Six-week chronic 2 bottle choice (2BC) alcohol drinking in female C57BL/6J mice used for the various experiments are shown in Table 1. All groups of mice showed an increase in preference and intake as previously shown [3]. Ethanol drinking remained constant during the last four-weeks when the mice had access to 10% EtOH.

Table 1 Ethanol preference and consumption Full size table

Female mice developed affective disturbances in abstinence from 6-week 2BC ethanol drinking

The effect of forced abstinence on affective disturbances was evaluated in female C57BL/6J mice following chronic 6-week alcohol drinking (Fig. 1a). In previous studies, we and others have shown that prolonged forced abstinence (i.e., 2 weeks or later) induces significant differences in depressive-like behaviors, such as FST, sucrose preference test, and NSFT, but not in tests more sensitive to anxiety such as the EPM and light-dark box [3,4,5]. In these previous studies, however, anxiety-like behavior was not assessed during early forced abstinence ([3]; Pang et al. 2016; Pang et al. 2016; [2]). Thus, in the present study, 22 water-drinking mice, and 35 ethanol-drinking mice were tested on the EPM 24-h after the onset of forced abstinence (day 43). In this study, mice with a history of ethanol drinking spent significantly less time on the open arms (Fig. 1b; Student’s t-test, t(55) = 3.719, p < 0.0001). Student’s t-test revealed no significant effect of drinking on distance traveled on the EPM (data not shown; Student’s t-test, t(55) = 0.8194, p < 0.42) indicating that locomotor-activity was not impaired in this group of mice. Thirteen water-drinking mice, and 18 ethanol-drinking mice were tested for a depressive phenotype two-weeks (day 56) after the onset of forced abstinence using NSFT, all of these mice were exposed to the EPM two-weeks prior. This experiment revealed that mice with a history of ethanol drinking took significantly longer to feed (Fig. 1c; Student’s t-test, t(29) = 5.539, p < 0.0001).

Fig. 1 Female mice with a history of chronic six-week long ethanol drinking develop affective disturbances. Mice were tested on the EPM twenty-four hours after the onset of abstinence, and NSFT two weeks after the onset of abstinence (a). Mice with a history of ethanol drinking displayed less time spent on the open arms compared to mice with a history of water drinking (b). Mice with a history of ethanol-drinking displayed a longer latency to feed in NSFT compared to mice with history of water-drinking (c). Time and distance are shown as the mean ± SEM. ****p < 0.0001 compared to saline-ethanol drinkers; analysis of variance (t-test) Full size image

Ketamine administration at the onset of abstinence prevented ethanol abstinence induced affective disturbances

Previously, we have shown that an acute injection of ketamine is able to reverse affective disturbances induced by prolonged abstinence from 2BC ethanol drinking when administered 30-min prior to testing [3]. Our goal in the present study was to identify a time-course window in which ketamine administration is effective in preventing the development of affective disturbances. First, we tested whether ketamine given at the onset of abstinence (at the same time the ethanol was removed and changed to water: day 42) could prevent the development of affective disturbances. Nineteen mice underwent 2BC water drinking, and 25 mice underwent 2BC ethanol drinking for 6 weeks. Thirty-mice (19 water drinkers and 11 ethanol drinkers) received a saline injection at the onset of abstinence, and 14 ethanol-drinking mice received an injection of ketamine (3.0 mg/kg i.p.) at the onset of abstinence (day 42). Mice were subsequently tested for the development of affective disturbances (Fig. 2a).

Fig. 2 A single low dosage of ketamine (3.0 mg/kg i.p.) at the onset of abstinence after six-weeks of chronic drinking prevents the development of affective disturbances. Representative figure for 2BC ethanol drinking followed by chronic abstinence, ketamine was administered at the onset of abstinence (a). Ketamine-injected mice showed a significant increase in time spent on open arms compared to saline injected ethanol drinking mice (b). Distance traveled on the EPM did not differ amongst the three groups (c). Mice develop affective disturbances two-weeks after abstinence as measured by NSFT. This was reversed by an administration of ketamine at the onset of abstinence (d). Time and distance are shown as the mean ± SEM (n = 6–12/group). *p < 0.05, **p < 0.01,****p < 0.0001 compared to saline-ethanol drinkers; analysis of variance (one-way ANOVA with Dunnett’s post hoc test) Full size image

Twenty-four hours after the onset of abstinence (day 43), mice were tested on the EPM. One-way ANOVA revealed a significant effect of drinking on time spent in the open arm (F (2,41) = 6.443; p = 0.0037). Dunnett’s post hoc revealed that saline-injected ethanol-drinking mice spent less time in the open arm compared to saline-injected water-drinking mice (p = 0.0172), and ketamine-injected ethanol-drinking mice (p = 0.0172) (Fig. 2b). One-way ANOVA revealed no significant effect on drinking, or treatment on distance traveled on the EPM (F (2,41) = 1.086 p = 0.9993) indicating that locomotor-activity was not impaired in this group of mice (Fig. 2c).

Next, mice underwent the NSFT 2 weeks following the onset of abstinence (day 56). One-way ANOVA revealed a significant effect of drinking (F(2,34) = 24.48; p < 0.0001) in latency to feed. Dunnett’s post hoc test revealed that saline-injected ethanol drinkers had a longer latency to feed compared to saline-injected water drinkers (p = 0.0001), and a longer latency to feed compared to ketamine-injected ethanol-drinkers (p = 0.0001). Saline-injected water drinkers, and ketamine-injected ethanol drinkers did not show a significant difference (p = 0.42) (Fig. 2d).

Ketamine administration two-days after the onset of abstinence failed to prevent ethanol abstinence induced affective disturbances

Next, we sought to determine whether ketamine administered two-days (day 44) into abstinence could also prevent the development of affective disturbances. Ten-mice underwent water 2BC and 20 mice underwent ethanol 2BC for six-weeks. Two-days into forced abstinence, 11 mice (five water drinkers and six ethanol drinkers) received an i.p. injection of saline, and six ethanol-drinking mice received an i.p. injection of ketamine (3.0 mg/kg). Mice were subsequently tested for the development of affective disturbances (Fig. 3a).

Fig. 3 A single low dosage of ketamine (3.0 mg/kg i.p.) 2 days into abstinence after six-weeks of chronic drinking failed to prevent the development of affective disturbances. Representative figure for 2BC ethanol drinking followed by chronic abstinence. Ketamine was administered 2 days after the onset of abstinence (a). Ketamine failed to prevent abstinence induced anxiety (b). Distance traveled on the EPM did not differ amongst the three groups (c). Ketamine failed to block abstinence induced affective disturbances as measured by NSFT (d). Time and distance are shown as the mean ± SEM (n = 6–12/group). *p < 0.05, **p < 0.01,****p < 0.0001 compared to saline-ethanol drinkers; analysis of variance (one-way ANOVA with Dunnett’s post hoc test) Full size image

Twenty-four hours after the saline or ketamine injection (3 days after the onset of abstinence; day 45) mice were tested on the EPM. One-way ANOVA revealed a significant effect of drinking on time spent in the open arm (F(2,17) = 4.904; p = 0.0208). Dunnett’s post hoc revealed that saline-injected ethanol-drinking mice spend less time in the open arm compared to saline-injected water-drinking mice (p = 0.0174) but had no significant difference in open time with ketamine-injected ethanol-drinking mice (p = 0.78) (Fig. 3b). One-way ANOVA revealed no-significant effect on water drinking, or treatment on distance traveled on the EPM (p = 0.9960) indicating that locomotor-activity was not impaired in this group of mice (Fig. 3c).

Next, a total of 23 mice (six water drinkers and 17 ethanol drinkers) underwent NSFT two-weeks after the onset of abstinence (day 56). One-way ANOVA revealed no significant effect of drinking in latency to feed (Fig. 3d; F(2,19) = 1.337; p, 0.28).

Ketamine administration six-days after the onset of abstinence failed to prevent ethanol abstinence induced affective disturbances

To further develop a timeline, ketamine or saline was administered six-days into abstinence (day 48) to determine if it is effective in preventing the development of affective disturbances. Nine mice underwent water 2BC and 30 mice underwent ethanol 2BC. Six-days into forced abstinence, 22 (9 water drinkers and 13 ethanol drinkers) received an i.p. injection of saline, and 17 mice received an i.p. injection of ketamine (3.0 mg/kg). Mice were subsequently tested for the development of affective disturbances (Fig. 4a).

Fig. 4 A single low dosage of ketamine (3.0 mg/kg i.p.) 6 days into abstinence after six-weeks of chronic drinking failed to prevent the development of affective disturbances. Representative figure for 2BC ethanol drinking followed by chronic abstinence. Ketamine was administered 6 days after the onset of abstinence (a). Ketamine failed to prevent abstinence induced anxiety (b). Distance traveled on the EPM did not differ amongst the three groups (c). Ketamine failed to block abstinence induced affective disturbances as measured by NSFT (d). Time and distance are shown as the mean ± SEM (n = 6–12/group). *p < 0.05, **p < 0.01, ****p < 0.0001 compared to saline injected-ethanol drinkers; analysis of variance (one-way ANOVA with Dunnett’s post hoc test) Full size image

Twenty-four hours after the saline or ketamine injection (day 49) mice were tested on the EPM. One-way ANOVA revealed a significant effect of drinking on time spent in the open arm (F (2,36) = 4.42; p = 0.0192). Dunnett’s post hoc revealed that saline-injected ethanol-drinking mice spent less time in the open arm compared to saline-injected water-drinking mice (p = 0.0116) but no difference was observed in time spent in the open arms with the ketamine-injected ethanol-drinking mice (p = 0.587) (Fig. 4b). One-way ANOVA revealed no significant effect on drinking, or treatment on distance traveled on the EPM (p = 0.7114) indicating that locomotor-activity was not impaired in this group of mice (Fig. 4c).

Next, mice were tested 2 weeks following the onset of abstinence using the NSFT (day 56). One-way ANOVA revealed a significant effect of drinking (F(2,41) = 11.67; p < 0.0001) in latency to feed. Dunnett’s post hoc test revealed that saline-injected ethanol drinkers had a longer latency to feed compared to saline-injected water drinkers (p = 0.0002), but not a significant change in latency to feed compared to ketamine-injected ethanol-drinkers (p = 0.82) (Fig. 4d).

Ketamine administration at the onset of abstinence prevents ethanol abstinence induced disturbances as assessed by the FST

The FST, a pharmacologically established proxy for depression-like behavior, was used as an additional test for measuring affective disorders. We repeated earlier findings showing that FST immobility in EtOH mice following 18 days of forced abstinence increases FST immobility time [3]. One-week after the NSFT test, mice underwent the FST test. One-way ANOVA shows that ethanol drinking increases immobility time (Fig. 4b. *p < 0.0001). Here we assessed the effectiveness of ketamine administration during the three-time points used above on FST performed in later abstinence (Fig. 4a). Ketamine prevented the development of FST disturbances only when it was administered at the beginning of forced abstinence (day 42; Dunnett’s post hoc #p < 0.0001) (Figure 5).

Fig. 5 Ketamine prevented affective disturbances in the FST only when administered at the onset of abstinence. Representative figure for two-bottle choice ethanol drinking followed by chronic abstinence. Mice received an injection of ketamine at three different time followed by a FST three weeks after the onset of abstinence (a). Ketamine administration at the beginning of abstinence prevented affective disturbances as assessed by the FST (b). Immobility time is shown as the mean ± SEM (n = 6–29/group). *p < 0.0001 and #p < 0.0001 compared to saline injected-ethanol drinkers; analysis of variance (one-way ANOVA with Dunnett’s post hoc test) Full size image

Ketamine administration at the onset of abstinence persistently increases the capacity for plasticity within the BNST

The BNST is a region heavily involved in affective behavior that also is thought to play an important role in alcohol withdrawal-related phenotypes [24, 25]. In parallel with the time-dependent ketamine injection studies on affective behavior, we also assessed the time dependency of ketamine administration on the capacity for LTP induction within the BNST. We thus assessed the effect of early abstinence ketamine (day 42), and ketamine administered six-days (day 48) into abstinence on BNST LTP induction two to 3 weeks after the onset of abstinence. Fifteen mice underwent 2BC ethanol drinking followed by forced abstinence, and four mice underwent 2BC water drinking for 8 weeks. At the onset of abstinence, five ethanol drinking mice received an injection of saline, and another five received an injection of ketamine (3.0 mg/kg). Six days into abstinence (day 48), another five ethanol drinking mice received an injection of ketamine. As a control, we performed LTP recordings in mice with a history of 2BC water drinking. Four mice underwent 2BC water drinking for six-weeks and received an injection of saline on the 42nd day of drinking. We recorded LTP within the BNST in all mice two to three weeks into abstinence (day 56–63) (Fig. 6a).

Fig. 6 Ketamine administration at the onset of abstinence persistently increases the capacity for plasticity within the BNST. Representative figure for 2BC ethanol drinking followed by chronic abstinence and field potential electrophysiology (a). Averaged time course of synaptic field potentials after high-frequency stimulation (arrow depicts two 1-s rains at 100 Hz) in the dlBNST from water-drinkers (white circles) or ethanol-drinkers (black squares) (b). Summary bar graph of averaged field potentials 21–30 min (First Ten) post-tetanus (c) and 51–60 min (Last Ten) post-tetanus (from time course in b) (d). Averaged time course of synaptic field potentials after high-frequency stimulation (arrow depicts two 1-s rains at 100 Hz) in the dlBNST from ethanol-drinkers injected with ketamine at the onset of abstinence (white circles) or ethanol-drinkers injected with ketamine 6 days into abstinence (black squares) (e). Summary bar graph of averaged field potentials 21–30 min (First Ten) post-tetanus (f) and 51–60 min (Last Ten) post-tetanus (from time course in e) (g). %Baseline N2 are shown as the mean ± SEM (n = 8–13/slices between 10 mice) ****p < 0.0001 compared to ethanol drinkers with an injection of saline (analysis of variance t-test) Full size image

Tetanization produced LTP in the BNST of mice with a history of water 2BC, consistent with previous studies [23, 26]. LTP was also induced in slices obtained from ethanol abstinent mice. A marked decrease in an early component of LTP (referred to here as Short-term Potentiation, STP) was observed in these experiments, along with a very modest but statistically significant reduction in the later component. We compared the first ten minutes after tetanization and observed that animals with a history of ethanol drinking showed significantly lower STP compared to animals with a history of ethanol 2BC (Fig. 6c: Student’s t-test t(18) = 5.732, p < 0.0001). When we compared the last ten minutes of the recording, we found a very modest but significant decrease in LTP in slices from the ethanol abstinent mice. (Fig. 6d; Student’s t-test t(18) = 3.57, p < 0.0022).

We next examined the impact of ketamine administration at either the onset of abstinence (day 42), or 6 days (day 48) into abstinence on measured BNST LTP two to three weeks after abstinence (day 56–63). We found that mice with a history of ethanol drinking that received an injection of ketamine at the onset of abstinence (day 42) had a robust increase in LTP compared to ethanol drinking mice that received an injection of ketamine six-days into abstinence (day 48) (Fig. 6e). We again compared the first ten minutes after tetanization and found that ethanol drinking mice that got an injection of ketamine 6 days into abstinence had significant lower STP compared to ethanol drinking mice that got an injection of ketamine at the onset of abstinence (Fig. 6f; Student’s t-test t(18) = 10.56, p < 0.0001). When we compared the last ten minutes of the recording, we found that ethanol drinking mice that got an injection of ketamine at the onset of abstinence-maintained potentiation to a higher degree compared to animals that got ketamine on the 6th of abstinence (Fig. 6g; Student’s t-test t(18) = 15.02, p < 0.0001). We were also interested to see if potentiation differed in 2BC ethanol drinking animals that got an injection of saline at the onset of abstinence differed from the ethanol drinking animals that got an injection of ketamine on the 6th day of abstinence. Student’s t-test showed no significant difference 10-min after the onset of tetanization (Student’s t-test t(18) = 1.351, p = 0.58 nor a significant difference in maintain potentiation (Student’s t-test t(18) = 0.1933, p = 0.19) (data not shown).