A Novel Sulfonamide, 4-FS, Reduces Ethanol Drinking and Physical Withdrawal Associated With Ethanol Dependence

Abstract

Carbonic anhydrase (CA) is abundant in glial cells in the brain and CA type II isoform (CA II) activity in the hippocampus plays an important role in buffering extracellular pH transients produced by neural activity. Chronic ethanol exposure results in respiratory and metabolic acidosis, producing shifts in extracellular pH in the brain and body. These neurophysiological changes by ethanol are hypothesized to contribute to the continued drinking behavior and physical withdrawal behavior in subjects consuming ethanol chronically. We explored whether chronic ethanol self-administration (ethanol drinking, 10% v/v; ED) without or under the influence of chronic intermittent ethanol vapor (CIE-ED) experience alters the expression of CA II in the hippocampus. Postmortem hippocampal tissue analyses demonstrated that CA II levels were enhanced in the hilus region of the hippocampus in ED and CIE-ED rats. We used a novel molecule-4-fluoro-N-(4-sulfamoylphenyl) benzenesulfonamide (4-FS)-a selective CA II inhibitor, to determine whether CA II plays a role in ethanol self-administration in ED and CIE-ED rats and physical withdrawal behavior in CIE-ED rats. 4-FS (20 mg/kg, i.p.) reduced ethanol self-administration in ED rats and physical withdrawal behavior in CIE-ED rats. Postmortem hippocampal tissue analyses demonstrated that 4-FS reduced CA II expression in ED and CIE-ED rats to control levels. In parallel, 4-FS enhanced GABA_A receptor expression, reduced ratio of glutamatergic GluN2A/2B receptors and enhanced the expression of Fos, a marker of neuronal activation in the ventral hippocampus in ED rats. These findings suggest that 4-FS enhanced GABAergic transmission and increased activity of neurons of inhibitory phenotypes. Taken together, these findings support the role of CA II in assisting with negative affective behaviors associated with moderate to severe alcohol use disorders (AUD) and that CA II inhibitors are a potential therapeutic target to reduce continued drinking and somatic withdrawal symptoms associated with moderate to severe AUD.

Keywords: Fos; GABA; GluR; carbonic anhydrase; ethanol self-administration; hippocampus.

Figure 1

(a) Chemical structure of 4-FS. (b) Schematic of the experimental timeline and experimental group information.

Figure 2

Ethanol self-administration and blood alcohol levels. (a) Amount of ethanol consumed prior to chronic intermittent ethanol (CIE) initiation and over the 6 weeks period of CIE or air exposure. Weeks of air/vapor exposure are indicated over a pale pink background. (b) Blood alcohol levels measured once a week in animals experiencing CIE. CIE-ED, n = 7 and ED, n = 8. $, interaction; #, main effect of groups; * p < 0.05 vs. CIE-ED by two-way analysis of variance (ANOVA). Data are expressed as mean ± S.E.M.

Figure 3

Carbonic anhydrase type II (CA II) expression in the adult rat hippocampus. (a) Photomicrograph of CA II immunohistochemistry in the hippocampus and cortex from one control rat. CA II+ cells appeared as single cells; each immunoreactive cell is pointed with an arrowhead. 1- CA II+ cell in the hilus (Hil); 2-CA II+ cell in the molecular layer (Mol); 3-CA II+ cell in the corpus callosum (cc); 4-CA II+ cell in the cortex. (b–i) 100× images of the hilus used for quantitative analyses of CA II cells. (e) Zoomed in image shown in (d) to indicate the morphology of CA II+ cells in the hilus. Scale bar in (e) is 20 µm; scale bar in (i) is 50 µm (applies b–d and f–i). (j) Number of CA II+ cells in the hilus. n = 5 controls, n = 5 ED, n = 4 CIE-ED, n = 3 vehicle controls, n = 3 4-FS controls, n = 8 4-FS ED rats, n = 7 4-FS CIE-ED rats. * p < 0.05, compared to controls; # p < 0.05 compared to 4-FS control. Data are expressed as mean ± S.E.M.

Figure 4

Physical withdrawal behaviors in ethanol drinking (ED) and CIE-ED rats with vehicle and 4-FS treatment. Withdrawal behaviors are evident in CIE-ED rats. Signs of ethanol withdrawal, i.e., aberrant body posture, locomotor activity and tail stiffness were reduced by 4-FS in CIE-ED rats. n = 7–8/group. * p < 0.05 compared to baseline and vehicle days, within-subject. # p < 0.05 vs. CIE-ED rats, between-subject. Data are expressed as mean ± S.E.M.

Figure 5

4-FS reduces drinking in ethanol drinking (ED) rats. (a) Ethanol intake expressed as g/kg and (b) active lever responses. (c) Inactive lever responses and (d) lever responses during timeout. n = 8 4-FS ED rats, n = 7 4-FS CIE-ED rats. * p < 0.05 vs. vehicle day, within-subject. Data are expressed as mean ± S.E.M.

Figure 6

(a,b,e,f) Schematic of tissue punches from the dorsal hippocampus (a,b) and ventral hippocampus (e,f). (c,g) sample western blots from dorsal (c) and ventral (g) hippocampus tissue punches. (d,h) Densitometric analysis of tissue for levels of GluA1, GluN2A, GluN2B, ratio of GluN2A/2B and GABA_A from the dorsal (d) and ventral (h) hippocampus tissue homogenate. n = 8 4-FS ED rats, n = 7 4-FS CIE-ED rats. Values are mean ± S.E.M. expressed as % control, where control is represented by the dotted line in each graph. * p < 0.05, compared to controls.

Figure 7

Fos expression is altered by 4-FS. (a) Photomicrograph of Fos immunohistochemistry in the hippocampus and cortex from one control rat. Fos+ cells appeared as single cells; each immunoreactive cell is pointed with an arrowhead. 1-Fos+ cell in the hilus (Hil); 2-Fos+ cell in the granule cell layer (GCL); 3-Fos+ cell in the CA1 region; 4-Fos+ cell in the cortex. Mol, molecular layer. Scale bar is 100 µm. (b,c) Number of Fos+ cells in the dorsal (b) and ventral (c) hippocampus. n = 8 4-FS ED rats, n = 7 4-FS CIE-ED rats. * p < 0.05, compared to controls. Data are expressed as mean ± S.E.M.