3.1 Alcohol consumption is markedly reduced in GLAST KO mice
Alcohol consumption was analyzed with genotype as between subjects factor, and two hierarchical within subjects factors, alcohol concentration (8, 12 and 16%), and measurement day (1-6) within the respective concentration. There was a significant main effect of genotype (F2,126=6.9, P<0.01), concentration (F2,252=88.9, P<0.001), as well as a genotype × concentration interaction (F4,252=10.9, P<0.001). Post hoc analysis showed significantly reduced alcohol consumption in GLAST KO compared to WT mice at 16% alcohol (). To examine a possible contribution of sex, data were also evaluated following introduction of this factor into the model above, but there was no genotype × sex, or genotype × concentration × sex interaction. To rule out the possibility that a contribution of sex went undetected because of the inherently lower power of an interaction test, we also carried out follow up analyses within each sex. These followed the same pattern as the global model presented, making a confound of sex highly unlikely. The decrease in consumption associated with GLAST deletion was similar in both sexes, although it was most pronounced in females (genotype: F2,57=9.8, P<0.001; concentration: F2,114=72.3, P<0.001; genotype × concentration: F4,114=6.7, P<0.001; post-hoc analysis: significantly lower consumption at 12 and 16% alcohol). In males, the main effect of genotype did not reach significance (F2,66=0.7, P>0.05), but in addition to a significant main effect of concentration (F2,132=31.0, P<0.001) there was a genotype × concentration interaction (F4,132=5.5, P<0.001); post-hoc analysis did not reach significance at any individual alcohol concentration.
Figure 1 Decreased 2-bottle free choice consumption of alcohol in GLAST KO mice. GLAST KO mice showed decreased alcohol consumption at the highest alcohol concentration tested, 16% (A), and showed a decreased preference for alcohol vs water both at 12 and 16% (more ...)
Similarly, alcohol preference was significantly affected by genotype (F2,104=10.4, P<0.01) as well as concentration (F2,208=5.5, P<0.001), and there was a significant genotype × concentration interaction (F4,208=11.1, P<0.001). Post hoc analysis showed a significantly reduced alcohol preference for GLAST KO mice at 16% (). Once again, introduction of sex as factor did not produce any significant interactions. Analysis by sex showed the same pattern as the consumption data, although the reduction of alcohol preference reached significance in both male and female GLAST KO mice (males: genotype: F2,51=2.7, P=0.07, concentration: F2,102=13.1, P<0.001, genotype × concentration: F4,102=6.4, P<0.001; post-hoc analysis: significantly lower preference at 16%; females: genotype: F2,50=9.0, P<0.001, concentration: F2,100=0.2, P=0.03, genotype × concentration: F4,100=5.0, P<0.01; post-hoc analysis: significantly lower preference at 12 and 16%).
3.2 GLAST KO mice lack conditioned place preference for alcohol
During habituation to the CPP apparatus, no side preference was present in any of the genotypes, and there was no different between genotypes (F1,50=1. 9, P>0.05; ). Similarly, no genotype difference in locomotor activity was found (WT=1439.9±92.9, HET=1498.9±78.6, KO=1551.2±81.0 beam breaks; mean±SEM; F2,52=0.4, P>0.05).
Figure 2 Loss of alcohol reward in GLAST KO mice. There was no significant difference between genotypes in preference for side going to become alcohol-paired in the initial habituation session (A). WT and GLAST HET mice showed robust place preference for the alcohol-paired (more ...)
During preference testing, WT and HET mice displayed robust CPP for alcohol, and spent about 80% time in the alcohol-associated compartment. In contrast, this measure was at chance level for GLAST KO:s. Accordingly, there was a significant effect of genotype for %time spent on the alcohol associated side (F2,50=5.6, P<0.01), and post hoc analysis showed that GLAST KO:s spent significantly less time on this side compared to both HET and WT mice (). There was no significant effect of sex when this factor was included in the analysis.
Locomotor activity was higher in the GLAST KO:s during alcohol-conditioning trials, but not during saline trials (data not shown), and also during preference testing, both in the saline and the alcohol associated compartment (alcohol associated compartment: WT=369.2±59.5, HET=770.5±77.8, KO=800.9±62.0; saline associated compartment: WT=469.5±55.0, HET=407.9±45.2, KO=731.5±85.5 beam breaks; mean±SEM; n=17-18; main effect of genotype F2,100=5.95, P<0.001; main effect of compartment: F1,100=14.2, P<0.001; no genotype × compartment interaction). However, in the GLAST KO:s, activity was not correlated with preference (r=-0.26, n=17, P=0.32).
3.3 GLAST KO mice do not have a general impairment of associative learning
Classical fear conditioning was unaffected by the GLAST deletion. First, freezing did not differ between genotypes during acquisition of conditioned fear (WT=59.1±10.5, HET=47.2±6.7, KO=43.6±10.4 % of observations; mean±SEM; n=10-15,). Second, no genotype differences were observed when conditioned fear was recalled by re-exposure to the shock-associated auditory cue (WT=77.8±6.3, HET=70.2±6.0, KO=55.8±7.1) or the shock-associated context (WT=35.3±6.1, HET=25.2±3.9, KO=33.0±8.3).
3.4 Sensitivity to depressant effects of alcohol is not altered by deletion of GLAST
There was no effect of sex in any of the analysis performed for the sensitivity to depressant effects of alcohol. For rotarod training, there were no difference between genotype at the final level, nor during baseline performance on the test day (data not shown). There was a robust effect of alcohol dose (F1,86=11.6, P<0.001), but no effect of genotype, or genotype × dose interaction with regard to the alcohol-induced impairment on the rotarod, measured as delta in the latency to fall off the dowel (2g/kg: WT=-51.7±9.2, HET=-63.5±8.8, KO=-61.3±12.2; 2.25 g/kg: WT=-90.1±12.2, HET=-87.5±8.8, KO=-93.7±10.2 sec; mean±SEM; n=13-17).
Baseline body temperature did not differ between genotypes (not shown). There was a significant effect of alcohol dose (F1,86=12.0, P<0.001), but no genotype effect, or genotype × dose interaction (delta temperature at 3g/kg alcohol: WT=-1.5±0.2, HET=-1.1±0.2, KO=-1.2±0.1; 4g/kg alcohol: WT=-1.7±0.2, HET=-1.8±0.2, KO=2.0±0.3°C; mean±SEM).
There was a robust effect of alcohol dose (F1,79=93.9, P<0.001), but no effect of genotype or genotype × dose interaction effect on the time to regain the righting reflex (3g/kg alcohol: WT=28.6±4.0, HET=34.2±4.0, KO=46.1±10.7; 4g/kg alcohol: WT=101.7±9.0, HET=96.5±8.8, KO=99.3±8.6 min; mean±SEM).
As expected, BACs declined significantly over time after the bolus injection (F3,78=45.8, P<0.001), but there was no effect of genotype or genotype × time interaction (5 min: WT=392.9±24.1 mg/dL, HET=390.3±42.0, KO=416.9±38.3; 30 min: WT=337.6±16.3, HET=331.6±45.0, KO=328.4±22.8; 360 min: WT=102.53±32.7, HET=89.3 ±29.0, KO=126.2 ±27.3 mg/dl; mean±SEM).
3.5 Extracellular glutamate levels are not altered in GLAST KO mice
Extracellular concentrations of glutamate in the nucleus accumbens were analyzed with genotype as between subjects, and sample number, a measure of time, as within subjects factor. There was a significant effect of time (F10,160=2.5, P<0.01) but no difference between genotypes (F1,16=0.003, P>0.05), or genotype × time interaction (F10,160=1.0, P>0.05; ).
Extracellular glutamate in the Nc. Accumbens was not affected by the GLAST deletion, neither at baseline, nor following systemic administration of alcohol (2g/kg) n=7-8 / genotype / time point. Data are mean±SEM μM.
3.6 Endocannabinoid signaling is impaired in striatal slices from GLAST KO mice
The basal properties of striatal synaptic transmission were not significantly altered in slices from GLAST KO mice relative to WT controls. Using paired pulses (50 ms interpulse interval) delivered every 20 sec, we observed that decay time of evoked EPSCs was 19±2.3 ms (EPSC 1), and 20±2.6 ms (EPSC 2) in slices from GLAST KO mice (n=16), and 17±1.6 ms (EPSC 1), and 18±1.7 (EPSC 2) in slices from WT mice (n=14). The paired pulse ratio (PPR) of EPSC 2 and EPSC 1 amplitudes (EPSC 2/EPSC1) did not differ in MSNs from GLAST KO (1.2±0.1, n=16), compared to WT (1.2±0.1, n=13; KO vs. WT, P>0.05), nor was the AMPA/NMDA ratio altered (WT: 2.3±0.9 (n=10), KO: 3.2±0.9 (n=11), F = 2.84, p > 0.05).
Repetitive activation of afferent fibers by high frequency stimulation (HFS) was insufficient to induce LTD in slices from KO mice (EPSC amplitude at t=20-25 min: 126±22% of control, n=7, P<0.05), while the same stimulation protocol induced a robust depression of EPSC amplitude in slices from WT mice (EPSC amplitude at t=20-25min: 76±15% of control, n=8, P<0.05; KO vs. WT, 15-20 min after HFS, P<0.01; ). To determine if a possible depression at these synapses could be unmasked by blockade of NMDA receptors, we perfused slices with picrotoxin (50 μM) and APV (50 μM). Blockade of NMDA receptors with APV prevented the small but significant (p<0.05) potentiation detected after HFS in slices from KO mice. More importantly, it did not unmask a depression, suggesting that striatal LTD is significantly impaired in slices from GLAST KO mice (). EPSC amplitude 20-25 min after HFS was 93±8.8% of control, n=9, P>0.05 in GLAST KO slices, and 60±8.7% of control in slices from WT mice, n=8, P<0.001; KO vs. WT, P<0.001. PPR was increased compared to baseline after HFS in slices from WT (106±1.4% of control, n=13, P<0.001), while no change was detected in slices from GLAST KO mice (99±3.1% of control, n=16, P>0.05).
Treatment with the L-type calcium channel activator FPL64176 has previously been shown to induce mGluR and D2R independent LTD (FPL-LTD) at glutamatergic synapses (Adermark and Lovinger, 2007a
). Similar to HFS, treatment with FPL64176 induced a robust LTD in slices from WT mice (EPSC amplitude=59±15% of control, n=5, P
<0.05), while no depression was detected in slices from KO mice (EPSC amplitude=104±11.9% of control, n=5, P
>0.05; ). In contrast, activation of presynaptic CB1 receptors by WIN55,212-2 (1 μM) induced a robust depression of EPSC amplitude in MSNs from GLAST KO mice (EPSC amplitude at t=20-25 min: 46±9.3% of control, n=5, P
<0.001; ), and significantly enhanced PPR (109±4.2% of control, n=6, P
3.7 LTP-induction is normal in striatal slices from GLAST KO mice
Field potential recordings dorsomedial striatum were carried out to examine possible changes in LTP-formation in GLAST KO mice. HFS-induced LTP was not significantly altered in GLAST KO mice (Fisher’s exact test, two tailed, p=0.43). Similar to whole-cell recordings from the dorsolateral striatum, LTD was only detected in slices from WT mice (). Striatal output (0-1 mV), evaluated by varying the stimulation intensity (0-150μA), was not altered in slices from GLAST KO mice (data not shown).