Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation

Abstract

Huntington's disease (HD) is an autosomal dominant progressive and fatal neurodegenerative brain disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the gene. Presymptomatic Huntington's disease patients often exhibit cognitive deficits before the onset of classical symptoms. To investigate the possibility that changes in synaptic plasticity might underlie cognitive impairment in HD, we examined hippocampal synaptic plasticity and spatial cognition in a transgenic mouse (R6/2 line) expressing exon 1 of the human Huntington's disease gene containing an expanded CAG repeat. This mouse exhibits a progressive and fatal neurological phenotype that resembles Huntington's disease. We report that R6/2 mice show marked alterations in synaptic plasticity at both CA1 and dentate granule cell synapses, and impaired spatial cognitive performance in the Morris water maze. The changes in hippocampal plasticity were age dependent, appearing at CA1 synapses several weeks before they were observed in the dentate gyrus. Deficits in synaptic plasticity at CA1 synapses occurred before an overt phenotype. This suggests that altered synaptic plasticity contributes to the pre-symptomatic changes in cognition reported in human carriers of the Huntington' disease gene. The temporal and regional changes in synaptic plasticity within the hippocampus mirror the appearance of neuronal intranuclear inclusions, suggesting a relationship between polyglutamine aggregation and dysfunction.

Fig. 1.

HD mice have near-normal synaptic physiology in area CA1. a, Input/output relationships for control and transgenic mice aged 5–18 weeks [control slices denoted by ○ (pooled data from 24 slices, from 17 animals); transgenic slices denoted by ● (pooled data from 27 slices, from 18 mice)]. Field EPSPs were evoked by stimulation of the Schaffer collateral–commissural pathway at 0.067 Hz and recorded in the stratum radiatum. b, Transgenic synaptic responses are unaffected by age and severity of the phenotype. Individual responses evoked at 400 μA are plotted against age [control (r² = 0.051,p > 0.1) and transgenic (r² = 0.004,p > 0.5) responses denoted by ○ and ●, respectively]. c, Analysis of the population action potential also revealed a normal input/output curve for transgenic responses. Data points are pooled values from nine control slices (9 animals, aged 5–18 weeks; ○) and 13 transgenic slices (from 11 animals, aged 5–18 weeks; ●). Potentials were evoked by stimulation of the Schaffer–commissural pathway and recorded in the stratum pyramidale. d, Paired-pulse facilitation of field EPSPs was similar in both control and transgenic slices. The mean slope of the paired EPSP (expressed as percentage change with respect to the first response) is plotted against interpulse interval. Data points are pooled values from nine control slices (9 animals, aged 5–18 weeks; ○) and 26 transgenic slices (from 17 animals, aged 5–18 weeks; ●).e, Paired-pulse facilitation of population action potential. Transgenic responses are enhanced at an interval of 20 msec (p < 0.05) but otherwise are normal [9 control slices (from 9 animals aged 5–18 weeks; ○) and 22 transgenic slices (from 15 animals, aged 5–18 weeks; ●)]. f, PTP is normal in transgenic slices. Experiments were performed in 25 μmd-AP5 and responses were evoked at 0.2 Hz. Tetanic stimulation (3× 100 pulses at 100 Hz; denoted by ▴) induced a transient potentiation rapidly decaying to baseline within 90 sec (7 control slices from 7 animals and 16 transgenic slices from 12 slices, aged 7–14 weeks, denoted by ○ and ●, respectively).g, NMDA receptor-mediated transmission is normal in transgenic mice ≥12 weeks of age. Input/output curves are superimposed for normal synaptic responses (round symbols) and NMDA receptor-mediated potentials (square symbols) recorded in the same afferent pathway [6 control and 6 transgenic slices (open and filled symbols, respectively)]. NMDA receptor potentials were isolated pharmacological in nominally magnesium-free ACSF containing 10 μm CNQX. h, Analysis of the somatically recorded EPSP and population action potential revealed a decrease in the likelihood for a transgenic EPSP to generate an action potential at lower stimulation intensities. Data points are pooled values of somatic EPSP slope/population action potential ratio plotted against stimulation strength [9 control slices (9 animals, aged 5–18 weeks; ○) and 13 transgenic slices (11 animals, aged 5–18 weeks; ●)]. Points indicated by an asterisk are significantly different compared with controls (p < 0.05 in each case; Mann–Whitney test). Comparison of the transgenic and control input/output relationships of somatically recorded EPSPs revealed no significant differences between the two groups [slope values (V/s) at 50, 100, and 400 μA were 0.72 ± 0.1, 1.85 ± 0.15, 4.59 ± 0.39, and 0.79 ± 0.01 (p > 0.6), 1.41 ± 0.24 (p > 0.1), 4.7 ± 0.41 (p > 0.8) for transgenic and control slices, respectively].

Fig. 2.

HD mice express abnormal synaptic plasticity at CA1 synapses. a, Left, Transgenic synapses show reduced LTP and exhibit activity-dependent synaptic depression (depotentiation) of the field EPSP. EPSP slope is plotted as percentage change against time and expressed as a pooled mean [13 control slices (○) from 12 animals, and 17 transgenic slices (●) from 15 animals; aged 5–18 weeks]; SEM was plotted for every fifth datum. Tetanic stimulation (denoted by ▴) induced robust LTP (56.5 ± 6.6%; measured 1 hr after induction over 5 min) in control slices but a smaller LTP at transgenic synapses (26.8 ± 3.4%; p < 0.001, Welch t test). In contrast, low-frequency conditioning applied 1 hr after the induction of LTP failed to depotentiate the control slices (−4.9 ± 1.7%; measured 1 hr after conditioning) but induced depression at transgenic synapses (−18.5 ± 2.8%; p < 0.001).Right, Representative traces showing averages of five consecutive EPSPs taken immediately before tetanic stimulation (indicated as control), 1 hr after the tetanus (indicated as LTP), and 1 hr after low-frequency stimulation (indicated as depot.) are superimposed. b, Transgenic slices exhibit normal LTP of the population action potential but show marked depotentiation.Left, Tetanic conditioning induced LTP of similar magnitude in control (○) and transgenic (●) slices [229.7 ± 36.6% (15 slices from 13 animals) and 246.1 ± 38.3% (18 slices from 17 animals), respectively; aged 5–18 weeks; p= 0.76]. The mean population action potential amplitudes were similar for control and transgenic slices before conditioning (2.23 ± 0.29 mV and 2.52 ± 0.29 mV for control and transgenic slices, respectively; p > 0.4) and 1 hr later (6.79 ± 1.05 and 8.06 ± 0.94 mV for control and transgenic slices, respectively; p >0.3). Low-frequency conditioning applied 1 hr later failed to depotentiate control responses (−7.0 ± 2.9%) but induced a profound depression in the transgenic potential (−46.9 ± 3.9%; p < 0.001).Right, Representative traces showing averages of five consecutive population action potentials taken immediately before tetanic stimulation, 1 hr after the LTP, and low-frequency stimulation are superimposed and indicated as in a.c, LTD is only seen in transgenic slices. Low-frequency stimulation (bar) induced LTD in transgenic (●) EPSPs but not in control (○) responses (−19.2 ± 1.2%, 7 slices, and 6.34 ± 4.9%, 5 slices, respectively; p = 0.0076) (Welch t test). Tetanic stimulation (▴) applied 1 hr after low-frequency conditioning successfully induced LTP in both control and transgenic slices. Note that for experiments inc and d, the test shock was set to elicit responses at 50% of maximum. d, Low-frequency conditioning (bar) also selectively induces LTD of transgenic population action potentials [−56.2 ± 4.1% (n = 7) and 3.7 ± 13.7% (n = 6) for transgenic (●) and control (○) slices, respectively; p = 0.0087 (Welcht test)]. Calibration (a,b): 5 msec, 5 mV.

Fig. 3.

Spatial cognition is impaired in HD mice.a, Mean escape latency is plotted against day of training in the Morris water maze, commencing at 7 weeks of age [22 control (○) and 9 transgenic (●) mice]. Mice did not differ in initial escape latency, indicating that transgenic mice did not exhibit a nonspecific sensory or motor impairment. Over the course of training, control mice rapidly learned the location of the submerged platform, whereas transgenic mice showed little improvement (ANOVA genotype × day interaction F_(9,261) = 2.45,p < 0.02). b, Representative swim paths illustrate the impairment of spatial cognition during the probe trial in transgenic mice. Although the controls concentrated their search in the location at which the platform had been placed during training, the transgenic mice showed less focused swim paths.c, Probe test analysis reveals that transgenic (filled bar) mice spend less time in the platform quadrant than controls (open bar; p< 0.02 using two-tailed t test to compare time spent in platform quadrant).

Fig. 4.

Photomicrographs of ubiquitinated NIIs in CA1 neurons in a section of R6/2 mouse brain (13 weeks of age) under bright-field (a, b) or fluorescence (c, d) illumination. The areasoutlined in a and c are shown at higher magnification in b and d. Inclusions were immunostained for ubiquitin, and the section was counterstained with the fluorescent dye Hoechst 33258 to visualize nuclei. Small arrows in b andd show that the same inclusions can be seen in both fields. Numerous neuronal nuclei can be seen in the pyramidal cell layer (c). At higher magnification, inclusions can be seen clearly localized to the nucleus of CA1 neurons. Thelarge arrow in b and dindicates an inclusion in a nucleus stained with Hoechst dye (arrowheads). Scale bar (shown in a):a, c, 100 μm; b,d, 33 μm.

Fig. 5.

The temporal and regional pattern of NII distribution in HD mouse hippocampus. Ubiquitinated inclusions are seen in CA1 pyramidal neurons (a, b,e, f) and granule cells of the dentate gyrus (DG) (c, d,g, h) in hippocampi of mice killed at 3 weeks (a, b, d), 7 weeks (c), or 10 weeks (e,f, g, h) of age. Nuclei inb, f, and h were visualized by staining with Hoechst 33258 (a,e, g). Inclusions are clearly localized to the nuclei of CA1 pyramidal cells (arrows ina, b, e,f) and dentate gyrus (g,h). Inclusions are present in CA1 neurons, but not granule cells, at 3 weeks of age. Some inclusions can been seen in the dentate gyrus at 7 weeks but are not present throughout the stratum granulosum until 10 weeks of age. Scale bars (shown ind): a–f, 100 μm; (shown inh): g, h, 100 μm.

Fig. 6.

Age- and region-dependent changes in plasticity in the HD mouse. a, Left, The magnitude of LTP expressed at transgenic CA1 synapses over three age ranges is compared with that seen in the control slices. LTP is significantly impaired in transgenic slices from 5 weeks onward [5–6 weeks, 26.2 ± 3.9% (n = 5); 7–9 weeks, 30.9 ± 7.2% (n = 5); ≥10 weeks, 22.9 ± 4.54 (n = 7); controls slices, 56.5 ± 6.6% (n = 13); level of significance determined using Bonferroni multiple comparisons test]. Right, Lasting activity-dependent synaptic depression is enhanced in transgenic slices from 5 weeks onward [5–6 weeks, −38.8 ± 5.5% (n = 5); 7–9 weeks, −47.4 ± 11.4% (n = 5); ≥10 weeks, −51.6 ± 4.2% (n = 8); control slices, −7.0 ± 2.9].b, The induction of LTP at granule cell synapses is age-dependent in transgenic mice. Top, LTP was induced in both control and transgenic slices in animals aged 4–7 weeks [57.8 ± 15.7% (n = 8) and 30.0 ± 9.4% (n = 11), respectively; p< 0.05]. Bottom, LTP is absent at transgenic granule cell synapses in animals aged ≥12 weeks [68.2 ± 13.4% (n = 5) and 15.8 ± 5.4% (n = 7) in control and transgenic slices, respectively; p < 0.01]. c, The induction of LTD at transgenic CA1 synapses is NMDA receptor-dependent. Low-frequency conditioning (open bar) failed to induce LTD in the presence of 25 μmd-AP5 (filled bar) (1.8 ± 0.1%, 3 slices from 3 animals; measured 1 hr after conditioning). Subsequent low-frequency conditioning after washout of AP5 successfully induced LTD (−31.8 ± 0.2%; p < 0.01, pairedt test).