Chronic alcohol exposure differentially affects activation of female locus coeruleus neurons and the subcellular distribution of corticotropin releasing factor receptors

Abstract

Understanding the neurobiological bases for sex differences in alcohol dependence is needed to help guide the development of individualized therapies for alcohol abuse disorders. In the present study, alcohol-induced adaptations in (1) anxiety-like behavior, (2) patterns of c-Fos activation and (3) subcellular distribution of corticotropin releasing factor receptor in locus coeruleus (LC) neurons was investigated in male and female Sprague-Dawley rats that were chronically exposed to ethanol using a liquid diet. Results confirm and extend reports by others showing that chronic ethanol exposure produces an anxiogenic-like response in both male and female subjects. Ethanol-induced sex differences were observed with increased c-Fos expression in LC neurons of female ethanol-treated subjects compared to controls or male subjects. Results also reveal sex differences in the subcellular distribution of the CRFr in LC-noradrenergic neurons with female subjects exposed to ethanol exhibiting a higher frequency of plasmalemmal CRFrs. These adaptations have implications for LC neuronal activity and its neural targets across the sexes. Considering the important role of the LC in ethanol-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis, the present results indicate important sex differences in feed-forward regulation of the HPA axis that may render alcohol dependent females more vulnerable to subsequent stress exposure.

Keywords: CRFr; Chronic alcohol; Locus coeruleus; Sex differences; c-Fos.

Figure 1

Graphs illustrating metrics analyzed during a 10 min exposure to the elevated zero maze (EZM). A illustrates distance traveled. Significant main effects were observed for treatment (p<0.001) and sex (p<0.05), with ethanol treated subjects traveling less than controls and females traveling more than males. B illustrates the percentage of trial time spent in the open arms. Significant main effects of sex (p<0.05) and treatment (p<0.001) were seen, with ethanol treated animals spending less time in the open arms, and female control animals spending more time in the open arms than male control animals. C illustrates the average number of head dips. A significant interaction between sex and treatment was seen (p<0.05), with control animals exhibiting this behavior more than the ethanol treated subjects, and female control subjects performing more of this behavior than male control subjects. D illustrates the average number of standing episodes exhibited during the trial. A significant difference was seen between treatment groups (p<0.001) with controls animals exhibiting this behavior more often.

Figure 2

Graphical depiction of c-Fos IR labeling in LC. A–D show representative labeling of c-Fos in males and females of both control and treatment groups. As shown in E, the LC of female ethanol treated subjects had significantly more c-Fos IR than controls (p<0.05), while no difference was seen between male groups. F is a schematic from the Rat Brain Atlas of Paxinos and Watson (1998) illustrating the location of the LC, with a high magnification representation of the boxed area. G illustrates c-Fos labeling (red, arrows) in the peri-LC and LC core of a female ethanol treated subject, which can be localized in NE neurons as seen by tyrosine hydroxylase (TH; green). G’ is a higher magnification representation of the boxed portion of G. 4v, fourth ventricle

Figure 3

Graph depicting a significant interaction in the level of c-Fos protein across sex and treatment. The female ethanol treated group had significantly more c-Fos protein than control counterparts (p<0.01), while no difference was seen between male control and ethanol treated groups.

Figure 4

Sections from control and alcohol-treated rats with immunogold-silver labeling for CRFr within the cytoplasm (arrows) and along the plasma membrane (arrowheads). Dendrite from a male control rat shows predominant localization along the plasma membrane (A), while CRFr labeling is increased in the cytoplasm following alcohol exposure (B). In contrast to control male rats, CRFr labeling is prominent in the cytoplasm (arrows) in control female rats (C), while following alcohol exposure, CRFr labeling is seen predominantly at the plasma membrane (arrowheads). A significant interaction between sex and treatment (p<0.01) was observed with respect to the ratio of cytoplasmic to total CRFr labeling. Unlabeled axon terminals (ut) are shown contacting the CRFr-containing dendrites (A,B,D). m, mitochondria. Scale bars, 0.5μm.

Figure 5

Schematic diagram depicting a working model of ethanol-induced adaptations in LC neurons. In male subjects chronic ethanol is activating the neural stress systems and increasing CRF release onto the dendrites of LC neurons. Upon CRFr activation, the receptor is internalized and deactivated, as proposed by previous studies looking at agonist-induced CRFr activity. This alteration in the CRFr system then decreases subsequent neuronal activation, and is accompanied by desensitization followed by habituation of the LC-NE-CRFr system to chronic ethanol exposure. In females, the working model posits that CRFrs are not internalized as seen in males, and this increased number of CRFrs on the plasma membrane is then hypothesized to promote repeated CRF signaling induced by chronic ethanol. This continued signaling may be responsible for the increased LC activation seen in the FEtoh group via c-Fos IR, and for the anxiety-like phenotype seen in females in the EZM. Further, the CRFr system dysregulation has implications for altered stress vulnerability seen in withdrawal and abstinence, making the CRFr system an attractive target for therapeutic investigation.