2 mouse model of Huntington’s disease

Neuroscience 146 (2007) 1571–1580

DOPAMINE-DEPENDENT LONG TERM POTENTIATION IN THE DORSAL STRIATUM IS REDUCED IN THE R6/2 MOUSE MODEL OF HUNTINGTON’S DISEASE V. W. S. KUNG,a R. HASSAM,a A. J. MORTONb AND S. JONESa*

Key words: synaptic plasticity, dorsolateral striatum, glutamate, NMDA receptor, dopamine D1 receptor, habit formation.

a

Department of Physiology, Development and Neuroscience, Anatomy School, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

The basal ganglia are a collection of forebrain nuclei involved in goal-directed actions, motor learning, and habit formation (Packard and Knowlton, 2002; Graybiel, 2004; Yin and Knowlton, 2006). The importance of the basal ganglia is highlighted in neurological disorders where function is compromised, such as Huntington’s disease (HD), which is characterized by motor, cognitive and psychiatric impairments (Bates et al., 2002). Glutamatergic inputs from almost all areas of the cerebral cortex as well as dopaminergic inputs from the midbrain converge on the striatum of the basal ganglia (Graybiel, 2004; Yin and Knowlton, 2006). The dorsolateral striatum is implicated in procedural learning. For example, dorsolateral striatal neurons change their activity during a procedural learning task in rats (Jog et al., 1999; Graybiel, 2004; Barnes et al., 2005) and monkeys (Schultz et al., 2003), and dopamine agonists and N-methyl-D-aspartic acid (NMDA) glutamate receptor antagonists influence procedural learning task performance in rats (Packard and Knowlton, 2002). Activity changes in dorsal striatum are also detected during procedural learning tasks in human subjects, and notably HD patients are impaired in procedural learning tasks (Packard and Knowlton, 2002). Dopamine-dependent forms of glutamatergic synaptic plasticity in the striatum are proposed as cellular models of procedural learning (Wickens et al., 2003; Mahon et al., 2004). In rats, long term potentiation (LTP) has been observed at corticostriatal synapses in vivo (Charpier and Deniau, 1997), while LTP as well as long term depression (LTD) has been observed in the dorsolateral striatum in vitro in response to high frequency stimulation (HFS) (Calabresi et al., 1996; Partridge et al., 2000). Recently, low frequency stimulation (LFS) has been shown to evoke LTD (Ronesi and Lovinger, 2005), and frequency-dependent bidirectional synaptic plasticity has been demonstrated in striatal slices from young rats (Fino et al., 2005). Together, these studies indicate that, as in other brain regions, corticostriatal synapses can increase or decrease their strength in response to different patterns of afferent input activity. Corticostriatal LTP and LTD have not been extensively studied in the mouse striatum, although there is increasing interest in deficits in corticostriatal synaptic plasticity in transgenic mice lacking key corticostriatal proteins (for example, Allen et al., 2006; Wang et al., 2006).

b

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK

Abstract—The striatum is critically important in motor, cognitive and emotional functions, as highlighted in neurological disorders such as Huntington’s disease (HD) where these functions are compromised. The R6/2 mouse model of HD shows progressive motor and cognitive impairments and alterations in striatal dopamine and glutamate release. To determine whether or not dopamine-dependent neuronal plasticity is also altered in the dorsolateral striatum of R6/2 mice, we compared long term potentiation (LTP) and long term depression (LTD) in striatal slices from R6/2 mice with that seen in slices from wild type (WT) mice. In adult WT mice (aged 8 –19 weeks), frequency-dependent bidirectional plasticity was observed. High frequency stimulation (four 0.5 s trains at 100 Hz, inter-train interval 10 s) induced LTP (134ⴞ5% of baseline), while low frequency stimulation (4 Hz for 15 min) induced LTD (80ⴞ5% of baseline). LTP and LTD were significantly blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D(ⴚ)-2-amino-5-phosphonopentanoic acid (D-AP5) (to 93ⴞ6% and 103ⴞ8% of baseline respectively), indicating that they are both dependent on NMDA glutamate receptor activation. LTP was significantly blocked by the dopamine D1 receptor antagonist R(ⴙ)-7-chloro-8hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390) (98ⴞ8% of baseline), indicating that LTP is dependent on activation of dopamine D1-type receptors, whereas LTD was not significantly different (90ⴞ7%). In adult R6/2 mice (aged 8 –19 weeks), LTP was significantly reduced (to 110ⴞ4% of baseline), while LTD was not significantly different from that seen in WT mice (85ⴞ6%). These data show that R6/2 mice have impaired dopaminedependent neuronal plasticity in the striatum. As dopaminedependent plasticity is a proposed model of striatum-based motor and cognitive functions, this impairment could contribute to deficits seen in R6/2 mice. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹44-0-1223-333795; fax: ⫹44-0-1223333786. E-mail address: [email protected] (S. Jones). Abbreviations: ACSF, artificial cerebrospinal fluid; D-AP5, D(⫺)-2amino-5-phosphonopentanoic acid; HD, Huntington’s disease; HFS, high frequency stimulation; LFS, low frequency stimulation; LTD, long term depression; LTP, long term potentiation; NMDA, N-methyl-Daspartic acid; SCH-23390, R(⫹)-7-chloro-8-hydroxy-3-methyl1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride; WT, wild type.

0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.03.036

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R6/2 mice are transgenic for exon 1 of the human HD gene carrying an expanded CAG repeat (Mangiarini et al., 1996; Bates et al., 2002). They show progressive motor and cognitive impairments (Carter et al., 1999; Lione et al., 1999), and alterations in neuronal plasticity in the hippocampal formation (Murphy et al., 2000; Gibson et al., 2005). One feature of HD is a dramatic loss of medium spiny neurons in the striatum (Bates et al., 2002), although increasing evidence suggests that a progressive decline in neuronal function precedes the overt symptoms and neuronal death in HD patients and in R6/2 mice (Lione et al., 1999; Li et al., 2003; Smith et al., 2006). Neuronal function has been examined in pre- and post-symptomatic R6/2 mice, and changes include alterations in corticostriatal synaptic transmission (Levine et al., 1999; Lafloret et al., 2001; Cepeda et al., 2001; Zeron et al., 2002; reviewed by Levine et al., 2004) and compromised dopaminergic function (Cha et al., 1998; Reynolds et al., 1999; Bibb et al., 2000; Hickey et al., 2002; Johnson et al., 2006). Alterations in glutamatergic and dopaminergic function in the striatum are likely to disrupt both synaptic activity and plasticity in the corticostriatal network, and impaired synaptic plasticity at corticostriatal synapses could contribute to the progressive motor and cognitive symptoms seen in R6/2 mice and in HD patients. Therefore, in this study we have compared LTP and LTD in wild type (WT) and R6/2 mice. We found that dopamine-dependent LTP, but not LTD, is impaired in R6/2 mice compared with that seen in WT mice.

EXPERIMENTAL PROCEDURES Animals R6/2 transgenic mice were taken from a colony established in the Department of Pharmacology, University of Cambridge and maintained by backcrossing R6/2 males to CBA⫻C57BL/6 F1 females. Genotyping was confirmed by polymerase chain reaction as reported in Gibson et al. (2005). A common set of mice was used by Gibson et al. (2005) and in the present study both for electrophysiology and for measurement of the repeat lengths of the mutation carried by R6/2 mice, which were 243⫾3 (n⫽26 mice). The present experiments were carried out using 67 slices from 41 WT mice (23 slices from 20 mice for HFS in control conditions; 7 slices from 7 mice for HFS in D(⫺)-2-amino-5-phosphonopentanoic acid (D-AP5); 7 slices from 7 mice for HFS in R(⫹)-7-chloro-8-hydroxy3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390); 12 slices from 12 mice for LFS in control conditions; 8 slices from 8 mice for LFS in D-AP5; 10 slices from 9 mice for LFS in SCH-23390), and 31 slices from 24 R6/2 mice (22 slices from 19 mice for HFS in control conditions; 9 slices from 9 mice for LFS in control conditions). All experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986 and conformed to UK Home Office and local guidelines on the ethical use of animals. All attempts were made to minimize the suffering and the number of animals used for this study.

Brain slice preparation Brain slices were prepared from adult male WT and R6/2 littermates aged between 8 and 19 weeks. Mice were killed by cervical dislocation and the brains were rapidly removed into ice-cold sucrose-based Ringer of the following composition (mM): sucrose 75, NaCl 70, KCl 2.5, NaH2PO4 1.25, NaHCO3 25, CaCl2 0.5, MgCl2 14, and D-glucose 10. Horizontal slices (400 ␮m) containing

the dorsal striatum were prepared (DTK-1000 microslicer, Dosaka, Kyoto, Japan) and held after cutting on a net in a submersion chamber containing artificial cerebrospinal fluid (ACSF) of the following composition (mM): NaCl 119, KCl 2.5, NaH2PO4 1.0, NaHCO3 26, CaCl2 2.5, MgCl2 7, D-glucose 10 at 30 °C and saturated with 95% O2/5% CO2.

Electrophysiological recordings Slices were transferred to a submersion recording chamber 1– 6 h after preparation, suspended between two nylon nets and continuously perfused at 2.5–3.5 ml min⫺1 with ACSF as detailed above but containing 1.3 mM MgCl2, at 29 –31 °C, and saturated with 95% O2/5% CO2. All experiments were carried out in brain slices taken dorsal to the anterior commissure and in the middle third of the anterior–posterior axis. A bipolar stainless steel electrode (Frederick Haer and Co., Bowdoin, ME, USA) was placed in the white matter on the lateral edge of the striatum to stimulate glutamatergic afferents to striatum. Test stimuli (100 ␮s duration) were applied at 10 s intervals at an intensity that evoked approximately 50% of the initial maximum compound field potential response. Stable field potential responses were monitored for at least 10 min to ensure there was no movement of the recording electrode or variability in the response. Movement of the electrodes occurred infrequently and was readily detected by sudden changes in field potential amplitude. HFS (four 0.5 s trains at 100 Hz, inter-train interval 10 s) was used to induce LTP and LFS (4 Hz for 15 min) was used to induce LTD; separate slices were used to study the effect of HFS and LFS. LTP and LTD experiments were carried out in the absence of GABA receptor blockers in order to study plasticity under nominally physiological conditions. Glass microelectrodes (2– 6 M⍀ when filled with ACSF) were placed in the dorsolateral striatum to record compound field potentials. Field potentials were amplified using an Axoprobe 1A amplifier (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA), low-pass filtered at 1–2 kHz and digitally sampled to a PC at 10 –20 kHz using a Micro1401 interface (Cambridge Electronic Design, Cambridge, UK). The amplitude of the field potential was measured using the computer program Spike 2 (version 4, Cambridge Electronic Design). To determine whether or not a significant change in field potential amplitude followed HFS or LFS in individual experiments, field potentials were analyzed using ANOVA with a post hoc Dunnett’s test (GraphPad Prism, version 4, San Diego, CA, USA); the criterion for stating that LTP or LTD had occurred was a significant increase or decrease (P⬍0.05) in amplitude in more than two 10 min periods occurring at least 20 min after HFS or LFS. To determine whether or not significant LTP or LTD had occurred within the combined data set for each experimental group, paired t-tests were used to compare field potential amplitudes during the 10 min baseline period with field potential amplitudes 30 – 40 min post-HFS/LFS. To compare the magnitude of LTP and LTD between slices from different experimental groups, the field potential amplitudes were measured 30 – 40 min postHFS/LFS and compared across groups using the unpaired t-test (with Welch’s correction if the variances between the groups were unequal). The ‘n’ values reported refer to the number of slices. All combined data are shown as mean⫾the standard error of the mean (S.E.M.). A critical P value of P⬍0.05 was considered significant for the statistical tests used throughout this study.

Materials All standard laboratory salts were obtained from BDH Laboratory Supplies (Poole, Dorset, UK). Drugs were obtained from SigmaAldrich (Gillingham, Dorset, UK).

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RESULTS Frequency-dependent bidirectional synaptic plasticity in the striatum A recent study described frequency-dependent bidirectional synaptic plasticity in individual medium spiny neurons of the striatum from young rats (Fino et al., 2005). To determine whether frequency-dependent bidirectional plasticity could be evoked in the striatum of adult mice, we measured compound field potentials from populations of striatal neurons in the dorsolateral region of the striatum, evoked by stimulation of the overlying white matter. The field potential responses are likely to be driven by cortical glutamatergic synaptic input to striatal neurons, because

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the responses were reduced by the non-NMDA receptor antagonist DNQX (WT: to ⫺2⫾13%, n⫽6; R6/2: to 8⫾12%, n⫽5; P⫽0.55). These data are summarized in Fig. 1. The time course of the field potential responses (Figs. 1 and 2) also agrees with a previous report of a peak inward negativity occurring 2– 4 ms following stimulation (Malenka and Kocsis, 1988). The effects of HFS (four 0.5 s trains of 100 Hz) and LFS (4 Hz for 15 min) on compound field potentials in WT mice were measured. HFS evoked LTP of striatal field potentials that lasted at least 50 min (Fig. 2A). In slices from WT mice, LTP was 134⫾5% of baseline (n⫽23; Fig. 2B). LTP was significant (paired t-test, P⬍0.001), and was present in 23/23 slices (ANOVA). LFS evoked LTD of

Fig. 1. Compound field potentials in adult mouse striatum. (A, a) Example compound field potentials (FP) in the striatum of a slice (average of 10 sweeps, taken from a WT mouse) recorded in control conditions (C) and in the presence of DNQX (D). The subtracted (DNQX-sensitive) response is shown below. (b) Bar graph shows the field potential amplitude (mV) before and after DNQX application in six slices from WT mice (* P⬍0.05). (B, a) Example compound field potentials in the striatum of a slice (average of 10 sweeps, taken from an R6/2 mouse) recorded in control conditions (C) and in the presence of DNQX (D). The subtracted (DNQX-sensitive) response is shown below. (b) Bar graph shows the field potential amplitude (mV) before and after DNQX application in five slices from R6/2 mice (* P⬍0.05).

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Fig. 2. Frequency-dependent bidirectional synaptic plasticity in adult mouse striatum. (A) Normalized compound field potentials (FP) in the striatum of slices taken from WT mice. Circles (mouse aged 11.9 weeks), HFS (arrow) induced LTP of 141% of baseline (measured 30 – 40 min post-HFS). Diamonds (mouse aged 13.4 weeks), LFS (arrow) induced LTD of 63% of baseline (measured 30 – 40 min post-LFS). Insets show example field potentials (average of 10 sweeps) recorded before (1) and after (2) HFS or LFS and (3) in the presence of DNQX. (B) Combined data showing HFS induced LTP of normalized compound field potentials (FP) recorded from the striatum of WT mice (n⫽23 slices). LTP measured at 30 – 40 min post-HFS was 134⫾0.05%. LFS (arrow) -induced LTD of normalized compound FP recorded from the striatum of WT mice (n⫽12 slices). LTD measured at 30 – 40 min post-LFS was 80⫾0.05%.

striatal field potentials that lasted at least 50 min (Fig. 2A). In slices from WT mice, LTD was 80⫾5% of baseline (n⫽12; Fig. 2B). LTD was significant (paired t-test, P⬍0.01), and was present in 11/12 slices (ANOVA). NMDA receptor- and dopamine D1 receptordependence of striatal LTP Several forms of striatum-based learning are dependent on NMDA receptors and dopamine D1 type receptors. To determine whether HFS-induced LTP is dependent on NMDA and D1 receptors, we applied HFS in the presence of selective antagonists. In the presence of the NMDA receptor antagonist D-AP5 (50 ␮M), HFS did not induce significant LTP (paired t-test, P⫽0.29). LTP was present in one of seven slices (ANOVA), and the magnitude of LTP

was 93⫾5.7% of baseline (n⫽7, P⬍0.0001 compared with control LTP; Fig. 3A, B). In the presence of the D1-type dopamine receptor antagonist SCH-23390 (2 ␮M), HFS also failed to induce significant LTP (paired t-test, P⫽0.72). LTP was present in two of seven slices (ANOVA), and the magnitude of LTP was 98⫾8.4% of baseline (n⫽7, P⬍0.005 compared with control LTP; Fig. 3A, B). Thus, HFS-induced LTP depends on activation of NMDA receptors and dopamine D1-type receptors. NMDA receptor- and dopamine D1 receptordependence of striatal LTD To determine whether LFS-induced LTD is dependent on NMDA and D1 receptors, we applied LFS in the presence of selective antagonists. Like HFS-induced LTP, in the

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ceptor activation is required for LTP, the role of dopamine in LTD is more complex. Striatal LTP, but not LTD, is impaired in slices from R6/2 mice Neuronal dysfunction at the level of both glutamate and dopamine has been reported in the striatum of R6/2 mice. This might be expected to adversely affect glutamate- and dopamine-dependent neuronal plasticity. To determine whether frequency-dependent bidirectional plasticity was altered, we applied HFS and LFS in striatal slices from R6/2 mice. Under nominally physiological conditions, there was no significant difference in the amplitude of field potential responses (at 50% of the maximum response) in slices from R6/2 mice (0.17⫾0.03 mV, n⫽5) compared

Fig. 3. LTP in adult mouse striatum is dependent on NMDA receptor and D1 dopamine receptor activation. (A) Normalized compound field potentials (FP) in the striatum of slices taken from WT mice recorded before and after HFS in different experimental conditions: slices perfused with D-AP5 (50 ␮M; light gray circles; n⫽7); slices perfused with SCH-23390 (2 ␮M; dark gray circles; n⫽7), with WT LTP shown for comparison (from Fig. 2B; black circles). (B) Bar graph shows LTP (% of baseline field potentials measured at 30 – 40 min post-HFS) in slices from WT mice recorded in control conditions, in the presence of D-AP5, and in the presence of SCH-23390 (*** P⬍0.001).

presence of the NMDA receptor antagonist D-AP5 (50 ␮M) LFS did not induce significant LTD (paired t-test, P⫽0.70). LTD was present in three of eight slices (ANOVA), and the magnitude of LTD was 103⫾7.9% of baseline (n⫽8, P⬍0.05 compared with control LTD; Fig. 4A, B). Thus, NMDA receptors are required for LFS-induced LTD of field potentials in the striatum. The effect of the D1 receptor antagonist on LTD was more complex. In the presence of SCH-23390 (2 ␮M), LFS did not induce significant LTD (paired t-test, P⫽0.18). However, the magnitude of LTD (90⫾7% of baseline, n⫽10) was not significantly different to control LTD (P⫽0.25; Fig. 4A, B), and the number of slices showing LTD in the presence of SCH-23390 (5 of 10 slices as determined by ANOVA) was not significantly different to control (P⫽0.56, Fisher’s exact test). Thus, while D1 re-

Fig. 4. LTD in adult mouse striatum is dependent on NMDA receptor but not dopamine D1 receptor activation. (A) Normalized compound field potentials (FP) in the striatum of slices taken from WT mice recorded before and after LFS in different experimental conditions: slices perfused with D-AP5 (light gray diamonds; n⫽8); slices perfused with SCH-23390 (dark gray diamonds; n⫽9), with WT LTD shown for comparison (from Fig. 2B; black diamonds). Single direction S.E.M. bars only are shown for clarity. (B) Bar graph shows LTD (% of baseline field potentials measured at 30 – 40 min post-LFS) in slices from WT mice recorded in control conditions, in the presence of D-AP5, and in the presence of SCH-23390 (* P⬍0.05).

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with slices from WT mice (0.26⫾0.06 mV, n⫽6, P⫽0.24). The magnitude of HFS-induced LTP was significantly reduced (to 110⫾4.3% of baseline, n⫽22, P⬍0.0005 compared with WT; Fig. 5A, C). LTP was present in 11/22 slices from R6/2 mice (ANOVA). By contrast with HFSinduced LTP, LFS-induced LTD was not significantly altered in R6/2 mice (85⫾5.9% of baseline, n⫽9, P⫽0.55 compared with WT LTD; Fig. 5B, C). LTD was present in six of nine slices from R6/2 mice (ANOVA). Thus, the range of plasticity in the striatum was reduced in R6/2 mice. Although LTP was significantly reduced in slices from R6/2 mice compared with LTP in slices from WT mice, it was not completely blocked: R6/2 slices had significant if reduced LTP (P⫽0.03, paired t-test). Therefore, the agedependence of LTP in slices from R6/2 mice was examined (Fig. 6). The magnitude of HFS-induced LTP in slices from 8 to 13 week old R6/2 mice (105⫾5%, n⫽12) was not significantly different from the magnitude of LTP in slices from 13 to 17 week old mice (107⫾8%, n⫽10, P⫽0.8; Fig.

Fig. 5. Frequency-dependent bidirectional striatal synaptic plasticity is disrupted in R6/2 mice. (A) Combined data of HFS-induced LTP of field potentials recorded from the striatum of R6/2 mice (n⫽22 slices, open circles) with WT LTP shown for comparison (from Fig. 2B; black circles). In R6/2 mice, LTP at 30– 40 min post-HFS was 110⫾0.04%. (B) Combined data of LFS-induced LTD of field potentials recorded from the striatum of R6/2 mice (n⫽9 slices, open diamonds) with WT LTD shown for comparison (from Fig. 2B; black diamonds). In R6/2 mice, LTD at 30 – 40 min post-LFS was 85⫾0.06%. (C) Bar graph shows LTP and LTD (% of baseline field potentials at 30 – 40 min post-HFS or LFS) in slices from WT and R6/2 mice recorded in control conditions (*** P⬍0.001).

Fig. 6. Age-dependence of impaired LTP in R6/2 mice. (A) Normalized compound field potentials recorded before and after HFS in slices from R6/2 mice aged 8 –13 weeks (light gray circles; n⫽12) and 13–17 weeks (dark gray circles; n⫽10). (B) Bar graph shows LTP (% of baseline field potentials at 30 – 40 min post-HFS) in slices from WT (black bars) or R6/2 (white bars) mice aged either 8 –13 weeks old or 13–17 weeks old (* P⬍0.05, *** P⬍0.001).

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6A). The magnitude of LTP was significantly reduced when compared with LTP in slices from WT mice in the two age groups (8 –13 weeks, P⬍0.001; 13–17 weeks, P⬍0.05; Fig. 6B).

DISCUSSION We have found that striatal neurons of adult mice exhibit frequency-dependent bidirectional plasticity, with HFS inducing NMDA receptor- and dopamine D1 receptor-dependent LTP, and LFS inducing NMDA receptor-dependent LTD. Furthermore, we have found that LTP, but not LTD, is impaired in the striatum of adult R6/2 mice. Our observation that evoked field potentials in the striatum of adult mice undergo frequency-dependent bidirectional plasticity is in agreement with the recent findings of Fino et al. (2005), that excitatory postsynaptic potentials of individual medium spiny neurons in the striatum of juvenile rats show frequency-dependent LTP and LTD. Our graded compound field potentials recorded from populations of striatal neurons in the dorsolateral region of the striatum in response to stimulation of the overlying white matter are likely to be driven by glutamatergic inputs to the striatum, based on their time course (Malenka and Kocsis, 1988) and sensitivity to the non-NMDA receptor antagonist, DNQX. However, in some recordings a residual DNQXinsensitive component remains, and the residual component may be mediated by NMDA receptors, due to removal of Mg2⫹ ion block during local field depolarization, or by non-glutamatergic synapses, for example cholinergic synapses. Glutamatergic inputs to the striatum activate striatal interneurons, as well as medium spiny neurons, and the responses of these cells may contribute to the population responses, although these cells comprise only 5–10% of the striatal cell population (Kawaguchi et al., 1995). Furthermore, changes in somatodendritic membrane potential, due to local depolarization following electrical stimulation, cannot be ruled out when extracellular recording is used. The most likely source of glutamatergic input to the striatum is the cerebral cortex, suggesting that the measured responses are driven by corticostriatal input, although glutamatergic inputs to the striatum also come from the thalamus. Early reports of LTP and LTD in the striatum reported that HFS could induce either form of plasticity, depending on the level of activation of NMDA receptors. Under physiological conditions, HFS induced LTD, but when NMDA receptor activity was facilitated by the absence of Mg2⫹, HFS induced LTP (reviewed by Calabresi et al., 1996). More recently, Fino et al. (2005) have reported that corticostriatal synaptic plasticity in rats shows the frequencydependence observed in other brain regions, with HFS inducing LTP and LFS inducing LTD. Ronesi and Lovinger (2005) also reported LFS-induced LTD at rat corticostriatal synapses. The HFS and LFS paradigms we used to induce LTP and LTD respectively resemble cortical firing patterns observed in vivo (Kasper et al., 1994; Charpier et al., 1999). Our data support the idea that corticostriatal synapses in the dorsolateral striatum, like glutamatergic syn-

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apses in other brain regions, can increase or decrease their strength under different physiological conditions of afferent activity. This flexibility in the range of synaptic strength is likely to be important in the behavioral plasticity of the striatum, including motor learning and habit formation (Packard and Knowlton, 2002; Wickens et al., 2003; Graybiel, 2004; Mahon et al., 2004). Normal striatal function, including procedural learning, depends on both glutamate and dopamine (Smith-Roe and Kelley, 2000; Reynolds et al., 2001; Packard and Knowlton, 2002; Wise, 2004; Faure et al., 2005), notably in the form of NMDA receptor– dopamine D1 receptor interactions. Glutamatergic and dopaminergic inputs to striatal cells co-localize at asymmetric synapses (Smith and Bolam, 1990), suggesting that an important role of dopamine is to modify cortical glutamatergic input. Previously described forms of corticostriatal LTP and LTD, induced by HFS under different experimental conditions that promote or reduce NMDA receptor activation respectively, showed a dependence on both NMDA receptors and dopamine D1 receptors (Calabresi et al., 1996; Spencer and Murphy, 2000; Kerr and Wickens, 2001; Wickens et al., 2003; Gerdeman et al., 2003; Centonze et al., 2003). In our study, LTP and LTD were blocked by the NMDA receptor antagonist D-AP5, indicating that they both require NMDA receptor activation. By contrast, although the D1 receptor antagonist SCH-23390 blocked LTP, LTD was not significantly reduced, suggesting that D1 receptors are required for strengthening corticostriatal synapses in response to strong afferent input but not for weakening corticostriatal synaptic strength in response to weaker afferent input. Our finding that LTD was variably dependent on D1type receptors suggests that the role of dopamine in the striatum is complex. The dopamine-dependence of LTD may depend on the location of the recorded neurons, since in striosomes (biochemically distinct compartments within the striatum; Graybiel, 1990), there is a different dopamine receptor expression pattern compared with that of the surrounding matrix, with D1 receptors being more prevalent in striosomes (Gerfen, 1992; Brene et al., 1995; Caille et al., 1995; Piggott et al., 1999). In addition, sub-populations of medium spiny neurons express D1-type receptors, while others express D2-type receptors (Gerfen et al., 1990; Gerfen, 2000). It is possible that all medium spiny neurons express LTD, but that LTD is only dependent on D1-type receptors in the D1-expressing sub-population, leading to variation in the population responses. By contrast to the LFS-induced LTD seen in this study, the LTD seen in response to HFS is dependent on the D5 member of the D1-type dopamine receptor family (Centonze et al., 2003). Interestingly, the LFS (4 Hz) used to induce LTD mimics the tonic activity of dopaminergic neurons, which causes a low and sustained pattern of dopamine release, while HFS (100 Hz) resembles phasic activity which releases rapid peaks of dopamine (Schultz, 2002). The two neuronal patterns are thought to sub-serve distinct functions. For example, phasic release may serve as a reward prediction error signal that is important for learning, while

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tonic release may act as an enabling signal for striatal functions (Schultz, 2002), for example learned habitual responses. Thus, our data are consistent with the idea that functions depending on tonic dopaminergic activity and involving LTD may be less critically dependent on dopamine D1 receptor activation than functions requiring phasic dopaminergic activity and involving LTP. R6/2 mice show a number of cellular deficits, including alterations in glutamate receptor expression (Cha et al., 1998, 1999; Luthi-Carter et al., 2003), abnormalities in synaptic machinery (Morton and Edwardson, 2001), decreased glutamate release (Li et al., 2003; Cepeda et al., 2003), reduced spines in medium spiny neurons of the striatum (Klapstein et al., 2001), and abnormal responses to NMDA (reviewed by Levine et al., 2004). Deficits in synaptic plasticity have been observed in the hippocampus of HD mouse models (Usdin et al., 1999; Murphy et al., 2000; Gibson et al., 2005). We have observed that, in addition to this catalogue of cellular dysfunction, NMDA receptor- and D1 receptor-dependent LTP, but not LFSinduced LTD, is impaired in the striatum of R6/2 mice. Basal field potential amplitude is not significantly different in R6/2 mice. In our original studies using R6/2 mice (Carter et al., 1999; Lione et al., 1999; Murphy et al., 2000), the CAG repeat length of mice we used was around 150. In the past few years, the mean repeat length of our colony has expanded. Since 2002 we have selected breeders for experiments so that their offspring have repeat lengths of around 230 –280. However, in all of our published behavioral studies we have repeat-length matched our experimental groups of R6/2 mice. The increase in repeat length is highly significant because it correlates with an improvement in overt phenotype. In our mice, the increased repeat length correlates with a delay in onset of signs. While mice with a repeat length of around 250 CAGs show measurable cognitive and motor dysfunction before 12 weeks of age, they do not show the overt signs (hind limb grooming, muscle wasting, lordokyphosis, body weight, and infertility) that are present in mice with shorter repeats (A.J.M., unpublished observations). Mice with a CAG repeat of ⬃250 survive for around 24 weeks, compared with a survival of around 15 weeks in mice with a repeat of 150 CAGs (A.J.M., unpublished observations). It is not known why there is a delay in signs in mice with longer CAG repeats. Indeed, this is paradoxical, because in humans longer CAG repeats correlate with earlier onset of disease. We do not know how the change in repeat length affects the electrophysiology of neurons, since this and our previous study (Gibson et al., 2005) used groups of mice with the same length repeat (243⫾3). We have not made a direct comparison of electrophysiological parameters in mice with different repeat lengths, although this would clearly be very interesting. Similarly, we do not know the nature of the relationship between the expanded CAG repeat and the abnormalities in synaptic function we have observed. However, it is notable that even in the absence of overt signs, synaptic plasticity is impaired very early. This was the case for animals with repeats of around 150 CAGs

(Murphy et al., 2000) and at around 250 (Gibson et al., 2005; this study). The R6/2 mice used by Gibson et al. (2005) showed marked impairments in LTP in the hippocampus by 4 weeks of age, although mice with similar length repeats do not show overt signs until around 15 weeks of age. We have hypothesized that the changes in synaptic plasticity seen in R6/2 mice are deleterious and contribute to cognitive impairments (Murphy et al., 2000; Gibson et al., 2005). However it is also possible that the changes in synaptic plasticity are adaptive changes that allow the animal to function apparently normally, albeit at the expense of normal cognitive function. The deficit in corticostriatal synaptic plasticity in R6/2 mice observed in this study could contribute to deficits in motor and cognitive functions (Lione et al., 1999; Carter et al., 1999) that are dependent on intact corticostriatal circuits. A significant deficit in striatal LTP was seen at the earliest age group tested (8 –13 weeks), and did not significantly progress with age, indicating that this deficit is already well established by 8 weeks. However, LTP is not impaired in R6/2 mice at 6 weeks of age; Picconi et al. (2006) recently reported no deficit in LTP in medium spiny neurons of the striatum in slices taken from R6/2 mice aged 6 weeks old, while LTP in cholinergic interneurons in the striatum was impaired. Speculatively, impaired LTP in cholinergic interneurons may form part of the presymptomatic decline in normal physiological function in R6/2 mice, with a decline in plasticity of medium spiny neurons occurring subsequently, as symptoms develop. Currently, there are a number of speculative explanations for the deficit in LTP in R6/2 mice. The fact that LTP but not LTD is impaired suggests that NMDA receptor function is intact in the striatum of R6/2 mice, at least in its role of inducing LTD. If NMDA receptor function is only partially compromised in R6/2 mice, it is possible that LTP, perhaps requiring higher and faster rates of Ca2⫹ entry through NMDA receptors, would be more sensitive to such an impairment whereas LTD, possibly requiring lower and slower increases in Ca2⫹, would not. On the other hand, based on existing evidence for dopaminergic dysfunction in R6/2 mice, a loss of normal dopamine release or a decrease in D1-type receptor number or function might account for the significant and selective reduction of LTP in R6/2 mice, because LTP was significantly reduced by a D1-type receptor antagonist. Thus, dopamine release is progressively compromised in R6/2 mice between 6 and 14 weeks (Johnson et al., 2006), dopamine levels are reduced in the striatum of R6/2 mice by at least 9 –12 weeks (Reynolds et al., 1999; Hickey et al., 2002), and D1 receptor binding is reduced in the striatum of R6/2 mice by at least 8 weeks of age (Cha et al., 1998, 1999). Dopamine depletion can also lead to a loss of corticostriatal synapses (Arbuthnott et al., 2000), which is consistent with the reduction in spine density in the R6/2 striatum observed by Klapstein et al. (2001). It would be interesting to attempt to restore LTP in the R6/2 mouse striatum with exogenous pro-dopaminergic agents in future studies, although functions that depend on phasic dopamine release cannot be readily restored with a dopamine agonist or dopamine precursor treatment (Schultz, 2002).

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Although an impairment in dopaminergic function might account for the observed deficit in LTP in R6/2 mice, we cannot rule out functional impairment in other neurotransmitter receptors in the striatum that might also be required for LTP, for example metabotropic glutamate receptors, adenosine receptors, or cholinergic receptors (Lovinger et al., 2003). Dopamine-dependent impairments in synaptic plasticity have been observed in the cerebral cortex of the R6/1 mouse model of HD (Cummings et al., 2006), indicating that altered dopaminergic function might be a widespread feature of HD. However, the meso-striatal and meso-accumbens dopaminergic pathways are particularly important in the motor, cognitive and emotional functions of the brain, and deficits here are likely to contribute to many of the frontostriatal deficits observed in HD mice. Alterations in dopaminergic function have also been observed in HD patients (Reisine et al., 1977; Antonini et al., 1996; Augood et al., 1997; Jakel and Maragos, 2000). Together with existing data, our findings suggest that dopamine-dependent neuronal plasticity may be compromised in mouse models of HD. The loss of dopamine-dependent striatal LTP in R6/2 mice in particular may contribute to impairments in motor and cognitive functions that depend on intact corticostriatal circuits. If this deficit extends to HD patients, this could account for observed deficits in procedural learning tasks (Packard and Knowlton, 2002). Acknowledgments—This work was supported by the Hereditary Disease Foundation USA, HighQ Foundation and CURE HD, USA; the Physiological Society and the Durham Fund, Kings College Cambridge (V.W.S.K.) and the Wellcome Trust (R.H.) provided vacation studentships.

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(Accepted 23 March 2007) (Available online 2 May 2007)