Attenuation of ethanol withdrawal by ceftriaxone-induced upregulation of glutamate transporter EAAT2

Abstract

Alcohol withdrawal syndrome (AWS) is a potentially fatal outcome of severe alcohol dependence that presents a significant challenge to treatment. Although AWS is thought to be driven by a hyperglutamatergic brain state, benzodiazepines, which target the GABAergic system, comprise the first line of treatment for AWS. Using a rat model of ethanol withdrawal, we tested whether ceftriaxone, a β-lactam antibiotic known to increase the expression and activity of glutamate uptake transporter EAAT2, reduces the occurrence or severity of ethanol withdrawal manifestations. After a 2-week period of habituation to ethanol in two-bottle choice, alcohol-preferring (P) and Wistar rats received ethanol (4.0 g/kg) every 6 h for 3-5 consecutive days via gavage. Rats were then deprived of ethanol for 48 h during which time they received ceftriaxone (50 or 100 mg/kg, IP) or saline twice a day starting 12 h after the last ethanol administration. Withdrawal manifestations were captured by continuous video recording and coded. The evolution of ethanol withdrawal was markedly different for P rats vs Wistar rats, with withdrawal manifestations occurring >12 h later in P rats than in Wistar rats. Ceftriaxone 100 mg/kg per injection twice per day (200 mg/kg/day) reduced or abolished all manifestations of ethanol withdrawal in both rat variants and prevented withdrawal-induced escalation of alcohol intake. Finally, ceftriaxone treatment was associated with lasting upregulation of ethanol withdrawal-induced downregulation of EAAT2 in the striatum. Our data support the role of ceftriaxone in alleviating alcohol withdrawal and open a novel pharmacologic avenue that requires clinical evaluation in patients with AWS.

Figure 1

Different timecourse and severity of withdrawal manifestations in alcohol-preferring (P) and Wistar rats. Following 3-5 day gavage of high-dose ethanol (a) stereotypy peaked between 12-15h after the final ethanol dose for Wistar rats, while in P rats, such behavior did not occur until 26-43 h of alcohol deprivation. (b) Similarly, myoclonic body jerks were displayed earlier and to a lesser extent in Wistar rats, resolving after 16 h of withdrawal, while P rats displayed more frequent body jerks over the majority of the second day of withdrawal. (c) Myoclonic head jerks were markedly more frequent in P rats than Wistar rats, peaking during the beginning of day 2 of alcohol deprivation. (d) P rats did not show spontaneous tonic/clonic seizure activity, and Wistars showed it infrequently from 12-21 h after alcohol cessation. (e) Wistar rats showed very infrequent unprovoked startle responses at 12 h, while P rats displayed startle primarily in the middle of day 2 of withdrawal. ^#P<0.05 for main effect of rat type by two-way ANOVA; *P<0.05 by Tukey posthoc test; n=6–10 per rat type. Data is expressed as mean±SEM.

Figure 2

Amelioration of alcohol withdrawal by ceftriaxone 200 mg/kg/day in alcohol-preferring (P) and Wistar rats. Stereotypy was almost completely eliminated by two-day ceftriaxone (200 mg/kg) treatment in P rats (a) and substantially reduced in Wistar rats (b) compared to saline-treated counterparts. Myoclonic body jerks were abolished in P rats (c) and diminished in Wistar rats (d) by ceftriaxone. Similarly, myoclonic head jerks were absent in ceftriaxone-treated P rats (e) and greatly reduced in Wistar rats (f) by ceftriaxone. (g) Ceftriaxone eliminated unprovoked startle responses in P rats, but (h) did not affect the relatively low frequency of spontaneous tonic/clonic seizures in Wistar rats. ^#P<0.05 by two-way ANOVA; *P<0.05 by Tukey posthoc test; n=6–10 per rat type. Data is expressed as mean±SEM.

Figure 3

Two-day ceftriaxone (200 mg/kg) administration abolished the increase in ethanol consumption following withdrawal in P rats (a) Two-way ANOVA ^#P<0.0001, Tukey's post-hoc test *P<0.0004. Ceftriaxone (200 mg/kg/day) treatment also reduced post-withdrawal ethanol consumption by Wistar rats (b) Two-way ANOVA ^#P=0.032, Tukey's post-hoc test *P<0.018. Note that ceftriaxone 100 mg/kg/day did not show significant effect on post withdrawal ethanol consumption in P rats (P>0.05), but with significant increase compared to saline-treated rats (P<0.05). Data is expressed as mean±SEM.

Figure 4

Ethanol withdrawal is associated with significant down regulation of the expression of EAAT2 in the striatum (a) One-way ANOVA followed by posthoc unpaired t-test (*P=0.002), and the mPFC (b) (*P=0.017). Two-day ceftriaxone (200 mg/kg) treatment upregulated EAAT2 protein levels in the striatum (a) (*P<0.05), but not in the mPFC (P>0.05), (b) of rats seven days following alcohol deprivation. Data is expressed as mean±SEM.