criswell

doi:10.3389/fncel.2020.580583

Abstract

Chemokines such as chemokine (C-C motif) ligand 2 (CCL2) play a role in several behaviors, including anxiety-like behavior, but whether neurons are an important source of CCL2 for behavior and how neuronal CCL2 may work to affect behavior are still debated. When a herpes simplex virus (HSV) vector was used to knockdown CCL2 mRNA in neurons of the central nucleus of the amygdala (CeA) in rats experiencing multiple withdrawals from low dose ethanol, anxiety-like behavior appeared in the social interaction task. To examine this finding further Fractalkine (CX3CL1), a chemokine that is often found to have an opposing function to CCL2 was measured in these rats. Both alcohol withdrawal and CCL2 knockdown increased the levels of the anti-inflammatory protein CX3CL1. The combination of alcohol withdrawal and CCL2 knockdown decreased CX3CL1 and may alter pro-inflammatory/anti-inflammatory balance, and thus highlights the potential importance of CCL2 and CCL2/CX3CL1 balance in anxiety. To find a mechanism by which neuronal chemokines like CCL2 could affect behavior, retrograde tracing with fluorescent nanobeads was done in two brain regions associated with anxiety the bed nucleus of the stria terminalis (BNST) and the ventral periaqueductal gray (VPAG). These studies identified CeA projection neurons to these brain regions that contain CCL2. To demonstrate that CCL2 can be transported via axons to downstream brain regions, the axonal transport blocker, colchicine, was given and 24 h later, the accumulation of CCL2 in CeA neuronal cell bodies was found. Finally, CCL2 in CeA neurons was localized to the synapse using confocal microscopy with enhanced resolution following deconvolution and electron microscopy, which along with the other evidence suggests that CCL2 may be transported down axons in CeA neurons and released from nerve terminals perhaps into brain regions like the BNST and VPAG to affect behaviors such as anxiety. These results suggest that neurons are an important target for chemokine research related to behavior.

Highlights

Alcohol withdrawal increases brain neuroimmune proteins such as chemokine C-C motif ligand 2 (CCL2; He and Crews, 2008; Ehrlich et al, 2012; Freeman et al, 2012; Vetreno et al, 2013; Whitman et al, 2013; Kane et al, 2014; Harper et al, 2015) in a brain-region-specific fashion (Knapp et al, 2016; BaxterPotter et al, 2017)
CCL2 Containing Neurons in the central nucleus of the amygdala (CeA) Are Involved in Alcohol Withdrawal-Induced Anxiety-Like Behavior
CX3CL1 staining was higher in the rats that underwent alcohol withdrawal and in the rats that received the herpes simplex virus (HSV) vector for CCL2 siRNA whereas rats that were exposed to both alcohol withdrawal and received the HSV vector for CCL2 siRNA had the lowest levels of CX3CL1 staining (CD-Sc vs. control liquid diet (CD)-CCL2 p < 0.05; ethanol diet (ED)-CCL2 vs. ED-Sc p < 0.05)

Summary

Introduction

Alcohol withdrawal increases brain neuroimmune proteins such as chemokine C-C motif ligand 2 (CCL2; He and Crews, 2008; Ehrlich et al, 2012; Freeman et al, 2012; Vetreno et al, 2013; Whitman et al, 2013; Kane et al, 2014; Harper et al, 2015) in a brain-region-specific fashion (Knapp et al, 2016; BaxterPotter et al, 2017). Given that chemokines like CCL2 are thought to act as neuromodulators (Adler et al, 2006; Gruol, 2016), it has been suggested that CCL2 and similar chemokines can alter alcohol withdrawal-related behaviors. CCL2 has been linked to important alcohol-related behaviors, drinking (Blednov et al, 2005; June et al, 2015; Valenta and Gonzales, 2016), and alcohol withdrawal-induced anxiety-like behavior (Knapp et al, 2011; Harper et al, 2017) making it an important candidate target for the treatment of alcohol use disorder. The role of CCL2 extends beyond alcohol-related behaviors and is related to other types of anxiety (Wohleb et al, 2013; Sawada et al, 2014).

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Results

Conclusion