Basic membrane properties
Basic membrane properties () of pyramidal neurons were examined at each age using CsMeth as electrolyte. Although there was an age-dependent increase in membrane resistance and decreases in membrane time constant and cell capacitance, no significant differences were observed between the genotypes at any age. When K-Gluconate was used as the internal electrolyte to record in current clamp mode, significant differences were observed in basic membrane properties in neurons from the 80 day group (), including an increase in membrane input resistance (p=0.0038) and a decrease in membrane capacitance (p=0.0024) in R6/2 pyramidal neurons. The difference between membrane time constants failed to reach significance (p=0.15). At this age the resting membrane potential (RMP) was significantly depolarized in cells from R6/2 mice compared to cells from WT mice (p=0.025; ). Action potential firing threshold was unaltered. The differences between values obtained using CsMeth and K-Gluconate were probably due to the Cs+
blocks voltage-dependent inwardly-rectifying K+
currents (Uchimura et al., 1989
; Nisenbaum and Wilson, 1995
; Reyes et al., 1998
Basic membrane properties pyramidal neurons in slices from R6/2 mice
Excitatory synaptic transmission
Spontaneous EPSCs were recorded in voltage clamp mode at −70 mV. The majority of currents were <30 pA amplitude () and were almost entirely blocked by co-application of the ionotropic glutamate receptor antagonists CNQX (10 μM) and DL-AP5 (50 μM) (), indicating that the majority of these currents were due to activation of ionotropic glutamate receptors (n=5). The few remaining events were likely to be GABAergic in origin as application of BIC reduced the frequency of spontaneous currents by approximately 5% in slices from animals aged 40 and 80 days (see section ‘Effects of BIC’ below, and ). In cells from WTs, there was a significant age-dependent decrease in mean frequency of spontaneous EPSCs from 21 to 40 days (p=0.039), which remained decreased at 80 days (p<0.001 compared to 21 days; p>0.5 compared to 40 days) (). In contrast, R6/2 cells showed approximately the same mean frequency across all ages. At 21 days R6/2 neurons appeared to have a lower mean frequency of EPSCs than WTs but this difference failed to reach statistical significance (p=0.11). At 40 days there was no difference between WTs and R6/2s (p>0.4). At 80 days, the difference between these means was statistically significant, reflecting a greater frequency of spontaneous EPSCs in cells from R6/2 mice (p=0.031). Amplitude-frequency histograms () revealed that, at 21 days in cells from R6/2 mice, mid-amplitude events showed a significant reduction (p<0.05) in frequency compared to WTs, but this only reached significance for events between 10–15 pA. No significant differences were obtained at 40 days. At 80 days, a significant increase in the frequency of small amplitude (<10 pA) events occurred (p<0.05) and this was reflected as an increase in the cumulative probability plot for inter-event intervals in R6/2s ().
Figure 1 A: Typical voltage clamp traces are shown for both WT and R6/2 cells from mice in each age group. B: CNQX/AP5 application completely blocked all synaptic currents at a membrane holding potential of −70 mV. Insets show traces on a magnified time (more ...)
Figure 3 A: Example traces showing EPSCs recorded in the presence of BIC (20 μM) for each age group. B: Mean frequency of EPSCs (±S.E.M.) for each group. C: Mean percent change in frequency from ACSF to BIC for each group. D: Amplitude-frequency (more ...)
The kinetics of EPSCs were analyzed by averaging events between 7 and 50 pA for each cell (). No differences in mean rise times, decay times or half-amplitude durations were observed at 21 days. At 40 days, the reductions in the decay-time (p=0.085) and half-amplitude duration (p=0.097) in cells from R6/2 mice did not reach significance. By 80 days, both decay time (p=0.037) and half-amplitude duration (p=0.02) were significantly reduced and this was accompanied by a reduction in area (p=0.045) (WT: 200.4±25.9 pA × ms, n=12; R6/2: 153.2±8.5 pA × ms, n=18; p=0.045). There were no consistent differences between mean EPSC amplitudes at any age. Although there were more excitatory events in cells from 80 day R6/2 mice, they were of shorter duration and smaller overall area.
Figure 2 A: Traces show examples of average EPSCs with an exponential fit for WT (black) and R6/2 (grey) cells at each age (n>100 per trace). B: Bar graphs show rise times, decay times and half-amplitude durations of spontaneous EPSCs at each age. Note (more ...)
Effects of BIC
At a holding potential of −70 mV, and in the absence of GABA receptor blockers, a proportion of currents could be mediated by GABAA receptors. Also, inhibition by interneurons within the cortex could be suppressing excitatory synaptic activity. We therefore applied the GABAA receptor antagonist BIC (20 μM) to eliminate GABAA receptor-mediated transmission and subsequently recorded in gap-free mode at −70 mV to determine the frequency of pure EPSCs (). At 21 days, BIC increased EPSC frequency in 90% of cells from both genotypes. However, there was a significantly larger increase in frequency from cells of R6/2 than those from of WT mice (p=0.035) (). At both 40 and 80 days, most cells (75%) responded with a small decrease in EPSC frequency in response to BIC. At these ages, there was no differential effect of BIC. Importantly, in the presence of BIC, the higher frequency of EPSCs in R6/2 cells from 80 day animals remained and was more evident in the small amplitude (<10 pA) events (). Again, this was reflected as an increase in the cumulative frequency plots ().
Effect of TTX on EPSCs
To determine whether the higher frequency of EPSCs in cells from R6/2 mice at 80 days is dependent upon presynaptic action potentials, TTX (1 μM) was applied to a subset of slices (). To maximize the data obtained from these slices and permit recordings at a holding potential of +10 mV (see below), BIC was not applied. While BIC was not used for this subset of slices, the application of BIC in the absence of TTX suggested that the majority of events (>80%) at this holding potential are EPSCs. TTX reduced the frequency of EPSCs in all cells from both WT and R6/2 mice (). Moreover, the higher frequency of EPSCs was maintained in cells from R6/2s (3.4±1.0 Hz, n=6) compared to WTs (0.9±0.3 Hz, n=6; p=0.025; ). This was particularly evident for the smallest amplitude events (<11 pA), as shown in the amplitude-frequency histograms ( right graph). Cumulative amplitude plots showed no difference between the genotypes, while cumulative frequency plots were altered in R6/2s ().
Figure 4 Miniature EPSCs in slices from 80 day mice were isolated with TTX. A: Typical traces from cells from WT and R6/2 mice, prior to and following the application of TTX. B: Amplitude-frequency histograms revealed that the increases in spontaneous EPSC frequency (more ...)
The altered frequencies of spontaneous and miniature EPSCs suggest presynaptic modification. Two presynaptic alterations could be a reduction in the number of release sites (n
) or the probability of release (Pr
). A classical method to assess changes in Pr
is to examine paired-pulse ratios, which are independent of n
; Choi & Lovinger, 1997
). Increases in paired-pulse ratio are associated with a reduced Pr
, or vice-versa.
EPSCs were isolated by voltage clamping membranes at −70 mV and blocking GABAA receptors using 10 μM PTX (see ‘Evoked IPSCs” below and , which demonstrates complete IPSC blockade by this concentration of PTX). Initially the input-output function was assessed using stimulation intensities between 0.05 and 0.14 mA. At 21 days, no significant differences were identified in amplitude at any intensity, although larger EPSC amplitudes occurred in R6/2 neurons (p=0.086 at 0.14 mA stimulation intensity, ). By 80 days, there was an age-dependent reduction in the amplitude of evoked EPSCs in both genotypes. Moreover, EPSC amplitudes were significantly larger in neurons from R6/2 mice than in age-matched WTs and this reached significance at stimulation intensities between 0.04 and 0.14 mA (p<0.05-p<0.003; ).
Figure 10 Evoked IPSCs were isolated by voltage clamp at +10 mV and application of the ionotropic glutamate receptor antagonists CNQX and AP5. A: i) Altering (Δ) probability of release (Pr) affected paired-pulse ratio. Decreasing extracellular calcium ion (more ...)
Figure 5 Evoked EPSCs were isolated by voltage clamp at −70 mV and application of PTX (10 μM) A. Left: The relationship between stimulus intensity and EPSC amplitude was identical in WT and R6/2 mice at 21 days. Right: Typical traces from WT and (more ...)
Paired-pulse stimuli were then applied at intervals between 25 and 400 ms to assess probability of release. At 21 days, the paired-pulse profiles were identical between the genotypes and were characterized by paired-pulse facilitation at intervals of 25, 50 and 100 ms, no difference at 200 ms and paired-pulse depression at 400 ms (). At 80 days, a similar profile was identified in WTs, while ratios were reduced in neurons from R6/2s (). At 25 and 100 ms, there was no difference between the second and first response in R6/2 cells. At 200 and 400 ms, paired-pulse depression occurred. At the 50 ms interval, the ratio remained the same as WTs. The differences between the genotypes were statistically significant (25 ms: p=0.0011; 100 ms: p=0.043; 200 ms: p=0.00059; 400 ms: p=0.0016), indicating an increase in probability of glutamate release in R6/2 cells.
Bath application of 20 μM BIC induced large-amplitude discharges in cells from both genotypes and at all ages (). These discharges consisted of a large amplitude (>1 nA) fast inward current that included a prolonged decay (). No difference was observed between the two genotypes in the proportion of cells displaying these discharges at any age (21 days: WT 76%, R6/2: 85%; 40 days: WT: 78%, R6/2 91%; 80 days: WT: 69%, R6/2: 67%; p>0.7 at all ages, z-test). There was a decrease in the frequency of these discharges with age in both genotypes but no differences were observed between cells from WT and R6/2 mice at each age (). There was, however, a progressive change in the complexity of discharges in cells from R6/2 mice (). In cells from WTs, discharges were restricted to large inward currents occurring as singlets or doublets (, left column), while in R6/2 cells more complex discharges were apparent, with between 2 and 6 discharges occurring in a train, or cells showing high-frequency activity following the initial inward deflections (, right column). In WT slices, the percentage of cells displaying complex discharges decreased with age and complex discharges were never seen in the 80 day group. In contrast, the percentage of R6/2 cells showing complex discharges increased with age. The increase in complex discharges in cells from R6/2 mice and the decrease in cells from WTs is reflected in the histograms showing duration of events (). While durations of events tended to be longer in cells from R6/2 mice than in those from WT mice at all ages, this difference only reached significance at 80 days (p<0.001, Fisher exact test).
Figure 6 A: Voltage clamp recordings show that application of BIC induced large amplitude discharges in both genotypes. R6/2 cells showed more complex wave forms than cells from WT mice at each age. B: Proportion of cells displaying large amplitude discharges (more ...)
As differences were observed in EPSC frequency only at 80 days of age, current clamp recordings were obtained from another population of pyramidal cells at this age using K-Gluconate as the electrolyte. Initially, we recorded in gap free mode under voltage-clamp (−70 mV) and found a similar higher frequency of EPSCs in R6/2s (10.2±2.2 Hz, n=7) compared to WTs (5.8±0.8 Hz, n=9; p<0.05) as observed in voltage-clamp using CsMeth as the internal solution. When cells were recorded in current-clamp mode, discharges were again observed following the application of BIC for extended periods of time (5–20 min) (). Again, the voltage deflections recorded in cells from R6/2 mice were more complex than those in WTs. WTs showed repetitive membrane depolarizations, while cells from R6/2 mice also showed high-frequency discharges. Complex discharges occurred in 67% of R6/2 cells (n=12) and in 10% of WT cells (n=10; p=0.02, z-test).
Progressive changes in inhibitory cortical neurotransmission
The differential increase in EPSC frequency following BIC application to slices from 21 day mice suggests that an alteration in inhibitory inputs to the pyramidal cells may have occurred. Therefore, in a separate population of cells, voltage clamp recordings were made at a holding potential of +10 mV and in the presence of CNQX and AP5 in order to isolate GABAA receptor-mediated spontaneous IPSCs. Differences were observed from the earliest age recorded and a progressive, biphasic electrophysiological phenotype emerged in R6/2 cells (). At 21 days, two populations of cells were identified in WTs. One displayed IPSCs of low-frequency (LF; range 4.5–8.9 Hz), while the other displayed IPSCs of high-frequency (HF; range 14.1–19.9 Hz; ). No differences were found in the basic membrane properties between these two groups of cells for either genotype and therefore all cells were pooled in the 21 day group for . Pyramidal neurons from R6/2 mice could also be divided into LF (range 4.9–11.8 Hz) and HF (range 15.0–27.6 Hz) groups, but these groups were markedly different from the corresponding WT groups. Both populations of cells showed significantly greater IPSC frequencies (LF: p=0.038; HF: p=0.0012; insets). The increased frequency in both groups of cells from R6/2 mice is also shown by an increase in cumulative frequency plots (), while no differences were observed in cumulative amplitude plots (). In the R6/2 HF group, events no longer occurred randomly, but showed a bursting pattern consisting of large amplitude (>50pA) currents (). The increase in frequency was not due to an increased glutamatergic drive onto interneurons, as ionotropic glutamate receptors were blocked using CNQX and AP5.
Figure 7 A: Typical recordings of spontaneous (s) and miniature (m) IPSCs in cells voltage clamped at +10 mV. CNQX and AP5 were present throughout the recordings, except at 80 days in the presence of TTX. At 21 days, two populations of cells were evident: a low (more ...)
At 40 days, only one population of cells was present in both genotypes. The increase in frequency was maintained (p=0.032; inset). Cumulative frequency plots were significantly different (), while no differences were obtained when cumulative amplitudes were assessed ().
At 80 days the effect was reversed and R6/2 cells showed a statistically significant reduction in frequency of IPSCs (p=0.00078). Significant differences were obtained when both cumulative frequency () and cumulative amplitude () were assessed.
The kinetics of individual GABAA receptor-mediated currents were altered distinctly in cells from R6/2 mice (). Most notable was a distinct change in the form of the currents in the 21day HF cells. Events from all other groups showed typical fast rise times followed by an exponential decay, while currents of the R6/2 HF group displayed a fast rise time followed by an initial decay, a plateau phase and then a rapid decay (, inset). As this group of cells had such markedly different kinetics, exponential fits to the decay time were not performed and no difference was found in mean rise times between cells from WT and R6/2 mice. At 21 days, the LF group of cells from R6/2 mice showed the typical exponential decay but displayed a slower decay time (p=0.027) and a longer half-amplitude duration (p=0.059). No differences were observed in the kinetics at 40 days. At 80 days, the decay of R6/2 IPSCs was significantly slower (p=0.029) while the longer half-amplitude duration did not reach significance (p=0.072). This was accompanied by a significant reduction in area-under-the-curve of the IPSC from 925±71 pA×ms in cells from WTs to 682±64 pA×ms in those from R6/2s (p=0.016). There was no difference in mean amplitude at each age.
Figure 8 A: Traces show examples of average IPSCs with an exponential fit for WT and R6/2 cells at each age (n>100 per trace). Inset shows the different kinetics of IPSCs recorded in the HF group of R6/2 cells at 21 days. As the decay kinetics do not fit (more ...)
Effects of TTX on IPSCs
Miniature IPSCs were examined in an additional population of cells by applying TTX in the presence of CNQX and AP5 to slices from 21 days R6/2 mice. In this population of cells, TTX reduced the frequency of IPSCs in all groups ( and insets). Moreover, the differences between WT and R6/2 were eliminated in the presence of TTX in both the low- and high-frequency populations of cells (compare and )
Figure 9 A: Cumulative frequency probability plots showing that TTX abolishes differences in frequency at 21 days, but not 80 days. B: No differences were identified in cumulative amplitude probability plots at 21 days, but differences were maintained at 80 days. (more ...)
Following the recording at −70 mV in the presence of TTX to determine the action potential-dependency of EPSCs in slices from mice at 80 days, the holding potential was stepped up to +10 mV to isolate miniature IPSCs. CNQX and AP5 were not present at 80 days in experiments assessing the effect of TTX ( and ). TTX reduced the frequency of IPSCs in both genotypes but the difference in frequency was maintained (p=0.010; ). Cumulative probability plots revealed significant differences for both amplitudes and inter-event intervals (), suggesting contributions of both pre- and postsynaptic components.
Sensitivity of spontaneous IPSCs to GABAA receptor blockade
The prolonged kinetics of spontaneous IPSCs, the alterations in amplitudes of both spontaneous and miniature IPSCs and the prolonged kinetics of GABA-evoked currents and differential sensitivity to zolpidem in dissociated neurons (see below), suggests that postsynaptic alterations of GABAA
receptors may have occurred. As no change in the total expression levels of GABA receptors has been identified in HD cortex (Yorling, IV and Cha, 2002
), an alternative possibility is that the subunit composition of GABAA
receptors is altered. GABAA
receptor subunits are known to have varying sensitivities to GABAA
receptor antagonists, including BIC (Zhang et al., 1995
; Hansen et al., 1999
). The sensitivity of spontaneous IPSCs to a low concentration (1 and 2 μM) of BIC was therefore examined in slices. At 21 (both LF and HF groups of cells) and 40 days, there was no significant difference between genotypes in the percent block observed between cells from WT and R6/2 mice (data not shown). At 80 days, however, the sensitivity of IPSCs to blockade by BIC was reduced in cells from WT mice compared to earlier ages, while R6/2 mice showed a response similar to that at 21 and 40 days. When 1 μM BIC was applied, cells from R6/2 mice showed a greater reduction in IPSC frequency (−77.2±10.0%, n=11) than cells from WTs (−34.4±14.2%, n=9; p=0.015). Subsequent application of 2 μM BIC further reduced IPSC frequencies and the difference in percent change between the genotypes was still evident (WT: −66.6±10.1%; R6/2: −97.3±1.3%; p=0.0023). Application of 5μM BIC completely blocked all IPSCs in both genotypes and at each age.
The differences in the frequencies of spontaneous and miniature IPSCs suggest altered presynaptic function. At 21 days, the increased frequency of IPSCs was dependent on action potentials, while at 80 days, the reduction in IPSC frequency was insensitive to TTX, suggesting independence of action potentials. Two other presynaptic alterations could be a reduction in n
. Similar to glutamatergic synapses, Pr
can be assessed at GABAergic synapses by examining paired-pulse ratios. A second predictor of presynaptic function is the coefficient of variation (CV), which is dependent on both n
(Malinow & Tsien, 1990
; Bekkers & Stevens, 1990
; Choi & Lovinger 1997
). Increases in CV are associated with a decrease in either n
Evoked IPSCs were isolated by voltage clamping membranes at +10 mV and by blocking ionotropic glutamatergic currents with CNQX and AP5. Evoked IPSCs were completely blocked by the application of either 5 μM BIC or 10 μM PTX (n=4; ). Initially we studied the relationship between stimulation intensity and amplitude of evoked IPSCs in WT and R6/2 pyramidal neurons. A typical sigmoidal relationship was obtained from all cells studied. At 21 days, no difference was observed in the relationships between stimulation intensity and evoked IPSC amplitude between WT and R6/2 neurons (). Separating cells into those that displayed low- and high-frequency spontaneous IPSCs had no effect on experimental outcome and therefore data were pooled. At 80 days (), however, WTs showed an increase in evoked IPSC amplitude while R6/2s showed a decrease compared to amplitudes obtained from their respective genotypes at 21 days. Thus, evoked IPSCs in R6/2 neurons were smaller than in WTs at 80 days and this reached significance at intensities greater than 0.04 mA (p<0.01-p<0.001).
Stimulation intensities were subsequently set to evoke responses ~50% of the maximal amplitude and paired stimuli were applied at intervals between 25 and 400 ms. To demonstrate the specificity of this approach in determining presynaptic function, we initially made a number of manipulations known to alter synaptic transmission. We showed that when Pr
was reduced by halving extracellular calcium concentration from 2 to 1 mM, PPF at 25 ms was increased (a single example is shown in , which was repeated in two other cells, data not shown). Conversely, increasing Pr
by doubling the extracellular calcium concentration to 4 mM reduced PPF. In contrast, reducing n
by recruiting fewer afferent fibers at a lower stimulation intensity had no effect on PPF ratio. Postsynaptic manipulations also had no effect on experimental outcome. Altering postsynaptic electrochemical drive by changing the membrane holding potential from +10 mV to −70 mV (in the presence of CNQX and AP5), or partially blocking GABAA
receptors with 2 μM BIC, had no effect on paired-pulse ratios. Thus, these data demonstrate that only alterations in Pr
are expected to alter paired-pulse ratio (but see Clark et al., 1994
At 21 days, a biphasic function was observed between inter-pulse interval and paired-pulse ratio (). At an interval of 25 ms, PPF was consistently seen at WT synapses; no change was seen at an interval of 50 ms; while at 100–400 ms, PPD was observed. No difference in paired-pulse ratio was identified for R6/2 synapses at 21 days compared to WTs. Separating neurons into those displaying low-and high-frequency spontaneous IPSCs did not alter the outcome of this experiment and data have been pooled. At 80 days, the biphasic function was maintained at cortical synapses in WTs. PPF at the 25 ms interval was enhanced over that seen at 21 days, and PPF was now observed at 50 ms intervals, indicating a lower probability of release in WTs at older ages. PPD was maintained at intervals of 100–400 ms (). In contrast, 80 day R6/2 pyramidal cells did not display PPF but rather no difference and PPD at intervals of 25 and 50 ms, respectively (p<0.001 and p=0.007 with respect to WT). No significant differences were observed in the degree of PPD in WT and R6/2 neurons at intervals of 100–400 ms (p>0.2) at 80 days.
Coefficient of variance
A second measure of presynaptic function, CV, was also calculated from successively evoked IPSCs. CV is dependent on both Pr and n. To demonstrate the ability to identify changes in presynaptic function at GABAergic synapses using this technique, manipulations of pre- and postsynaptic function were again employed in 3 cells. Reducing Pr by reducing extracellular calcium increased the CV value (123±18%), whereas increasing Pr with a high extracelluar calcium reduced the CV (−50±2%). Also, reducing n using a lower stimulation intensity increased CV (406±118%), while increasing n using a higher stimulation intensity reduced CV (−28±2%). In contrast, postsynaptic manipulations had little effect on CV. Voltage clamping cells at −70 mV (7±1%) or reducing postsynaptic receptor number by partially blocking GABAA receptors with 2 μM BIC (7±8%) had no effect on CV. Thus, changes in both Pr and n would be expected to alter CV values in R6/2 neurons. Furthermore, a change in CV while paired-pulse ratio remained constant would indicate a change in n in the absence of a change in Pr.
At 21 days CV was statistically (p>0.6) identical in WT (0.19±0.02, n=13) and R6/2 (0.20±0.03, n=13) cells. Separating cells into those that displayed low- and high-frequency spontaneous IPSCs had no effect on the experimental outcome and data were pooled. CV was similar in WTs at 21 and 80 days (0.22±0.03, n=7, p>0.3). In contrast, CV was reduced at 80 days in R6/2 (0.15±0.05, n=7) compared to WT neurons (p=0.047).
GABA-evoked currents in dissociated pyramidal neurons
To assess purely postsynaptic function, we studied currents evoked by the application of GABA to dissociated cortical pyramidal neurons. GABA currents were evoked by application of GABA at increasing concentrations between 1 and 1000 μM (). At 21 and 40 days, no difference was observed in the amplitudes or current densities of evoked currents (). At 80 days, mean amplitude of evoked currents were significantly smaller in R6/2 neurons at GABA concentrations of 10 μM and higher. However, when current densities were calculated, these differences were normalized ().
Figure 11 A: Typical GABA-evoked responses in dissociated pyramidal neurons from WT (black) and R6/2 (grey) mice at each age and at 10 and 1000 μM GABA. Note that at 80 days, amplitudes are reduced in neurons from R6/2s. B: Peak current densities at each (more ...)
Desensitization of GABA currents was also assessed at GABA concentrations between 10 and 1000 μM. Similar results were obtained at each of these concentrations and are presented at 1000 μM GABA in . At 21 days, desensitization times were statistically identical in WT And R6/2 neurons. At 40 days, two populations of cells were detected in R6/2s: one had ‘fast’ desensitization times and were similar to WTs (p>0.2; n=8), while a second group had significantly slower desensitization times (p=0.045 vs WT and p=0.0016 vs R6/2 cells with ‘fast’ desensitization times; n=6). At 80 days, only one population of cells were identified and mean desensitization times were significantly slower than WT (p=0.044). Finally, as the presence or absence of certain GABAA
receptor subunits impacts on GABA current kinetics, we tested zolpidem, a type I benzodiazepine that binds more specifically to GABAA
receptors containing α1 subunits (Martinez-Torres et al., 2000
; Sanna et al., 2002
) in neurons from 40 day old mice. Potentiation of GABA (10 μM) peak by zolpidem (0.1 μM) was significantly larger (p=0.018) in WT (28.2±3.4%, n=9) pyramidal neurons compared to R6/2 (18.1±2.1%, n=11; ).
Increased susceptibility to seizures
The presence in R6/2 pyramidal neurons of complex discharges following the application of BIC and the relative changes in EPSC and IPSC frequencies suggest that these mice may be more susceptible to seizures. Indeed, seizures have been reported in R6/2 mice upon handling and may have been the cause of death in some mice (Mangiarini et al., 1996
). Furthermore, seizures are a prominent symptom of juvenile HD, which is caused by CAG repeat lengths greater than ~80 (for example see Gambardella et al., 2001
; Seneca et al., 2004
). To investigate the susceptibility to seizures in these mice, systemic administration of the GABAA
receptor antagonist PTX was employed. PTX was administered and latencies to seizure were recorded in R6/2 mice and their age-matched WT littermates. At all three ages, R6/2 mice showed significantly shorter latencies to onset of all stages of seizure activity compared to their WT counterparts ().
R6/2 mice were more susceptible to seizures induced by systemic administration of the GABAA receptor antagonist picrotoxin. Significant deceases in latencies occurred at all ages for both clonic and tonic-clonic seizures.
Spontaneous postsynaptic currents in YAC128 and CAG 140 mice
We performed electrophysiological recordings from layer II/III pyramidal cells in slices from YAC128 and CAG140 KI mice (). The slow progression of the HD phenotype in these models requires recording from older animals and we therefore opted not to perform an extensive longitudinal study similar to the one in the R6/2, but rather to restrict our analysis to 6 and 12 months in the YAC128 mice and to 12 months in CAG140 KI mice. At 6 months of age, YAC128 mice begin to show deficits on Rotarod performance and become hypoactive. Reductions in brain weight or volume are not detected until 9 or 12 months (Slow et al., 2003
). At 12 months, CAG140 KI mice display extensive motor and non-motor deficits, a reduction in brain weight and a reduction in DARPP-32 positive neurons (Menalled et al., 2003
; Hickey et al., 2008
Figure 13 Cortical spontaneous synaptic currents in 6 (left) and 12 (middle) month YAC128 (white bars) and 12 month CAG140 KI mice (right; grey bars) and their respective WTs littermates (black bars in all graphs). A: Spontaneous EPSCs were recorded at a membrane (more ...)
Initially, cells were voltage clamped at −70mV and basic membrane properties were recorded (). In pyramidal cells from both YAC128 (6 and 12 months) and CAG140 KI (12 months) mice, input resistance was significantly greater in mutants than in their respective WT controls. No significant differences were observed in membrane time constant or membrane capacitance. At a holding potential of −70mV spontaneous EPSC frequency was similar to WTs in pyramidal neurons from the 6 month old YAC128 mice (p>0.4) (, left panel). In WTs, there was a reduction in frequency with age, such that, by twelve months a significantly higher frequency of spontaneous EPSCs in YAC128 compared to WTs was evident (p=0.0097) (, middle panel). Similarly, pyramidal neurons of 12 month CAG140 KI mice displayed a significantly greater frequency of EPSCs than their respective WTs (p=0.037) (, right panel).
Basic membrane properties of pyramidal neurons in YAC128 and CAG140 KI mice
Subsequently, the membrane potential was stepped to +10mV and the ionotropic glutamate receptor antagonists CNQX and AP5 were applied to isolate spontaneous IPSCs (). At both ages studied, neurons from YAC128 mice displayed IPSCs of higher frequencies than their respective WTs (6 months: p=0.024; 12 months: p=0.00029). Pyramidal neurons of the 12 month CAG140 KIs also had a significantly greater frequency of IPSCs (p=0.0049).