Effects of chronic ethanol consumption on the expression of GLT-1 and neuroplasticity-related proteins in the nucleus accumbens of alcohol-preferring rats

Abstract

Chronic ethanol exposure induces impairments in CNS excitatory and inhibitory activity. These impairments are associated with glutamatergic dysfunction, including altered neuroplasticity. This study examined the effects of 6-week ethanol (15% and 30% v/v) consumption, by male alcohol-preferring P rats, on protein expression associated with neuroplasticity and glutamate transporter-1 (GLT-1) function. The latter regulates intra- and extra-synaptic glutamate levels. We focused on the shell and core subregions of the nucleus accumbens (Acb); i.e., shell (AcbSh) and core (AcbCo), for these measures. Chronic ethanol exposure increased the expression of BDNF, Arc and phosphorylated (p)-post-synaptic density protein-95 (p-PSD-95) in the AcbSh of P rats. Moreover, the ratio of phospho-neuronal nitric oxide synthase (p-nNOS) to total nNOS was also increased in the AcbSh. These changes in BDNF, Arc and p-nNOS/nNOS ratio were not observed in the AcbCo. Furthermore, chronic ethanol consumption reduced GLT-1 expression in the AcbSh. Alternatively, treatment with ceftriaxone (CEF), a known GLT-1 upregulator, abolished the effect of chronic ethanol consumption on BDNF expression in the AcbSh. Overall, the present findings confirm that chronic ethanol consumption modulates activity-associated synaptic proteins, including BDNF, Arc and nNOS in a subregion-specific (i.e., in the AcbSh but not AcbCo) manner. Thus, alterations in mesocorticolimbic glutamatergic homeostasis and neuroplasticity are possible functional targets for the treatment of alcohol use disorders.

Keywords: Arc; BDNF; Ethanol dependence; GLT-1; glutamate; nNOS; nucleus accumbens.

Figure 1.. Ethanol consumption in P-rats.

Average ethanol intake (g/kg/24hours) over 6 weeks in male P rats (n=8) with continuous free access to 0, 15% and 30% ethanol [with permission from the publisher (Alhaddad et al., 2020)]. P rats with intake of < 4 g/kg/day did not meet the criteria for the development of ethanol dependence and were excluded from the study.

Figure 2.. Effect of six weeks ethanol consumption on BDNF protein expression in the AcbCo and AcbSh.

(A) Representative immunoblots for BDNF and β-tubulin of water-control vs ethanol groups in AcbCo (left panel) and AcbSh (right panel). (B) Statistical analyses revealed no significant change in BDNF protein expression in the AcbCo of the ethanol group, as compared to the water-control group. A significant upregulation of BDNF protein expression in the AcbSh was observed following chronic ethanol drinking. Data are shown as mean ± SEM; (*p < 0.05); (n = 7–8 for each group).

Figure 3.. Effect of six weeks ethanol consumption on Arc protein expression in the AcbCo and AcbSh.

(A) Representative immunoblots for Arc and β-tubulin of water-control and ethanol groups in the AcbCo (left panel) and the AcbSh (right panel). (B) Statistical analyses revealed no significant change in Arc protein expression in the AcbCo of the ethanol group, as compared to the water-control group. A significant upregulation of Arc protein expression in the AcbSh was observed after chronic ethanol intake. Data are shown as mean ± SEM; (*p < 0.05); (n = 8 for each group).

Figure 4.. Effect of six weeks ethanol consumption on p-nNOS and t-nNOS protein expression in the AcbCo and AcbSh.

(A) Representative immunoblots for p-nNOS and β-tubulin in AcbCo and AcbSh respectively (upper panel) of the water-control and ethanol groups. Statistical analyses revealed no significant change in p-nNOS protein expression within either the AcbCo or AcbSh of the ethanol group, as compared to the water-control group (lower panel). (B) Representative immunoblots for t-nNOS and β-tubulin in the AcbCo and AcbSh, respectively (upper panel), of the water-control and ethanol groups. Statistical analyses revealed no significant change in t-nNOS protein expression in the AcbCo of the ethanol group, whereas t-nNOS protein expression in the AcbSh was significantly downregulated by chronic ethanol (lower panel). (C) Statistical analyses showed a significant increase in the p-nNOS/t-nNOS ratio following ethanol drinking in the AcbSh, but not AcbCo. The ratio is calculated by dividing the expression of p-nNOS by t-nNOS from the same western blot membranes. Data are shown as mean ± SEM; (*p < 0.05); (n = 8 for each group).

Figure 5.. Effect of six weeks ethanol consumption on p-PSD-95 and t-PDS-95 protein expression in the AcbCo and AcbSh.

(A) Representative immunoblots for p-PSD-95 and β-tubulin in AcbCo and AcbSh respectively (upper panel) of the water-control and ethanol groups. Statistical analyses revealed no significant change in the p-PSD-95 protein expression within the AcbCo, whereas there was a significant upregulation of p-PSD-95 protein expression in the AcbSh (lower panel). (B) Representative immunoblots for PSD-95 and β-tubulin in the AcbCo and AcbSh respectively (upper panel) of the water-control and ethanol groups. Statistical analyses revealed no significant change in PSD-95 protein expression in either the AcbCo or AcbSh following ethanol drinking (lower panel). (C) Statistical analyses revealed no significant change in the p-PSD-95/PSD-95 ratio of the ethanol group compared to water-control group in either the AcbCo or AcbSh. The ratio is calculated by dividing the expression of p-PSD-95 by PSD-95 from the same western blot membranes. Data are shown as mean ± SEM; (*p < 0.05); (n = 8 for each group).

Figure 6.. Effect of six weeks ethanol consumption on GLT-1 protein expression in the AcbCo and AcbSh.

(A) Representative immunoblots for GLT-1 and β-tubulin of water-control and ethanol groups in the AcbCo (left panel) and AcbSh (right panel). (B) Statistical analyses revealed no significant change in the GLT-1 protein expression in the AcbCo of the ethanol group as compared to the water-control group, while there was a significant downregulation of GLT-1 protein expression in the AcbSh of the ethanol group, as compared to the water-control group. Data are shown as mean ± SEM; (*p < 0.05); (n = 6–8 for each group).

Figure 7.. Effects of ceftriaxone treatment on ethanol and water drinking behavior and expression of BDNF and Arc in the AcbSh.

(A) CEF (200 mg/kg, i.p.) treatment significantly reduced ethanol consumption in Day 3 through Day 6 as compared to saline treated group (n=5–6/group). (B) Water consumption was significantly increased in CEF group compared to saline treated group in Day 3 through Day 6 (n=5–6/group). (C) Representative immunoblots for BDNF and β-tubulin of water-control, ethanol-saline (saline) and ethanol-CEF 200 (CEF 200) groups in the AcbSh (upper panel). Statistical analyses revealed that BDNF expression was significantly increased in ethanol-saline group compared to water-control group. There was no significant change in BDNF expression in CEF 200 group compared to ethanol-saline and water-control groups (lower panel). (D) Representative immunoblots for Arc and β-tubulin of water-control, saline and CEF 200 groups in the AcbSh (upper panel). Statistical analyses revealed that Arc expression was significantly increased in ethanol-saline and ethanol-CEF 200 group compared to water-control group. There was no significant change in the expression between CEF 200 and saline groups (lower panel). Data are shown as mean ± SEM; (*p < 0.05, **p < 0.01 and ****p<0.0001); (n = 6 for each group).

Figure 8.

Schematic diagram summarizes the effects of chronic ethanol consumption on GLT-1, NO pathway, BDNF, and Arc expression in the AcbSh. Chronic ethanol consumption increases synaptic glutamate concentration mainly due to downregulation of GLT-1 (both GLT-1a and GLT-1b isoforms) and cystine/glutamate exchanger transporter (xCT) expression (Alhaddad et al., 2014a; Das et al., 2015). Chronic ethanol consumption also increases NO system activity, BDNF-Arc expression and PSD-95. NO regulates the incorporation of postsynaptic GluA1 subunit of AMPA receptors probably through CREB-BDNF-Arc pathway and ubiquitination of PSD-95. In addition, chronic ethanol exposure was associated with increases in the inflammatory response through upregulation of inflammatory mediators such as high mobility group box 1 (HMGB1), a receptor for advanced glycation end products (RAGE) and TNF-α (Alasmari et al., 2020).