Glutamate synaptic transmission changes with age in WT mice
To measure glutamate synaptic transmission, we examined frequency of sEPSCs in the presence of the GABAA blocker Bic. In WT animals, both D1 and D2 cells showed significant differences with age, from 1.5 to 12 months (). Interestingly, glutamate transmission changed in opposite directions in the two types of MSSNs. In D1-WT cells, sEPSC frequency increased with age (one-way ANOVA F2,69=7.17, p=0.002). Frequency was the lowest at 1.5 months and significantly different from frequency at 6 (t=2.81, p=0.019) and 12 months (t=3.23, p=0.006). There was no difference in EPSC frequency between 6 and 12 months in D1-WT cells (t=0.81, p=1.0). In D2-WT cells, sEPSC frequency decreased with age (one-way ANOVA F2,62=4.82, p=0.011). Frequency was similar at 1.5 and 6 months (t=0.81, p=1.0) while it was significantly lower at 12 months compared to 1.5 (t=2.61, p=0.034) and 6 months (t=2.84, p=0.017).
Figure 1 Frequency of sEPSCs varies differentially with age in D1 and D2 cells in WTs. A. Traces show sEPSCs in D1- and D2-WT cells at 1.5, 6 and 12 months. B. Graph shows that the mean frequency of sEPSCs increases with age in D1 while it decreases with age in (more ...)
Glutamate transmission is increased at 1.5 months and decreased at 12 months in D1-YAC128 cells
In contrast to D1-WT cells, D1-YAC128 cells showed a decrease of frequency with age, with the highest frequency at 1.5 months (, one-way ANOVA F2,29=4.61, p=0.016). Frequency at 1.5months was significantly higher than at 12 months in D1-YAC128 cells (t=2.84, p=0.022). There were also differences between D1-WT and D1-YAC128 cells with age (one-way ANOVA F2,108=6.73, p=0.002). At 1.5 months, D1-YAC128 cells displayed a higher frequency than D1-WT cells (t=2.32, p=0.022) while at 12 months, it was the reverse (t=1.74, p=0.005). Frequencies at 6 months were similar in D1-YAC and D1-WT cells (t=0.42, p=0.67). At 1.5 months, cumulative inter-event interval histograms suggested a significantly increased probability of release ontoD1-YAC128 cells, while at 12 months, cumulative inter-event interval histograms suggested a decreased probability of release onto D1-YAC128 cells (). Miniature EPSCs were recorded in the presence of TTX at 1.5 months to determine if the increased glutamatergic transmission was action potential-dependent. In the presence of TTX, mEPSC frequency was still higher in D1-YAC128 than in D1-WT cells suggesting an increase in spontaneous glutamate release onto YAC128 cells that was action potential-independent ().
Figure 2 Biphasic alterations of excitatory synaptic transmission in D1-YAC128 cells. A. Mean sEPSC frequency and cumulative probability distributions of inter-event interval histograms for sEPSCs show that D1-YAC128 cells display higher sEPSC frequencies at 1.5 (more ...)
To provide further evidence that the differences in EPSC frequencies were due to altered glutamate release, we examined glutamate responses evoked by corticostriatal afferents (eEPSCs) and measured paired-pulse ratios (PPRs). In D1-WT cells, PPRs decreased from 1.5 to 12 months (two-way RM ANOVA F1,98=28.7, p<0.001, t-test p<0.01 at 25, 50 and 100 ms intervals), suggesting the initial probability of glutamate release onto D1-WT cells is increased in older mice (). At 1.5 months, PPRs were higher in D1-WT cells than in D1-YAC128 cells at 25, 50 and 100 ms intervals (two-way RM ANOVA F1,109=13.91, p<0.001, t-test p<0.05 at 25, 50 and 100 ms intervals). In contrast, at 12 months, D1-YAC128 cells displayed higher PPRs (two-way RM ANOVA F1,114=12.39, p<0.001, p<0.05 at 25,50and 100ms intervals). These findings suggest that the initial probability of glutamate release onto D1-YAC128 cells is higher than on D1-WT cells at 1.5 months, which could explain the higher frequencies of EPSCs in D1-YAC128 cells at that age. In contrast, at 12 months, probability of release onto D1-YAC128 cells is lower, correlating with a lower frequency of sEPSCs.
Glutamate release is not significantly altered inD2-YAC128cells
Although mean sEPSC frequency tended to be lower with age in D2-YAC128 cells, the difference failed to reach significance (; one-way ANOVA F2,39=2.52 p=0.094). However, the t-test between 1.5 and 12 months showed a decrease of sEPSC frequency in D2-YAC128 cells with age (t25=2.68p=0.013). There was no significant genotype difference between D2-WT and D2-YAC128 cells with age (two-way ANOVA F2,98=0.29, p=0.74). However, the cumulative inter-event interval distribution exhibited a significant rightward shift. To investigate further if the rightward shift could indicate a decrease in glutamate release, we measured mEPSCs and PPRs (). In the presence of TTX, mEPSC frequency was similar in D2-WT and D2-YAC128 cells at 1.5 months (). There were no differences in PPRs between D2-WT and D2-YAC128 cells at 1.5 (two-way RM ANOVA F1,109=0.06 p=0.79) or at 12 months (two-way RM ANOVA F1,99=0.28 p=0.6) () suggesting glutamate release ontocells of the indirect pathway is not altered in YAC128.
Figure 3 Excitatory synaptic transmission in D2-YAC128 cells. A. Mean sEPSC frequency was not different in D2-YAC128 and D2-WT cells at any age. Cumulative probability distributions of inter-event intervals only showed slight differences at 12 months when D2-YAC128 (more ...)
Evoked glutamate currents are increased in D2-YAC128 cells at 1.5 months and decreased in D1-YAC128cellsat 12 months
In WT cells, eEPSCs also differed with age. In D1-WT cells, eEPSCs were larger at 12 months than at 1.5 months (two-way RM ANOVA F1,139=5.092 p=0.033; ). In D2-WT cells, eEPSC peak amplitude did not change with age (two-way RM ANOVA F1,129=0.418 p=0.524). In addition, eEPSCs were larger in D1-WTcells than in D2-WTcells, both at 1.5 and 12 months(two-way RM ANOVA F3,269=5.084, p=0.004). D1-YAC128 cells displayed similar currents as D1-WT cells at 1.5 months (, two-way RM ANOVA F1,139=0.381, p=0.542). In contrast, D2-YAC128 cells had larger eEPSCs compared to D2-WT cells at that age (two-way RM ANOVA F1,75=9.326 p=0.005) (). At 12 months, D1-YAC128 cells displayed smaller currents than D1-WT while D2-YAC128 and D2-WT cells displayed similar currents ().
Figure 4 Mean amplitude of evoked EPSCs is increased in D2-YAC128 cells at 1.5 months and decreased at 12 months in D1-YAC128 cells. A. Traces show evoked EPSCs to stimulation in WT (black) and YAC128 (red) at 1.5 and 12 months. Numbers above traces are stimulus (more ...)
Glutamate transmission in BACHD mice
At 2 months, similar to D1-YAC128 cells at 1.5 months, D1-BACHD cells displayed a significant decrease in PPR (two-way RM ANOVA F1,89=15.6, p=0.001) at 25(t=3.8, p<0.001) and 50(t=3.57, p<0.001) ms () suggesting an increased probability of glutamate release onto cells of the direct pathway in BACHD mice at an early stage. D2-BACHD cells did not show any difference in PPR compared to their WT littermates (two-way RM ANOVA F1,83=0.19, p=0.662), again a result similar to findings in D2-YAC128 cells at the presymptomatic stage (). Alterations in eEPSC amplitudes were also similar in BACHD and YAC128 D1 and D2 cells:eEPSC peak amplitudes were similar in D1-BACHD and D1-WT cells (F1,89=1.18, p=0.293) while D2 cells from BACHD mice showed larger currents than in WT mice (F1,69=7.75, p=0.034)().
Figure 5 Evoked EPSCs and PPRs in BACHD mice at 2 months. A. PPRs are significantly decreased in D1-BACHD compared to D1-WT cells at 2 months. In contrast, PPRs are similar in D2-BACHD and D2-WT cells. B. Evoked EPSC amplitudes are significantly increased in D2-BACHD (more ...)
D1 and D2 modulation of glutamate synaptic activity and cell firing in YAC128
The D1 receptor agonist SKF81297 (5 and 10 μM) increased sEPSC frequency (paired t-test t20
=5.86, p<0.001) in D1-WT cells as published previously (Andre et al., 2010
). This effect was similar at 1.5, 6 and 12 months. In D1-YAC128 cells, the D1 receptor agonist did not change the frequency of sEPSCs (t9
=0.96, p=0.361) at 1.5 months (significantly different from D1-WT cells, t20
=3.23, p=0.004). However, SKF81297 increased sEPSC frequency in D1-YAC128 cells at 6 and 12 months and this effect was similar to that observed in D1-WT cells (). The D2 receptor agonist quinpirole (10 μM) decreased sEPSC frequency at 1.5 (t12
=5.34, p<0.001) and 6 (t11
=3.24, p=0.008) months in D2-WT cells (). At 1.5 (t11
=0.30, p=0.765) and 6 (t12
=0.72, p=0.480) months, quinpirole had no significant effect on sEPSC frequency in D2-YAC128 cells. At 12 months, the D2 receptor agonist had no effect on sEPSC frequency in D2-WT cells (data not shown), suggesting a floor effect because of the low sEPSC frequencies. Therefore, we tested the D2 antagonist remoxipride (10 μM) at 12 months and found that it increased sEPSC frequency in D2-WT cells (t10
=2.68, p=0.023) while it had no effect in D2-YAC128 cells(t8
=0.69, p=0.509) ().
Figure 6 DA modulation of sEPSCs in D1 and D2 cells is altered in YAC128 mice. A. Traces show that at 1.5 months, the D1 receptor agonist SKF81297 increased sEPSC frequency in D1-WT cells while it had no effect in D1-YAC128 cells. In contrast at 12 months, SKF81297 (more ...)
We also examined the effects of DA receptor agonists on cell excitability in current clamp in YAC128 mice at 1.5 months (supplementary Fig. 1
). In D1 and D2 cells for WTs or YAC128 mice, there were no differences in resting membrane potential, current-voltage relationship or rheobase. In contrast, the rheobase was significantly higher in D1-WT compared to D2-WT cells (data not shown) as previously published (Cepeda et al., 2008
; Gertler et al., 2008). In D1-WT cells, SKF81297 (5 μM) increased excitability by increasing the spike frequency (p=0.03) while it had no significant effect in D1-YAC128 cells(Supplementary Fig. 1C, 1D
). In D2-WT cells, quinpirole (10 μM) decreased excitability by decreasing spike frequency (p<0.01) while it had no significant effect in D2-YAC128 cells(Supplementary Fig. 1G, 1H
Our data suggest complex biphasic anomalies in DA neurotransmission, with loss of D1 and D2 receptor function in early symptomatic mice. D1 receptor function in fully symptomatic mice appears similar to that of WTs. This early alteration in DA neurotransmission might be induced by abnormal DA concentrations as elevated striatal DA concentrations decrease DA receptor function (Giros et al., 1996
; Dumartin et al., 2000
; Wu et al., 2007
). In addition, in HD patients, TBZ is the only drug that alleviates chorea. TBZ ultimately depletes DA stores, suggesting that decreasing DA might be beneficial for hyperkinetic symptoms. Most studies report a decrease in DA release in symptomatic HD (Hickey et al., 2002
; Johnson et al., 2007
), but it remains unknown whether striatal DA tone is increased in early HD and contributes to some of the hyperkinetic symptoms. To test whether DA function is rescued in early HD, we examined the effect of TBZ in early HD in vitro
and in vivo
TBZ restores the effect of exogenously applied D1 receptor agonist and PPRs
In YAC128 slices from 1.5 month mice, we bath-applied TBZ (10 μM) after establishing baseline glutamate sEPSC frequency for 15minin D1-WT and D1-YAC128 cells(sEPSC frequency was quantified for 3 min before TBZ application). In D1-WT cells, TBZ had biphasic effects, increasings EPSC frequency at 5 min (+25.3±6.0%, paired t-test t8=5.58 p<0.01, 8cells) (). At 10 min, the increase (+14.2±6.9%) was no longer significant. sEPSC frequency returned to baseline values by 20 min, then gradually decreased. The decrease was significant after 40 min (-13.3±3.8%, t8=2.68 p=0.028) and 65 min (-18.6±4.1%, t8=2.50 p=0.037). In D1-YAC128 cells, TBZ did not induce an increase in sEPSC frequency. The effect of TBZ at 5 min was significantly different between D1-WT and D1-YAC128 cells (t15=3.65 p=0.002). In D1-YAC128 cells, TBZ induced a decrease that was significant by 20 min (-16.8±5.5, p=0.016, 7 cells). The decrease in TBZ was significantly higher in D1-YAC128 than in D1-WT cells at 25, 40, 50 and 65 min (F1,6302=36.63, p<0.001). To test whether the decrease in TBZ could be non-specific and due to a run-down of the cell health during these long recordings, we assessed the effect of DMSO on sEPSC frequency. In D1-WT cells, DMSO did not change sEPSC frequency even after 60 min indicating that changes in sEPSC frequency are specific to TBZ.
Figure 7 A. Effect of TBZ on sEPSC frequency in D1-WT and D1-YAC128 cells at 1.5 months. In D1-WT cells, TBZ induced a transient but significant increase in sEPSC frequency at 5 min while at 40, 50 and 65 min TBZ induced a significant decrease in sEPSC frequency. (more ...)
We then tested the effect of the D1 receptor agonist in a different group of cells in slices from YAC128 at 1.5 months incubated in TBZ (10 μM) or in DMSO (0.1%) for 2–4 hours (). In YAC128 slices incubated in TBZ, SKF81297 (5 μM) significantly increased sEPSC frequency (t8=2.67, p=0.028) while in YAC128 slices incubated in DMSO, the D1 receptor agonist had no significant effect (t9=1.04, p=0.326). The difference between the two treatments was statistically significant(t17=2.65, p=0.017).
As in D1-YAC128, sEPSC frequency was significantly higher in D1-BACHD cells at 2 months (, t18=2.781, p=0.012) and the D1 agonist SKF81297 had no effect in D1-BACHD cells. To test whether the increased PPRs could be a result of increased presynaptic glutamate release probability due to elevated DA tone, we examined PPRs in D1 cells in BACHD slices that were incubated in TBZ (10 μM) or in DMSO (0.1%) for 2–4 hours () and we tested the effect of a D1 antagonist on sEPSCs. In BACHD slices incubated in TBZ, PPRs were significantly higher than in BACHD slices incubated in DMSO (t23=2.883, p=0.008). The D1 antagonist SCH23390 (20 μM) did not have any effect in D1-WT cells (), suggesting DA tone is low in WT animals. However, in D1BACHD cells, SCH23390 had a significant effect and decreased sEPSC frequency by −15.9±6.5% (). Effect of SCH23390 was significantly larger in D1-BACHD compared to D1-WT cells (t18=2.858, p=0.01).
Locomotor and repetitive behaviors in HD mice
We measured locomotor activity in the dark during the dark phase for 15 min in a novel open-field test in WT and YAC128 mice at 1.5, 6 and 12 months. In WT mice, locomotor activity slightly decreased with age, this was reflected by decreased distance traveled at 12 months compared to 1.5 and 6months (F2,55=7.38, p=0.001) (). In YAC128 mice, distance traveled also decreased with age and this was significant at 12 compared to 6 months (F2,55=7.38, p=0.001). YAC128 mice at 1.5months did not show any differences in locomotion compared to WTs while symptomatic 6 and 12 month old YAC128 mice displayed decreased distance traveled (). The number of stereotypic movements measured by the open field test decreased with age in YAC128 mice (t=3.9, p<0.001 at 6 vs 1.5 months) while it did not change with age in WT mice. YAC128 mice at 1.5 (t=4.5, p<0.001) and 6 (t=2.8, p=0.006) months showed more stereotypies than WT mice while at 12 (t=1.7, p=0.1) months, stereotypies were similar in WT and YAC128 mice().
Figure 8 Locomotor activity in YAC128 and BACHD mice. A. YAC128 mice at 6 and 12 months displayed decreased distance traveled during 15 min in the open-field apparatus compared to WT mice. In WT and YAC128 mice, the distance traveled slightly decreased with age. (more ...)
In BACHD compared to WT mice, the distance traveled was significantly lower at 2 months (t35=2.1, p=0.042; ). This decrease in locomotor activity was due to the occurrence of increased stereotypies (t33=2.0, p=0.48) detected by the open field test and visually, BACHD mice spent significantly more time grooming (t23=2.3, p=0.027) and floor sniffing (t23=2.1, p=0.043) (). About 40% of the BACHD mice (5/13) spent more than 20% of the time grooming and when those mice were excluded, the distance travelled was no longer different in the BACHD (4116±144 cm) compared to the WT group (4324±233 cm, p>0.05). The intensive grooming was not present in WT mice (n=12).
We chose to use 2.5 mg/kg TBZ for in vivo studies because it decreased distance traveled by about 50% after 90 min while mice injected with 5 mg/kg displayed almost total immobility (Supplementary Fig. 2A
). At this concentration, the maximal effect for TBZ was obtained 40 min post-injection (Supplementary Fig. 2B, 2C
). At 40 min, TBZ decreased distance traveled similarly (t9
=0.12, p=0.90) in WT and BACHD mice (). However, TBZ’s effect on stereotypies was significantly different in WT and BACHD mice (). TBZ did not have any significant effect on stereotypies in WT mice while it significantly decreased stereotypies in BACHD mice to WT levels. The percent change induced by TBZ was significantly different between WT and BACHD mice(t8
Expression of EGFP
Five to eight hundred neurons were counted for each animal. Neuron densities (NeuN-positive cells/mm2) were similar between WT and YAC128 mice at 1.5 and 12 months (). This does not exclude there is a loss of cells as reported by others (Slow et al., 2003
; Van Raamsdonk et al., 2005a
; Tang et al., 2007
). We did not perform stereology as our focus was to determine the ratio of EGFP-expressing neurons. We found there were no differences in EGFP expression between WT and YAC128 mice at any age (two-way ANOVA F3,33
=0.122, p=0.947). The only difference was in the ratio of neurons expressing D1- or D2-EGFP as reported before (Bertran-Gonzalez et al., 2008
). At 1.5 months, there were more neurons expressing D1-EGFP (D1-WT: 53.8±0.7 vs D2-WT: 48.2±1.4% t5
=3.68, p=0.014; D1-YAC128: 54.4±1.0 vs D2-YAC128: 47.7±0.5% t8=7.46 p<0.001). At 1 year, the difference was not significant but was in the same direction (D1-WT: 51.5±1.8 vs D2-WT: 46.5±2.1% t6
=2.14, p=0.075; D1-YAC128: 50.6±2.4 vs D2-YAC128: 45.2±2.0% t7
Figure 9 A. Photomicrographs of immunofluorescent staining for NeuN (red) combined with EGFP in D1 (top row) or D2 cells (bottom row), at 1.5 (2 left columns) and 12 (2 right columns) months in the dorsal striatum of WT and YAC128 mice. Pictures show overlay of (more ...)