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

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.

Keywords: CCL2; CCR2; CRF; CX3CL1; CeA; anxiety; ethanol; neurons.

Figure 1

Loss of neuronal CCL2 during withdrawal from subthreshold alcohol causes anxiety-like behavior. (A) Representative injection site derived from post-mortem histological assessments of CeA injections. Neurons infected with herpes simplex virus (HSV) express GFP. The CeA is outlined by a red dashed line. (B) Diagram of injections/alcohol withdrawal paradigm. Male Wistar rats were subjected to three 5 day blocks of 4.5% w/v ethanol diet (ED) and were tested for social interaction (SI) 5–6 h into the third withdrawal. An amplicon with siRNA for CCL2 was injected into the CeA 2 days before the final withdrawal. (C) Only rats that received ethanol diet and loss of neuronal CCL2 showed reduced SI indicating increased anxiety-like behavior. (D) Neither virus nor ethanol diet alone or used in combination caused changes in locomotor activity. N = 7–11 per group. Data presented as mean ± SEM. Post hoc *p < 0.05.

Figure 2

Alcohol withdrawal increases the anti-inflammatory CX3CL1 in the CeA, which is reversed by the loss of CCL2 in neurons. (A) Representative images are immunostained for CX3CL1 of the CeA of male Wistar rats that were microinjected with an amplicon with CCL2 siRNA. The CeA is outlined by a white dashed line. (B) Representative images are immunostained for CX3CL1 of the CeA of male Wistar rats that underwent alcohol withdrawal and microinjected with an amplicon with siRNA for CCL2. The CeA is outlined by a white dashed line. (C) The integrated density of CX3CL1 CeA images shows that alcohol withdrawal increases the levels of CX3CL1 which is reversed by the loss of CCL2. The images from panels (A,B) were converted into grayscale and then the same threshold was applied to both images to make the images in panels (D,E,), respectively. The images in panels (D,E) highlight differences in identifiable CXC3CL1 containing neurons in the images using the same threshold. (F) Cell counts of CX3CL1 CeA images show that alcohol withdrawal increases the levels of CX3CL1 positive cells which is reversed by the loss of CCL2. N = 6 per group. Data presented as mean ± SEM. Post hoc *p < 0.05.

Figure 3

CCL2 containing neurons co-localize with CCR2 and corticotropin-releasing factor receptor 1 (CRFR1) in the CeA. (A) Representative images of the CeA neurons of naïve rats immunostained for CCL2. (B) Same image is immunostained for CCR2. (C) The merged image containing both immunostains showing neurons containing both CCL2 and CCR2 in the CeA. (D) Representative images of the CeA neurons of naïve rats immunostained for CCL2. (E) The same image is immunostained for CRFR1. (F) The merged image containing both immunostains showing neurons containing both CCL2 and CRFR1 in the CeA. White arrow indicates co-localization.

Figure 4

CRFR1 is not found in many microglia in the CeA. (A) Representative images of the cells in the CeA of naïve rats immunostained for CCL2. (B) The same image is immunostained for CRFR1. (C) Image of immunostaining of Iba-1 as a marker of microglia. (D) The merged image containing all markers shows microglia containing both CCL2 and CRFR1 in the CeA. White arrow indicates co-localization.

Figure 5

CeA projection neurons to the bed nucleus of the stria terminalis (BNST) and the ventral periaquaductal gray (VPAG) contain CCL2. (A) Representative image of CeA neurons in a naïve rat that underwent unilateral injection of fluorescent nanobeads into the BNST. (B) Same image immunostained for CCL2. (C) Merged image showing neurons containing both the fluorescent nanobeads and immunostained for CCL2. (D) Injection site of the fluorescent nanobeads in the BNST of the same rat. The BNST is outlined by a white dashed line. (E) Representative image of CeA neurons in a naïve rat that underwent unilateral injection of fluorescent nanobeads into the VPAG. (F) Same image immunostained for CCL2. (G) Merged image showing neurons containing both the fluorescent nanobeads and immunostained for CCL2. (H) Injection site of the fluorescent nanobeads in the VPAG of the same rat. The VPAG is outlined by a white dashed line. White arrow indicates colocalization.

Figure 6

Blocking axonal transport increases CCL2 in neuronal cell bodies in the CeA. (A) Representative image of a rat that received an intracerebroventricular injection of artificial cerebral spinal fluid (aCSF) and (B) a rat that received colchicine, an axonal transport blocker.

Figure 7

Subcellular localization of CCL2 within CeA neurons. (A) Neuronal nuclei in the CeA of naïve rats were stained with DAPI (4′,6-diamidino-2-phenylindole). (B) The same neurons were immunostained with CCL2. (C) Neurons were immunostained with PSD-95 (postsynaptic density 95) as a marker of synapses. (D) Combined images with all three markers. White arrows indicate colocalization. (E) Electron micrographs show CCL2 was found in vesicles (red arrow) in neuronal processes near postsynaptic densities (white arrow). (F) Additionally, DAB precipitate was found along microtubules (white arrow). (G) The rough endoplasmic reticulum (red arrow) and the surrounding ribosomes (white arrow) also show DAB precipitate.