Chronic Ethanol Exposures Leads to a Negative Affective State in Female Rats That Is Accompanied by a Paradoxical Decrease in Ventral Hippocampus Excitability

Abstract

Alcohol use disorder (AUD) differentially impacts men and women and a growing body of evidence points to sex-dependent adaptations in a number of brain regions. In a prior study, we explored the effect of a chronic intermittent ethanol exposure (CIE) model of AUD on neuronal and molecular adaptations in the dorsal and ventral domains of the hippocampus (dHC and vHC, respectively) in male rats. We found the vHC to be particularly sensitive to CIE, showing an increase in neuronal excitability and synaptic proteins associated with augmented excitation. These findings were accompanied by a CIE-dependent increase in anxiety-like behaviors. To explore sex-dependent adaptations in the hippocampus, we conducted a similar study in female rats. CIE-treated female rats showed a relatively modest increase in anxiety-like behaviors along with a robust increase in depressive-like measures. Despite both sexes showing clear evidence of a negative affective state following CIE, the vHC of females showed a decrease, rather than an increase, in neuronal excitability. In line with the reduced sensitivity to neural adaptations in the dHC of male rats, we were unable to identify any functional changes in the dHC of females. The functional changes of the vHC in female rats could not be explained by altered expression levels of a number of proteins typically associated with changes in neuronal excitability. Taken together, these findings point to sex as a major factor in CIE-dependent hippocampal adaptations that should be explored further to better understand possible gender differences in the etiology and treatment of AUD.

Keywords: alcohol use disorder; chronic intermittent ethanol exposure; electrophysiology; hippocampus; negative affective behavior.

FIGURE 1

Female CIE-treated rats do not show anxiety-like behavior on the EPM. (A) Air (n = 8) and CIE-treated (n = 8) rats spend similar amount of time in the open arms, (B) make similar number of open arm entries, and (C) closed arm entries. All data is expressed as the mean ± SEM.

FIGURE 2

There is no evidence of CIE-induced anxiety-like behavior on the OFT. Air (n = 12) and CIE-treated rats (n = 12) showed no differences in (A) the total distance traveled, (B) the amount of time spent, or (C) in the distance traveled in the center of the arena. All data are expressed as the mean ± SEM.

FIGURE 3

The SAT revealed an anxiety-like phenotype in CIE-treated female rats. (A) CIE-treated female rats (n = 10) spent more time in the least anxiogenic zone (Black) than Air control rats (n = 10), but the two groups spent comparable amounts of time in all other zones. (B) CIE-treated rats had a longer latency to venture into the most anxiogenic open zone (White). (C) The total distance traveled was similar between Air and CIE-treated female rats. All data are expressed as the mean ± SEM, *p < 0.05.

FIGURE 4

Female CIE-treated rats show a depressive-like phenotype on the FST. (A) CIE-treated female rats (n = 10) spent more time immobile, (B) traveled a shorter distance, and (C) had a slower moving velocity than their Air control (n = 10) counterparts. All data are expressed as the mean ± SEM *p < 0.05.

FIGURE 5

There is a reduction in excitatory neurotransmission in the vHC of CIE-treated rats. There is a CIE-dependent reduction in the stimulus dependent (A) fV amplitude and (B) fEPSP slope. (C) The relative balance between vHC fV amplitude and fEPSP was not impacted by CIE-treatment. (D) Representative example traces of fEPSPs acquired from slices of an Air (upper trace) and CIE-treated (lower trace) rat. All data are expressed as the mean ± SEM, *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 6

Excitatory neurotransmission in the dHC was not impacted by CIE treatment. We found no effect of CIE on the stimulus-dependent (A) fV amplitude and (B) fEPSP slope, or (C) the relative balance between dHC fV amplitude and fEPSP. (D) Representative example traces of fEPSPs acquired from slices of an Air (upper trace) and CIE-treated (lower trace) rat. All data are expressed as the mean ± SEM.

FIGURE 7

CIE does not impact the expression levels of proteins commonly associated with alterations in excitatory neurotransmission in the vHC. However, in the dHC, the GluA1 subunit is upregulated. Representative western blot examples illustrating the expression of (A) the GluA1 and (B) GluA2 subunit of the AMPA receptor, (E) the GluN1, (F) and GluN2 subunit of the NMDA receptor and (G) SK2. In panels (A,B,E–G) the corresponding expression of actin (normalization protein) is shown in the lower blot panel of each protein of interest. (C,D,H–J) Group data, normalized to actin and the internal control group (Air), for the (C) GluA1 (Air n = 4, CIE n = 4), (D) GluA2 (Air n = 4, CIE n = 4), (H) GluN1 (Air n = 4, CIE n = 4), (I) GluN2B subunit (Air n = 4, CIE n = 4) and (J) the SK2 channel (Air n = 4, CIE n = 4) illustrating an increase in the expression of the GluA1 subunit (Air n = 4, CIE n = 4) in the dHC without changes in other proteins in the dHC and vHC of Air and CIE-treated rats. All data are expressed as the mean ± SEM, *p < 0.05.

FIGURE 8

Expression levels of proteins commonly associate with changes in inhibitory neurotransmission are not impacted by CIE-treatment in the dHC or vHC. Representative western blot examples illustrating the expression of (A) the GABA_ARα1 subunit or (B) Gephyrin. The corresponding expression of actin (normalization protein) is shown in the lower blot panel of each protein of interest. (C,D) Group data, normalized to actin and the internal control group (Air), for (C) the GABA_ARα1 subunit (Air n = 4, CIE n = 4) and for (D) Gephyrin (Air n = 4, CIE n = 4) illustrating no change in the expression of these proteins in the dHC and vHC following CIE-treatment. All data are expressed as the mean ± SEM.