Biphasic alterations in evoked corticostriatal synaptic currents
Excitatory postsynaptic currents (EPSCs) were evoked in MSNs from YAC128 and WT mice at 1 and 7 months to examine alterations in synaptic responses. The mean stimulation threshold required to evoke an EPSC was significantly lower in cells from YAC128 mice than those from WT mice at 1 month (0.06±0.01 mA for YAC128 vs. 0.11±0.02 for WT; p<0.05) but not at 7 months (0.14±0.02 mA for YAC128 vs. 0.16±0.02 mA for WT). At 1 month, the mean peak response amplitudes were significantly greater in cells from YAC128 mice compared to those of WTs at stimulation intensities between 0.3–1.0 mA (; p<0.05–0.01). At 7 months however, the mean peak amplitudes in cells from YAC128 mice were smaller than those of WTs, especially at the higher intensities of stimulation (; p<0.05). In cells from WTs, the mean peak current amplitudes increased with age (; p<0.05). In contrast, the mean current amplitudes in YAC128 cells showed a small decrement with age ().
Figure 1 AMPA (R) receptor-mediated synaptic responses in YAC128 mice. A, Representative traces showing inward currents evoked by the same two intensities of stimulation in cells from YAC128 and WT mice at each age. B, Graphs showing mean (±S.E.) peak (more ...)
Recordings of synaptic responses also were made in slices from WT and YAC128 mice at 12 months of age in which the effects of quinpirole were examined (see below). In this series we used low stimulation intensities (0.05–0.3 mA) because we wanted to concentrate on modulation by quinpirole. Peak amplitudes of responses in YAC128 mice at 1 year of age also were significantly smaller than those of WT mice at this age. The peak amplitudes displayed the greatest differences at stimulation intensities ≥0.15 mA (p<0.001; 2-way ANOVA). Mean values ± SE (in pA) at each intensity for cells from WT (n=9) and YAC128 (n=10) mice, respectively were: 0.05mA: 44.2±12.6 versus 21.7±2.9, p=0.6; 0.1mA: 171.0±35.6 versus 106.6±18.2, p=0.11; 0.15mA: 276.6±42.9 versus 179.1±18.2, p=0.02; 0.2mA: 294.3±42.5 versus 187.2±18.2, p=0.011; 0.3mA: 309.4±42.5 versus 184.6±18.1, p=0.004 (Fisher post-hoc test). These data indicate that the decreases in peak amplitude in YAC128 mice first observed at 7 months remain at 1 year. Thus, cells from YAC128 mice display a biphasic response to synaptic activation of AMPA receptors. Young YAC128 mice displayed increased AMPAR-mediated responses followed by a subsequent reduction in older mice partially due to increasing response amplitudes in cells from WTs. There were no significant differences between YAC128 and WT mice in mean rise or decay times or duration at half-amplitude (data not shown). We anticipated that the observed age-dependent changes in the corticostriatal synaptic response could be due to presynaptic alterations in glutamate release, alterations in postsynaptic AMPARs or a combination of both effects. Subsequent experiments examined each of these possibilities.
Biphasic alteration in evoked corticostriatal release
To examine presynaptic function, we assessed FM1-43 release from cortical terminals within the dorsal striatum (). Stimulation of axons or cell bodies of projection neurons in layers V–VI of the cortex overlying the motor striatum resulted in endocytosis of FM1-43 dye by recycling synaptic vesicles, revealing linear en passant
arrays of fluorescent puncta, characteristic of corticostriatal afferents () (Bamford et al., 2004a
; Bamford et al., 2004b
; Bamford et al., 2008
). Following dye loading, cortical re-stimulation resulted in exocytosis of FM1-43 dye from the terminals, which decreased in a manner approximated by a single exponent, characteristic of synaptic vesicle fusion () (Stevens and Tsujimoto, 1995
; Wolfel and Schneggenburger, 2003
). FM1-43 destaining was calcium dependent since cadmium prevented stimulated release of the dye from presynaptic terminals (). As FM1-43 destaining generally followed first-order kinetics, corticostriatal release was characterized by the halftime (t1/2
) of release, defined as the time required for terminal fluorescence to decay to half its initial value.
The effects of mutant huntingtin on corticostriatal release were examined in slices from YAC128 mice at 1, 7 and 12 months. We observed the effect of frequency-dependence by unloading corticostriatal terminals at 1 Hz, 10 Hz and 20 Hz. In slices from 1 month-old WT mice, destaining was dependent on the frequency of applied stimulation and exhibited the greatest slope at 10 Hz (), suggesting that this frequency would be sensitive for detecting responses to genotype and pharmacological manipulations (Stern et al., 1997
; Bamford et al., 2004b
). At this stimulation frequency, the mean halftime of release from corticostriatal terminals in slices from YAC128 mice was 17% lower at 1 month of age compared to WT (t1/2
= 180 sec for YAC128 vs. t1/2
= 211 sec for WT; ; p<0.001), indicated by a faster release of FM1-43. At 7 months however, there was a 6% increase in average release halftimes from YAC128 mice compared to WTs (t1/2
= 243 sec for YAC128 vs. t1/2
= 230 sec for WT; ; p=0.24). By 12 months, corticostriatal halftimes of release in slices from YAC128 mice significantly increased by 28% (t1/2
= 300 sec for YAC128 vs. t1/2
= 234 sec for WT; ; p<0.001). Thus, compared to WTs, corticostriatal release was enhanced in young YAC128 mice but subsequently declined with age.
Figure 3 Age-dependent changes in corticostriatal release. A, FM1-43 destaining is dependent on the frequency of cortical stimulation. n=89–425 puncta for each condition; ***p<0.001, Mann-Whitney. B, Time-intensity analysis of FM1-43 destaining (more ...)
An advantage of this optical technique is that we are able to examine and compare corticostriatal vesicular release kinetics from individual terminals. When the halftimes of individual terminals are presented relative to their standard deviation from the mean value, a straight line indicates a normally distributed (or single) population (Bamford et al., 2004b
). At one month, mutant huntingtin increased vesicular release from all terminals (). At 7 months in slices from the YAC128 mice, exocytosis from fast-releasing terminals remained potentiated, but terminals with halftimes lower than one standard-deviation below the mean became inhibited (). While the mean value of release was similar between WT and YAC128 at 7 months, there was a significant change in the distribution of terminals (p<0.001, Kolmogorov-Smirnov test). In 12 month-old YAC128 slices, all terminals became slower so that the faster releasing subpopulation became similar to WTs, whereas the slower population of terminals were further inhibited ().
In WTs, corticostriatal release decreased progressively with age. The halftime of release increased from t1/2= 211 sec at 1 month, to t1/2= 230 sec at 7 months, and to t1/2= 234 sec at 12 months (; F(2, 814)=46, p=0.01, ANOVA). In YAC128 mice, terminal release kinetics also declined with age but did so to a greater extent than in WTs, decreasing from t1/2= 180 sec at 1 month, to t1/2= 243 sec at 7 months, and to t1/2= 300 sec at 12 months (; F(2, 685)=59, p<0.001, ANOVA). Thus, while corticostriatal release in WT mice decreased by 11% between 1 and 12 months, YAC128 mice demonstrated a 67% decrease over the same period.
Although terminal release halftimes in WTs decreased with age, the population of terminals remained mostly normal (). Examination of individual terminals in slices from YAC128 mice at 7 months also demonstrated linearity, consistent with normally-distributed release kinetics (). At 1 month however, there was preferential excitation in those terminals with halftimes of release lower than one standard deviation below the mean. At 12 months those terminals appeared preferentially inhibited (). Thus, compared to WTs, cortical input to the YAC128 striatum was enhanced in young mice but showed a progressive decline with age, with time-dependent alterations in specific subpopulations of corticostriatal terminals at each age.
Functional activity of corticostriatal afferents
We determined if these age-related changes in corticostriatal activity might be produced through alterations in endocytosis, a reduction in active terminals, or by modifications in the fractional release of FM1-43. Changes in release of FM1-43 in slices from 1 month-old mice were not due to inadequate FM1-43 loading of the recycling synaptic vesicle pool as loading stimulation frequencies of 1 Hz, 10 Hz or 20 Hz (for 10 min) did not significantly affect unloading at 10 Hz, either in WT (t1/2= 210 sec at 1 Hz, 211 sec at 10 Hz, and 205 sec at 20 Hz; n=32–425 puncta; p>0.5, Mann-Whitney) or in YAC128 mice (t1/2= 177 sec at 1 Hz, 179 sec at 10 Hz, and 177 sec at 20 Hz; n=39–353 puncta; p>0.1, Mann-Whitney). Likewise, synaptic vesicle fusion was conserved in all age groups, as corticostriatal release in slices from both WT and YAC128 mice approximated first-order release kinetics (r2>0.99; ) and remained dependent on calcium (). However, while the number of active terminals in each slice was similar in WT and YAC128 mice at 1 month (32.4±4 puncta vs. 33.3±2 puncta for WT and YAC128 mice, respectively; n=34–35 slices; p=0.4, ANOVA) and at 7 months (36.9±3 puncta vs. 38.1±5 puncta for WT and YAC128 mice, respectively; n=16–34 slices; p=0.4, ANOVA), it was significantly reduced in YAC128 mice at 12 months (33.8±4 puncta for WTs vs. 21.9±3 puncta for YAC128 mice; n=19–21 slices; p=0.02, ANOVA), suggesting a reduction in functional corticostriatal afferents in older YAC128 mice.
In WTs, the mean fractional destaining of FM1-43 per stimulus (f
) at 1 month was 0.033±0.002% (), similar to previous reports (Bamford et al., 2004b
; Bamford et al., 2008
). There was a decline in the fractional release with age to f
= 0.032±0.002% at 7 months and to f
= 0.025±0.002% at 12 months (F(2,90)
= 3; p<0.05, ANOVA). Fractional destaining decreased more rapidly in slices from YAC128 mice, declining from f
= 0.038±0.003% at 1 month, to f
= 0.027±0.003% at 7 months, and to f
= 0.021±0.001% at 12 months (F(2,90)
= 11; p<0.001, ANOVA), with the greatest depression seen at 1 year (p<0.05, ANOVA). The reduced fractional release of label during exocytosis in slices from YAC128 mice could be due to a reduced probability of recycling synaptic vesicles that undergo exocytic fusion per stimulus, a reduced amount of FM1-43 released per exocytic event, or a combination of these mechanisms (Bamford et al., 2008
Dopamine filters corticostriatal release
Previous experiments using untreated adult mice demonstrated that dopamine filters cortical information to the striatum by inhibiting exocytosis from less active corticostriatal terminals via activation of D2 receptors (Bamford et al., 2004a
; Bamford et al., 2004b
; Bamford et al., 2008
). In slices from WTs at 1 month, amphetamine (which induces continuous dopamine efflux via reverse transport (Schmitz et al., 2001
)) decreased corticostriatal release by 35% at 10 Hz cortical stimulation (t1/2
= 284 sec vs. 211 sec for untreated WTs; p<0.001). Similar to amphetamine, the D2 receptor agonist quinpirole (0.5 μM) also depressed corticostriatal exocytosis (t1/2
= 258; p<0.001). As expected, the D2 receptor antagonist sulpiride (10 ΩM) completely blocked inhibition by amphetamine (t1/2
= 205 sec for amphetamine and sulpiride; p>0.5 compared to untreated slices). Both amphetamine and quinpirole created two reversible populations of terminals that diverged at one standard deviation below the mean (), preferentially inhibiting slow-releasing terminals (~80%).
Figure 4 D2 receptors modulate corticostriatal release in 1 month-old YAC128 mice. A, Release halftimes for 1 month-old WT slices following cortical stimulation at 1 Hz, 10 Hz and 20 Hz in the presence and absence of amphetamine in vitro (n=89–425 puncta (more ...)
In slices from 1 month-old YAC128 mice, amphetamine also inhibited FM1-43 destaining with the greatest effect seen at 10 Hz cortical stimulation (t1/2
= 250 sec vs. 180 sec for untreated slices from YAC128 mice; p<0.001). Quinpirole also inhibited release (t1/2
= 257; p<0.001), and amphetamine’s inhibitory effect was blocked by sulpiride (t1/2
= 179 sec for amphetamine and sulpiride; p>0.5 compared to untreated slices from YAC128 mice). Thus, in both WT and YAC128 slices, amphetamine or quinpirole produced a D2 receptor-dependent filter with filtering applied preferentially to terminals with the lowest probability of release (Bamford et al., 2004b
; Dani and Zhou, 2004
D2 receptor-dependent corticostriatal filtering was also seen at 7 months in slices from both WT and YAC128 mice. In WTs, quinpirole depressed release (t1/2= 277 vs. 230 sec for untreated slices; p<0.001) as did amphetamine (t1/2= 289 sec; p<0.001). Sulpiride blocked the inhibitory effect of amphetamine (t1/2= 221 sec for amphetamine and sulpiride; p=0.5 compared to untreated slices). Similar to 1 month-old WT mice, both quinpirole and amphetamine filtered corticostriatal release by selective inhibition of the slowest releasing terminals (). In YAC128 slices, quinpirole (t1/2= 311 vs. 243 sec for untreated slices from YAC128 mice; p<0.001) or amphetamine (t1/2= 308 sec; p<0.001) reduced release and did so through high-pass corticostriatal filtering (). Sulpiride alone did not alter release (t1/2= 246; n=287 puncta; p=0.8, Mann-Whitney) and blocked the inhibitory effect of amphetamine (t1/2= 235 sec for amphetamine and sulpiride; p=0.7 compared to untreated slices from YAC128 mice).
Figure 5 D2 receptor responses in 7 month-old YAC128 mice. A, In 7 month-old WTs, both Quin and Amph decreased FM1-43 destaining. The inhibitory effect of Amph was reversed by Sulp. B, Distribution of mean t1/2 of release for destaining curves shown in panel A. (more ...)
Dopamine filtering is less effective in older YAC128 mice
In 12 month-old WTs, both quinpirole (t1/2= 288 vs. 234 sec for untreated slices; p<0.001) and amphetamine (t1/2= 276 sec; p<0.001) inhibited corticostriatal release but did so through non-selective inhibition of all terminals (). Sulpiride alone had no effect on release (t1/2= 226 sec; n=198 puncta; p=0.4, Mann-Whitney) and blocked amphetamine-induced inhibition (t1/2= 240 sec for amphetamine and sulpiride; p=0.5 compared to untreated slices).
Figure 6 D2 receptor responses in 12 month-old YAC128 mice. A, In 12 month-old WTs, both Quin and Amph inhibited FM1-43 release. Sulp reversed the effect of Amph. B, Distribution of mean t1/2 of release for destaining curves shown in panel A. n=258, 56, 157, 127 (more ...)
Compared to WTs, exocytosis was reduced in slices from 12 month-old YAC128 animals (t1/2= 300 vs. 234 sec for same-aged WTs; ; p<0.001, Mann-Whitney), but both quinpirole (t1/2= 336 sec; p<0.05) and amphetamine (t1/2= 340 sec; p<0.01) remained inhibitory. Sulpiride alone did not change release (t1/2= 299 sec; n=96 puncta; p=0.8, Mann-Whitney) and prevented amphetamine-induced inhibition (t1/2= 307 sec for amphetamine combined with sulpiride; ; p=0.7 compared to untreated YAC128 slices). Thus, in slices from older WT and YAC128 animals, dopamine-dependent high-pass filtering found in slices from younger mice was absent as activation of D2 receptors more broadly inhibited cortical input.
To parallel the release experiments, modulation of synaptic responses by quinpirole were also tested at 1 year. D2 receptor activation produced a reduced effect in cells from YAC128 mice compared to those from WTs at 1 year of age. Mean percent change was averaged for each cell across stimulation intensities. Quinpirole reduced WT peak amplitudes by −18.7±5.8% and −29.1±5.5% from baseline at 1 and 10 μM concentrations, respectively. In contrast, peak amplitudes of cells from YAC128 mice increased by 15.8±6.7% (p=0.0012 compared to values from WTs) and 6.5±11.6% (p=0.016 compared to values from WTs) at quinpirole concentrations of 1 and 10 μM, respectively.
Although both amphetamine and quinpirole continued to inhibit release in older WT and YAC128 mice, corticostriatal filtering became impaired and the average response to D2 receptor manipulation declined with age. At 1 month, amphetamine decreased exocytosis (increased average terminal halftimes) by 35±8% in slices from WT mice and by 35±5% from YAC128 mice (). At 7 months, the response to amphetamine declined to 28±2% in WTs and to 22±6% in YAC128 mice. At 12 months, amphetamine decreased exocytosis by 20±1% in WTs, but by only 10±3% in YAC128 mice. While dopamine’s inhibition of corticostriatal release decreased in both WT and YAC128 slices, there was a greater reduction in YAC128 mice by 12 months (; p<0.05). Therefore, the expected decline in amphetamine’s ability to release synaptic dopamine in senescence (Gerhardt and Maloney, 1999
) was greater in YAC128 slices. Interestingly, in WT slices, quinpirole reduced release by 18%–21%, with little variability over the age of the mouse (), suggesting that D2 receptor sensitivity does not change as decreases in dopamine reuptake may compensate for the age-dependent decline in dopamine availability (Gerhardt and Maloney, 1999
; Hebert and Gerhardt, 1999
). However, in slices from YAC128 mice, quinpirole decreased exocytosis by 35±5% at 1 month, but by 26±4% at 7 months, and by only 9±2% at 12 months (; p<0.001, ANOVA). Therefore, while the response to direct D2 receptor stimulation in WTs remained constant, quinpirole’s effect in YAC128 mice was amplified at 1 month but became diminished at 12 months (p<0.05 interaction between YAC128 mutation and age, 2-way ANOVA).
To confirm that slices from older YAC128 mice did not respond to D2 receptor stimulation to the same degree as their WT littermates, we released striatal dopamine in a different manner using local bipolar stimulation (). Rodents exposed to behaviorally salient stimuli display a rapid pulsatile elevation of striatal dopamine that reaches 200–500 nM and declines to background levels in <1 sec (Robinson et al., 2001
). In the slice preparation, striatal stimulation at 0.1 Hz evokes local dopamine release in a manner similar to behavioral stimuli associated with activation of reward pathways, reaching a peak level of ~1–2 μM for the first stimulus, and eventually decreasing to a plateau of ~30% of initial concentration (Bamford et al., 2004b
). At this frequency, electrically-evoked synaptic dopamine inhibits glutamate release in normal mice to the same extent as amphetamine and striatal stimulation alone has no measurable effect on punctum fluorescence, suggesting negligible interactions with glutamatergic afferents (Bamford et al., 2004b
). In slices from 7 month-old YAC128 mice, striatal stimulation reduced exocytosis by 22% (t1/2
= 297 sec vs. 243 sec for un-stimulated sections; n=110 puncta; p<0.001, Mann-Whitney), a halftime of release similar to that observed in YAC128 slices with quinpirole (t1/2
= 311 sec; p=0.4, Mann-Whitney) or amphetamine (t1/2
= 308 sec; p=0.7, Mann-Whitney). In 12 month-old YAC128 mice, striatal stimulation reduced corticostriatal release by 14% (t1/2
= 343 sec vs. 300 sec for un-stimulated sections; n=110 puncta; p=0.02, Mann-Whitney), similar to that achieved by quinpirole (t1/2
= 336 sec; p=0.1, Mann-Whitney) or amphetamine (t1/2
= 340 sec; p=0.5, Mann-Whitney). As expected, sulpiride blocked the effect of striatal stimulation (t1/2
= 296 sec; n=110 puncta; p=0.2 compared to un-stimulated sections, Mann-Whitney). Thus, D2 receptor stimulation with quinpirole, dopamine released via amphetamine, or dopamine released by direct striatal stimulation were all less effective in modulating corticostriatal vesicular release in 12 month-old YAC128 mice.
Biphasic alterations in biophysical membrane properties and postsynaptic AMPAR-mediated currents
In order to more specifically examine postsynaptic changes in AMPAR function, we assessed changes in evoked currents in acutely dissociated MSNs at 1 and 7 months. Based on electrophysiological measurements and cell appearance, all electrophysiological recordings were from MSNs (). The mean cell capacitance, input resistance and membrane time constants were similar in YAC128 and WT mice at 1 month (). However, a decrease in mean membrane time constant (p<0.05) and an increase in mean input resistance (p<0.005) occurred in MSNs obtained from YAC128 mice compared to WTs at 7 months. The mean capacitance also decreased but this difference was not statistically significant (p=0.057). Increases in input resistance and decreases in capacitance and membrane time constants of MSNs have been observed in other mouse models of HD (Starling et al., 2005
Figure 7 Membrane properties of acutely dissociated striatal MSNs. A shows representative images of MSNs from YAC128 mice and their WT littermates at 7 months. B shows bar graphs of mean capacitance, input resistance and time constants (±S.E.) from cells (more ...)
In cells from both WT and YAC128 mice, AMPA (100 μM) application produced a rapid peak response that desensitized quickly (). The amplitude of the AMPA peak increased markedly in the presence of cyclothiazide (CTZ; 10 μM), an inhibitor of AMPAR desensitization () with little difference in the qualitative appearance of the response between age groups (). At 1 month in response to AMPA or AMPA+CTZ, cells from YAC128 mice displayed higher mean peak current (p<0.02 and p<0.025 for AMPA and AMPA+CTZ, respectively) and mean peak current density (p<0.005 and p<0.006 for AMPA and AMPA+CTZ, respectively) than cells from WT mice (Figs. 8C1,C2,D1,D2). Mean steady-state currents and current densities were also higher in cells from YAC128 mice at 1 month of age, but the difference was significant for only the steady-state current densities (Figs. 8C3,D3; p<0.05). At 7 months however, mean peak currents and peak current densities, as well as mean steady-state currents and steady-state current densities were similar in cells from YAC128 and WT mice. Thus, while AMPAR-mediated responses in WTs generally increased with age, such responses in YAC128 cells declined.
Figure 8 AMPA currents in acutely dissociated MSNs from YAC128 and WT mice. A and B, Representative traces showing inward currents evoked by 100 μM AMPA, either alone or in the presence of 10 μM CTZ, from MSNs from YAC128 and WT mice at 1 and 7 (more ...)
Contribution of Ca2+-permeable GluR2 subunits
It is possible that the higher AMPAR-mediated currents observed at 1 month in YAC128 mice were the result of an increased proportion of GluR2-lacking Ca2+
-permeable AMPARs. Philanthotoxin-433 (PhTX-433) is a polyamine toxin that selectively inhibits Ca2+
-permeable AMPAR, which lack Q/R edited GluR2 subunits (Toth and McBain, 1998
). We compared the contribution of Ca2+
-permeable AMPARs by using PhTX-433 inhibition of AMPA+CTZ induced peak currents (). At both ages and in both genotypes about 10–15% of the AMPA+CTZ peak current was blocked by PhTX-433 (10 μM). However, there were no differences in the mean percentage of the AMPA current blocked by PhTX-433 between YAC128 and WT cells at either 1 or 7 months.
Figure 9 Effects of PhTX-433 and desensitization of AMPAR responses and concentration-response functions in striatal MSNs. A, Bar graphs showing mean (±S.E.) fractional AMPA+CTZ peak currents after inhibition by 10 μM PhTX-433 in acutely dissociated (more ...)
Desensitization of AMPAR-mediated responses in YAC128 mice
In addition to its effects on peak AMPA currents, CTZ also differentially modulates AMPAR desensitization depending on the predominance of either the FLIP or the FLOP splice variants. The desensitization rates are slower when the FLIP version of AMPARs predominates and faster when the FLOP version predominates (Partin et al., 1994
; Partin et al., 1995
; Vorobjev et al., 2000
). Striatal MSNs mainly express FLIP variants of GluR1, GluR2 and GluR3 AMPAR subunits (Vorobjev et al., 2000
). As an index of the ratio of FLIP and FLOP variants in the composition of striatal AMPARs, we examined the ratio of AMPA+CTZ current at peak and 1.5 sec after the peak (at the halfway point of the 3 sec exposure) to calculate the extent of the decay in peak current of these responses. The desensitization induced decay in AMPA+CTZ currents was significantly lower in YAC128 mice both at 1 month (p=0.019) and 7 months (p=0.012; ). Thus, MSNs from YAC128 mice at both ages may express a higher proportion of FLIP variant compared to those of WTs. Moreover, the FLIP to FLOP ratio remained unchanged in both genotypes since the desensitization-induced decay in peak currents at 1 and 7 months were approximately the same.
Concentration-response functions of AMPAR-mediated currents
Finally, we determined if age-dependent alterations in glutamate release produced sensitivity changes in AMPAR-mediated responses. Different concentrations of AMPA (1–1000 μM) applied to a subset of neurons at 1 and 7 months from both YAC128 and WT mice produced similar sigmoid dose-response curves (). The EC50 and Hill coefficients were similar at each age in cells from WT and YAC128 animals suggesting that apparent affinity of the AMPA receptor for AMPA does not differ significantly.