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Human Molecular Genetics Advance Access originally published online on April 6, 2006
Human Molecular Genetics 2006 15(10):1690-1703; doi:10.1093/hmg/ddl092
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Early development of aberrant synaptic plasticity in a mouse model of Huntington's disease

Austen J. Milnerwood1,{dagger}, Damian M. Cummings1,{ddagger}, Glenn M. Dallérac1,2, Jacki Y. Brown2, Sarat C. Vatsavayai1,2, Mark C. Hirst1,2, Payam Rezaie1,2,3 and Kerry P.S.J. Murphy1,2,*

1Huntington's Disease Research Forum and 2Department of Biological Sciences, The Open University, Milton Keynes, UK and 3Department of Neuroscience, Institute of Psychiatry, King's College London, London, UK

* To whom correspondence should be addressed at: Department of Biological Sciences, The Open University, Milton Keynes MK7 6AA, UK. Tel: +44 1908652917; Fax: +44 1908654167; Email: k.murphy{at}open.ac.uk

Received December 14, 2005; Accepted March 29, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive motor, psychiatric and cognitive decline. Marked neuronal loss occurs in the cortex and striatum. HD is inherited in an autosomal dominant fashion and caused by a trinucleotide repeat expansion (CAG) in the gene encoding the protein huntingtin. Predictive genetic testing has revealed early cognitive deficits in asymptomatic gene carriers at a time when there is little evidence for cell death, suggesting that impaired cognition results from a cellular or synaptic deficit, such as aberrant synaptic plasticity. Altered hippocampal long-term potentiation has been reported in mouse models of HD; however, the relationship between synaptic dysfunction and phenotype progression has not previously been characterized. We examined the age-dependency of aberrant hippocampal synaptic plasticity in the R6/1 mouse model of HD. Long-term depression (LTD) is a developmentally regulated form of plasticity, which normally declines by early adulthood. Young R6/1 mice follow the same pattern of LTD expression as controls, in that they express LTD in the first weeks of life, and then lose the ability with age. Unlike controls, R6/1 synapses later regain the ability to support LTD. This is associated with nuclear localization of mutant huntingtin, but occurs months prior to the formation of nuclear aggregates. We present the first detailed description of a progressive derailment of a functional neural correlate of cognitive processing in HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) is an autosomal dominant, late-onset, progressive and fatal neurodegenerative disease. The causal mutation is a CAG triplet repeat expansion in the open-reading frame of exon 1 of the coding gene (IT15) (1Go), which results in an expanded tract of polyglutamine residues in the protein huntingtin. Clinically, HD is characterized by a severe movement disorder and cognitive and emotional disturbances (reviewed in 2Go). Currently, there is no cure and no effective treatment against onset or progression, and following 15–20 years of inexorable decline, the disease is terminal.

Progressive neurodegeneration occurs with selective cell loss seen in striatum (3Go), cortex (4Go) and hippocampus (5Go) as well as a generalized atrophy of the majority of brain structures (6Go). There is mounting evidence that cognitive deficits are apparent in HD gene carriers many years prior to the onset of classical symptoms (7Go–13Go). Furthermore, some postmortem studies have shown limited signs of pathology despite substantial clinical evidence of HD (3Go), suggesting that neuronal dysfunction rather than cell death may significantly underlie the neurological manifestation of the HD mutation.

Several transgenic and knockin mouse models of HD have been produced, which variously recapitulate many of the disease characteristics, in which the severity of phenotype is inversely proportional to HD transcript length and directly proportional to polyglutamine repeat load and expression levels (reviewed in 14Go).

The hippocampus is a brain region known to be critical to the formation of episodic memory in man, other primates (15Go) and rodents (16Go), and the hippocampo-fronto-striatal pathway is involved in higher cognitive tasks such as goal-directed behaviour and executive function (17Go). Hippocampal function has been assessed in transgenic (18Go,19Go) and knockin (20Go) mouse models of HD, and in each study, basal neurotransmission at hippocampal synapses (CA3–CA1 field excitatory post-synaptic potentials, field EPSPs) appeared normal, whereas long-term potentiation (LTP) was reduced (14Go). Activity-dependent alterations in synaptic efficacy (synaptic plasticity), such as LTP and long-term depression (LTD), are widely believed to underlie information processing and storage in the brain (16Go).

The R6/2 mouse (21Go) is one of the best characterized transgenic models of HD and has been widely employed in the search for potential therapeutics. We have previously reported altered synaptic plasticity in R6/2 mice (19Go); synaptic deficits were observed at all ages studied (5–18 weeks), as were impairments in spatial learning (7 weeks onwards) and the formation of neuronal intranuclear inclusions (NIIs) (3 weeks onwards). These events were observed prior to the onset of an overt phenotype (hind limb clasping and weight loss), consistent with the view that early cognitive decline in HD is due to dysfunctional plasticity (14Go). The most conspicuous characteristic of R6/2 plasticity was a N-methyl-D-aspartate (NMDA) receptor-dependent form of LTD induced by low-frequency stimulation (LFS), a form of plasticity that was entirely absent in age-matched controls. Taken together, these findings suggest that a tendency towards synaptic depression may underlie a synaptic phenotype and the cognitive decline seen in presymptomatic HD patients. However, the extremely swift progression of the R6/2 phenotype hinders dissection of the temporal relationship between the causal HD mutation and alterations in synaptic plasticity, behaviour, cognition and the formation of NIIs.

In the present study, we have further characterized the R6/1 line, in which the mice have a longer life span and exhibit a slower phenotypic progression compared with R6/2 mice (21Go). We have shown that R6/1 mice, after a period of near-normal synaptic development and maturation, develop a highly conspicuous form of NMDA receptor-dependent LTD that becomes evident at ~12 weeks of age. This occurs after nuclear accumulation of mutant huntingtin peptide (but before NII formation) and many weeks prior to the onset of hind limb clasping and weight loss. At advanced ages, intrinsic membrane properties and action potential (AP) characteristics were also altered in R6/1 hippocampal neurones.

This study demonstrates that HD synapses are dysfunctional in the mechanisms believed to underlie cognition and memory many months prior to onset of the classic phenotype and prior to the formation of intranuclear inclusions. Furthermore, this report demonstrates that the R6/1 mouse may be better suited than the R6/2 mouse as a model in which to assess the early effects of the HD mutation, while retaining the advantage of a progressive and well-defined phenotype. These findings have major implications for current drug development strategies, particularly those aimed at interfering with aggregate formation and modulation of NMDA receptor function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Development of the overt R6/1 phenotype
In agreement with the initial report (21Go), the presence of the transgene produced a delayed phenotype in R6/1 mice, appearing at ~20 weeks of age (5 months). As shown in Figure 1A, wild-type (WT) mice spread their hind- and forelimbs when suspended by the tail and generally attempted to effect an escape. Conversely, R6/1 mice developed a tendency to curl their torso and grasp their hind limbs for brief periods. Clasping became progressively more pronounced and, occasionally, older mice maintained the posture for brief periods following return to the home cage. The age profile of the development of clasping is shown in Figure 1B, the majority (>80%) of R6/1 mice demonstrated clasping at 19–23 weeks of age (19 of 23 mice), and by 26 weeks, nearly all animals (>90%) were recorded as clasping (63 of 66 mice). The earliest incidences were observed in one animal at 13 weeks and another at 15 weeks. A divergence in body weight was also observed as animals aged (Fig. 1C). By 20 weeks of age, R6/1 males weighed nearly 20% less than WT littermates. Weight loss in R6/1 mice was also progressive, with male R6/1 mice weighing significantly less at 32 weeks than at 20 weeks and nearly 40% less than aged-matched WT littermates.


Figure 0921
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  		Figure 1. Development of the overt R6/1 phenotype. (A) Hind limb clasping in R6/1 mice. Photographs show typical response of WT littermate and R6/1 mouse (both aged 32 weeks) to tail suspension. (B) Late onset and progressive development of clasping. The percentage of animals showing hind limb clasping (see Materials and Methods) is plotted against age. From 19 weeks >80% of R6/1 mice demonstrate clasping (filled circles, n=153 total observations). Clasping was first detected in one individual R6/1 animal at 15 weeks and another at 13.5 weeks. The trait was not observed in WT littermates (open circles, n=162 total observations). (C) Late onset and progressive weight loss. Mean animal weight (±SEM) is plotted against age. R6/1 mice (filled circles, n=79, males only) weighed significantly less than age-matched WT littermates (open circles, n=98, males only, ***P<0.001) from 20 weeks onwards. Weight loss was progressive ({dagger}{dagger}{dagger}P<0.001).

 
Region-specific, progressive nuclear accumulation of transgenic huntingtin peptide and the formation of NIIs
Figure 2A shows the pattern of localization of mutant huntingtin found within the hippocampus of R6/1 animals with progressive age, demonstrated using immunohistochemistry with the S830 antibody. In 1-month-old R6/1 animals, diffuse huntingtin immunoreactivity was detected largely within pyramidal cells of the CA1 area (stratum pyramidale), and in the subiculum, whereas little or no immunoreactivity was evident in other hippocampal regions. At 3 months, the mutant protein was also detected within a significant proportion of cells located in the CA3 region, whereas the granule cells of the dentate gyrus (DG) exhibited little immunoreactivity. By 7 months, there was clear staining observed within the stratum granulosum of the DG in R6/1 mice. Also, by this age, it was noted that cells within the thalamus and cerebral cortex were also heavily stained for the mutant protein (data not shown).


Figure 0922
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  		Figure 2. Progressive, age-dependent and cell-specific nuclear accumulation of mutant huntingtin and the formation of NIIs. (A) S830 immunolabelling in R6/1 hippocampus. Representative photomicrographs of the hippocampus immunolabelled with S830 antibody, from WT (1 month) and R6/1 (1, 3 and 7 months) mice as indicated. Nuclei counterstained with Harris' haematoxylin solution. CA1, CA3 and DG are marked for reference (top left). Sections from WT littermates showed a lack of immunoreactivity with S830 at all ages examined. In contrast, mutant huntingtin was detected as a dark (diffuse) reaction product, predominantly located within the stratum pyramidale of area CA1 in R6/1 mice aged 1 month, whereas little or no localization was detected in either the CA3 or the DG stratum granulosum. At 3 months, immunoreactivity was observed in a proportion of cells within area CA3, and by 7 months, labelling was apparent throughout all three principal regions of the hippocampus in R6/1 mice. (B) Nuclear localization of mutant huntingtin. Representative higher magnification photomicrographs of hippocampal regions immunoreacted with S830 antibody (brown). Nuclei counterstained with Harris' haematoxylin solution (blue). Mutant huntingtin was not detected in sections from WT littermates at any age (representative example top left panel, 1 month). Nuclear localization was observed in R6/1 CA1 cells at 1 (top right) and 3 months (middle left). At 7 months of age, in addition to a marked increased in nuclear staining within area CA1 (middle right), area CA3 and the DG, a significant proportion of cells in all three areas exhibited NIIs (arrows; CA1, middle right; CA3, bottom left; DG, bottom right) in R6/1 mice. (C) Corresponding images from the R6/1 hippocampal CA1 fields at 1 and 7 months of age (top and middle panels), indicating the optical density segmentation constant (used to quantify immunoreactivity) as red overlay. Note the specificity of the segmentation for the immunoreaction product (areas devoid of immunoreactivity are not highlighted by segmentation). Bar graph (lower panel) represents the mean % area of immunoreactivity per field [10 observations from each of three R6/1 and WT animals in each age group (n=18)±SD, ***P<0.001].

 
This progressive accumulation of mutant huntingtin within defined somatic subfields of the hippocampus (CA1>CA3>DG) is similar to that previously reported for the appearance of NIIs in R6/2 mice (19Go,22Go).We separately noted that pyramidal cells in layer II/III of the perirhinal cortex of R6/1 mice showed significant somatic staining at 3 months, and by 7 months, expression of the mutant protein had extended to all cortical layers within this region (data not shown).

Figure 2B shows higher magnification photomicrographs of immunoreactivity within hippocampal subfields. In contrast to the distinct lack of labelling in WT littermates, marked diffuse nuclear huntingtin immunoreactivity was demonstrated in ~20–30% of CA1 pyramidal neurones in 1-month-old R6/1 mice, but not in other hippocampal cell types. Immunoreactivity was also detected in primary apical dendrites of CA1 neurones. Diffuse nuclear immunoreactivity was detected in a greater percentage (~40–60%) of CA1 pyramidal neurones in 3-month-old R6/1 animals and also observed in a proportion (~20–30%) of nuclei within the stratum pyramidale of CA3. NIIs were not apparent in any hippocampal region at this age. By 7 months of age, in addition to the high prevalence of diffuse nuclear immunoreactivity (estimated at >90% of CA1, CA3 and DG granule cells imaged), dense spherical aggregates (NIIs, see arrows on Fig. 2B) were also apparent within the majority of immunolabelled cell nuclei in all three principal subfields. Likewise, NIIs were also localized within cortical pyramidal neurones, as well as neurones within the striatum, thalamus and midbrain of R6/1 mice at 7 months.

As shown in Figure 2C, quantitative image analysis clearly demonstrated a progressive accumulation of nuclear huntingtin [red–green–blue (RGB) threshold setting constant indicated as red overlays on corresponding images] within hippocampal area CA1, with increasing age in R6/1 mice. This (highly significant) increase in the fractional area of nuclear immunoreactivity describes the progressive increase in the number of CA1 cells exhibiting nuclear huntingtin staining, rather than increased nuclear staining within a subpopulation of cells.

Therefore, in R6/1 mice, diffuse nuclear accumulation of mutant huntingtin occurs at a very young age (before 1 month), and this accumulation is cell type and brain region specific. NIIs first appear at some time after 3 months within the hippocampus and cerebral cortex and have formed within the vast majority of neurones by 7 months of age.

Basal synaptic transmission
To ascertain whether gross phenotypic progression is associated with alterations in basic synaptic transmission, recording of field EPSPs was employed to assess input/output (I/O) relationships (a general measure of synaptic efficacy) and paired-pulse facilitation (PPF) ratios (a measure of presynaptic function and neuromodulation) in acute slices prepared from R6/1 mice compared with those from WT littermates over a variety of ages.

There were no significant differences between R6/1 and WT littermate I/O curves at 1, 3 and 6 months of age (Fig. 3A). However, at 8 months, there was a significant increase in the R6/1 field EPSP slope at stimulation intensities between 100 and 300 µA relative to age-matched control slices (Fig. 3A). Importantly, differences in the I/O relationship were only observed after the onset of a neurological phenotype.


Figure 0923
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  		Figure 3. Normal basic synaptic transmission and aberrant synaptic plasticity in slices from R6/1 mice. (A) Basic synaptic transmission. Left: graphs show pooled slope means±SEM plotted against stimulus intensity (I/O curve) for ages indicated. There was no significant difference between R6/1 (filled circles) and WT littermate (open circles) I/O curves at 1, 3 or 6 months of age. However, at 8 months, there was a significant difference between WT littermates and R6/1 [for WT and R6/1, respectively: 1 month n=12(2), 20(3); 3 months n=10(2), 20(5); 6 months n=16(3), 16(3) and 8 months n=18(6), 38(10), *P<0.05, **P<0.01]. Right: graphs show pooled PPF ratios (±SEM) plotted against the inter-pulse interval. PPF ratios were similar for both WT littermates (open circles) and R6/1 (filled circles) at all ages examined (for WT and R6/1, respectively: 1 month n=12(2), 17(3); 3 months n=11(2), 21(5); 6 months n=13(3), 21(8) and 8 months n=18(6), 38(10)]. (B) Reversible LTD in R6/1 adult slices. Graphs [in (B–D)] show pooled mean slope values expressed as a percentage change in slope with respect to the control period, plotted against time (for clarity, in this and all other figures, SEM is plotted for every fifth datum). Following baseline recording, LFS [indicated by a bar in (B–D)] induced LTD in slices from adult (6 months) R6/1 mice [filled circles, –10.4±2.3% (measured 55–60 min post-LFS), n=8(3); P<0.02], whereas no significant change was observed in age-matched WT slices [open circles, 6.2±3.6%, n=5(2); R6/1 versus WT, P<0.001]. At 1 h post-LFS, tetanic stimulation (arrow) induced LTP in both control (30.4±7.4%, P<0.05) and R6/1 slices (35.2±7.5%, P<0.001, relative to the last 5 min prior to tetanus). (C) LTD in adult R6/1 slices is long-lasting and not saturated. LFS induced stable LTD of the EPSP slope in slices from adult (6 months) R6/1 mice [filled circles, –14.3±5.5% (P<0.05) and –16.3±5.5% (P<0.001) measured 1 h and 2 h post-conditioning, respectively, n=6(2)]. A second period of LFS induced a further reduction of the EPSP slope [–12.8±3.2%, n=6(2), P<0.03, measured 55–60 min after the second LFS]. In contrast, in slices prepared from adult WT animals [open circles n=4(3), 8 months], LFS did not produce a change in EPSP slope after one episode or following a second episode of LFS. (D) LFS-induced LTD in R6/1 adult slices is input-specific. Field EPSPs were evoked in single slices by stimulation of two independent afferent pathways. LTD was only induced in the pathway that received LFS conditioning [filled circles; –10.4±2.4%, n=5(4), P<0.05]. No change was observed in control pathways [open triangle; 2.5±3.8%, n=5(4), P>0.6, slices from animals aged 3–6 months]. In (B–D), traces are averages of five consecutive responses corresponding to the time points indicated by roman numerals. The scale bars in (B and D) indicate 5 ms and 5 mV.

 
No significant differences in PPF ratios were seen between R6/1 and WT littermates at any age studied (1, 3, 6 and 8 months) (Fig. 3A). Statistical analysis of all factors by three-way ANOVA did establish significant age effects within genotype (P<0.0001, F21,1152=2.686, ANOVA with Fisher LSD post hoc test), due to highly significant reductions in the PPF ratio between 1 and 8 months of age for both R6/1 and littermate controls. Overall, these data suggest that basic synaptic transmission in R6/1 animals is similar to that of WT littermates, although at advanced ages (8 months), long after the onset of gross phenotype, I/O relationships differ.

LTD-induced in adult R6/1 slices is reversible, input-specific, stable, cumulative and expressed at both the population spike and dendritic EPSP
LFS of CA3–CA1 hippocampal synapses revealed that, similar to R6/2 animals (19Go), LTD was induced in slices prepared from adult (6 months) R6/1 animals, but not aged-matched WT littermates (Fig. 3B). LFS induced a significant reduction from baseline in R6/1 EPSPs, whereas no significant change was seen in EPSPs in WT slices. The outcome of LFS conditioning was significantly different between transgenic and non-transgenic slices.

The induced LTD was readily reversed by tetanic stimulation to give LTP in R6/1 slices of a similar magnitude to that achieved in WT animals. Although there is no significant difference in the magnitude of the relative increase produced by tetanic stimulation in R6/1 and WT slices (P>0.6), a direct comparison is hindered by the difference in the size of the EPSP at the point of LTP induction. Nonetheless, the actual increase in absolute EPSP slope induced by tetanic stimulation in R6/1 slices was not significantly different from that induced in WT slices (0.83±0.1 and 1.1±0.2 V/s, respectively, P>0.4). This observation confirms that LTD expressed in adult R6/1 slices is not attributable to afferent damage and, importantly, that the mechanisms responsible for LTP induction are, at least, partially functional in adult R6/1 mice.

As shown in Figure 3C, LFS-induced LTD in adult R6/1 slices is stable and lasts for at least 2 h following LFS and a second episode of LFS induced a further significant reduction in EPSP slope. As expected in slices prepared from adult WT animals, the first period of LFS failed to induce LTD, as did the second application of LFS 2 h later, suggesting that LFS in WT slices does not have an additive or priming effect. These data demonstrate that in slices from adult R6/1 animals LFS-induced LTD lasts for at least 2 h and that successive bursts of LFS can incrementally reduce synaptic weighting, whereas no such reductions are seen in WT slices with successive periods of LFS.

Independent pathways were isolated (determined by the absence of heterosynaptic PPF) in order to investigate the synapse specificity of LFS-induced LTD in adult R6/1 slices. As shown in Figure 3D, two pathway experiments demonstrated that LTD was only induced in the pathway that received LFS, whereas no change was seen in the control (unconditioned) pathway. The two pathways are significantly different from one another (paired t-test, P<0.005), demonstrating that LTD in R6/1 slices is not due to a generalized decrease in slice viability and that the reduction seen in the post-synaptic response is an input-specific result of LFS conditioning.

Delayed onset of the R6/1 synaptic phenotype
Having established that LTD was reliably induced in slices from adult R6/1 animals, experiments were performed to determine whether LFS-induced LTD in R6/1 slices was a persistence of early developmental LTD as seen in non-transgenic animals (23Go) or the emergence of abnormal plasticity following a period of normal hippocampal development.

As shown in Figure 4A, at 0.5 months, LFS induces a significant depression in both R6/1 and WT littermate slices and there is no difference between the resultant LTD between genotype (P>0.6). As predicted by the normal developmental profile of LTD (23Go), at 1 month of age, LFS does not induce a significant change in EPSP slope in WT littermate or R6/1 slices, and the result of LFS between the two genotypes is not significantly different (P>0.1), suggesting that at this age the developmental profile of synaptic plasticity is normal in R6/1 mice.


Figure 0924
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  		Figure 4. Delayed onset of the R6/1 synaptic phenotype. (A) Induction and expression of LTD. LFS (bar) induced LTD of EPSPs recorded in slices prepared from both R6/1 and WT littermate animals at 0.5 months of age [–18.0±3.7%, n=6(3), P<0.01 and –15.5±5.0%, n=7(3), P<0.05, respectively]. LFS (bar) did not induce significant LTD in slices from juvenile (1-month-old) R6/1 mice [–6.5±3.9%, n=16(3), P>0.3] or WT littermates [–0.5±1.8%, n=11(2), P>0.1]. In aged animals (6 months), LFS did not induce LTD in slices from WT littermates [4.7±3.1%, n=11(3), P>0.1], but did in R6/1 slices [–10.8±2.1%, n=23(8), P<0.001]. (B) LTD as a function of age. Top: the magnitude of LTD induced in slices from R6/1 [filled circles, n=119(40)] and WT littermates [open circles n=164(67)] is shown as a scatter plot against postnatal age (days). Middle: age-grouped data (mean LTD±SEM plotted against age) demonstrates that in WT slices LTD is initially expressed [0.5 months, n=83(37)] and then rapidly downregulated so that it is absent by the end of the first postnatal month [1 month, n=30(12)]. A similar pattern of LTD expression is seen in the R6/1 hippocampus [0.5 months, n=6(3); 1 month, n=16(3)]. However, at 2 and 3–4 months, the response to LFS is significantly altered in R6/1 slices with respect to WT [2 months: n=10(4) and n=6(3), respectively; 3–4 months: n=23(7) and n=12(4), respectively; ***P<0.001]. At 6 and 8 months, there are also highly significant differences in the response to LFS between R6/1 [6 months: n=23(8); 8 months: n=41(13)] and WT slices [6 months: n=11(3), 8 months: n=27(8), ***P<0.001] with transgenic slices exhibiting highly significant reductions from baseline ({dagger}{dagger}{dagger}P<0.001). Bottom: the probability of LTD induction is plotted against age (LTD is defined as depression of >10% measured 55–60 min post-conditioning).

 
In slices prepared from animals aged 6 months, there is no significant change produced by LFS in WT littermates, whereas at this age a highly significant reduction in EPSP slope is seen in R6/1 slices, an effect that is even more pronounced in slices prepared from animals aged 8 months [R6/1: –12.1±1.4%, n=41(13), P<0.0001; WT: 0.8±1.9%, n=27(8), P>0.4].

A population comparison of the outcome LFS conditioning between R6/1 and WT mice is shown as a scatter plot in Figure 4B (top). The plot shows the tendency for LFS to induce LTD in young animals regardless of genotype and to induce LTD only in adult mice that are transgenic. Age-grouped data (Fig. 4B, middle) demonstrates that there is no significant difference in the effects of LFS between R6/1 and WT animals at 0.5 months of age or 1 month of age. In slices prepared from animals aged 2 months onwards, there are highly significant differences in outcome between R6/1 and WT animals.

The probability of LTD induction is also shown in Figure 4B (bottom). In slices prepared from animals at 0.5 months, the probability of LFS inducing LTD is high for both genotypes (R6/1 83%; WT 68%). By 1 month of age, the probability of LTD induction fell dramatically for WT slices (10%) and the likelihood of LFS successfully inducing LTD in R6/1 mice was reduced by more than half (38% of experiments). LTD was not induced in any experiments in WT slices between 2 and 6 months of age and LTD was only observed in two experiments (<8%) in the 8 months age group. Conversely, in R6/1 slices prepared at 2 months, 20% of experiments resulted in LTD and the likelihood of LTD induction increased to ~50% from 3 months onwards (3–4 months, 43%; 6 months, 48%; 8 months, 61%).

Together these data suggest that the R6/1 hippocampus undergoes near-normal (though possibly protracted) developmental plasticity over the first month of life, as evidenced by the downregulation of LFS-induced LTD. However, in slices taken from animals aged 2 months, the response to LFS is altered in animals expressing the mutant peptide. By 3–4 months, markedly aberrant synaptic plasticity in transgenic mice is highlighted by the conspicuous re-emergence of LFS-induced depression, indicating overt synaptic dysfunction at this age that becomes progressively more pronounced.

Transgenic LTD is NMDA receptor-dependent
As shown in Figure 5A, in the presence of 50 µM of the NMDA receptor antagonist D-AP5 (24Go), LFS in R6/1 slices did not result in a significant reduction in EPSP slope. After washout of D-AP5, a second period of LFS induced a robust, stable and significant reduction in responses evoked in the same pathways that previously failed to express LTD. Thus, similar to LFS-induced LTD in the developing mouse hippocampus (23Go), LTD in R6/1 adult slices is reversibly blocked by NMDA receptor antagonism and is therefore NMDA receptor-dependent.


Figure 0925
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  		Figure 5. LTD in R6/1 adult slices in NMDA receptor mediated. (A) NMDA receptors and LTD in aged (8-month-old) mice. Mean EPSP slope (percentage change) is plotted against time. Blockade of NMDA receptors, in the presence of the antagonist, D-AP5 (50 µM, filled bar), prevents the induction of LTD in slices prepared from 8-month-old transgenic animals [2.2±5.4%, n=6(3), P>0.9, measured 55–60 min after first period of LFS]. Following washout of D-AP5, a second period of LFS conditioning successfully induced LTD [–17.9±1.5%, n=6(3), P<0.0001]. (B) NMDA receptor pharmacology and LTD in WT and R6/1 slices. Bar graph summarizes the action of the NMDA receptor antagonists D-AP5 and ifenprodil on the induction of LTD (**P<0.01, ***P<0.001).

 
A summary of the effects of pharmacological manipulation of the NMDA receptor and LFS in slices prepared from adult mice (6–8 months) is given in Figure 5B. In WT littermate control slices, the EPSP slope returned to a value that was not significantly different from the baseline by 30 min post-conditioning [–3.0±1.1%, n=38(11), P>0.1]. In contrast, in R6/1 slices, the EPSP slope was significantly depressed [–14.9±0.9%, n=62(21), P<0.00001]. In R6/1 slices, LTD was reversibly blocked by D-AP5 (in D-AP5: 3.6±5.6%, P>0.9; after washout of D-AP5: –18.0±1.4%, n=6(3), P<0.0001; wash versus D-AP5 P<0.001, 30 min post-LFS].

No difference was observed in the response to LFS by WT littermate slices in the presence of the NMDA receptor NR2B subunit-specific (25Go,26Go) antagonist ifenprodil [–3.7±2.0%, n=12(2), P>0.1]. In contrast, the degree of depression induced by LFS in R6/1 slices in the presence of ifenprodil [–8.7±1.9%, n=12(6), P<0.001] was significantly reduced compared with the level of depression seen in the absence of the drug (41% less depression, P<0.002). These results suggest that NR2B subunit containing NMDA receptors contribute to the induction of LFS-induced LTD in R6/1 transgenic mice.

Alterations in CA1 pyramidal cell intrinsic membrane properties and LTD
Intracellular recordings from hippocampal CA1 neurones demonstrated that in slices from 8-month-old R6/1 animals CA1 pyramidal cells have altered membrane and AP properties in comparison to age-matched control cells, as shown in Figure 6 and Table 1. (Recordings made in slices prepared from transgenic animals aged 4 months and younger were similar to those made in control slices; data not shown.) The passive membrane properties and AP characteristics of the WT cells recorded here are similar to those reported in the literature (19Go). Although there were no significant differences between the mean resting membrane potential of R6/1 and WT cells in this small sample, a population of R6/1 cells (57%) were depolarized (less than –65 mV). Input resistance was increased in R6/1 cells when calculated from both the maximal (28%) and the steady-state potentials (33%), but only approached significance (P=0.06). The membrane time constant was significantly reduced in R6/1 cells when calculated from both max (38%, P<0.003) and steady-state potentials (39%, P<0.002), as was the membrane capacitance (Cm) of R6/1 cells when calculated from both max (49%, P<0.005) and steady-state potentials (54%, P<0.002). Although there was no significant difference in AP threshold potential, APs were significantly smaller in R6/1 cells, as shown by a reduced AP overshoot (25%, P<0.05), amplitude (9%, P<0.05) and half-width (measured at 50% max amplitude; 16%, P<0.02). The data suggest that at this advanced age (8 months), CA1 neurones in R6/1 mice display a markedly altered physiology, including stunted APs, increased membrane resistance, reduced time constant and capacitance, and that a population of cells are depolarized. These observations are consistent with a global change in a membrane property such as altered potassium or/and sodium conductance. Interestingly, changes in potassium channel number have recently been reported in symptomatic R6/2 mice (27Go). Although alterations in sodium channel activity has not been described in HD models, mRNA analysis has suggested reductions in sodium channel expression in symptomatic R6/2 mice (28Go,29Go).


Figure 0926
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  		Figure 6. Membrane properties and AP characteristics are altered at 8 months. (A) Resting membrane potential. Bar graphs show the distribution of resting membrane potentials recorded in slices prepared from 8-month-old WT and R6/1 animals. (B and C) Input resistance. (B) Membrane responses (top) to injection of hyperpolarizing square current pulses (bottom); each trace is an average of three consecutive responses. WT cell (RMP of –69 mV) shown on the left and transgenic (RMP of –61 mV) shown on the right. Both maximal (filled arrow) and steady-state (open arrow) voltages were used to determine input resistance (Rin) as plotted in (C). (D) Response to depolarizing current injection. Typical responses (top) to a 0.4 nA square pulse current injection (bottom); same cells as (B and C).

 

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  		Table 1. Passive membrane and AP properties of CA1 hippocampal neurones are altered in slices from 8-month-old R6/1 mice
 
Intracellular examples of LTD recorded in R6/1 CA1 neurones in slices prepared from adult animals are shown in Figure 7. The first example in Figure 7A is recorded in a slice taken from a 4-month-old R6/1 mouse. The test shock intensity was set to produce an EPSP at ~60% of that required to elicit an othrodromic AP and subsequent LFS induced a stable depression (87.6% reduction in EPSP amplitude.). The second example in Figure 7B is a recording made in a CA1 neurone in a slice prepared from an 8-month-old R6/1 mouse. Note that in this example the resting membrane potential was depolarized (–58 mV). The test shock intensity was again set to produce a synaptic potential at ~60% of that required to reach the AP firing threshold, and in this instance, the resultant potential consisted of both an early EPSP followed by inhibitory post-synaptic potential. LFS induced a stable LTD in the early excitatory component of the synaptic potential (–56.9±11.0% SD change in amplitude). In this recording, a second recording electrode was placed in the stratum radiatum to monitor the corresponding field EPSP which also exhibited LTD (–28.4±2.4% SD change in EPSP slope). Importantly, these results demonstrate that LTD can be induced in transgenic cells that do not have a depolarized RMP and that the LTD induced is specific to glutamatergic neurotransmission.


Figure 0927
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  		Figure 7. Intracellularly recorded LTD in R6/1 adult hippocampus. (A) Intracellular LTD in a slice prepared from a 4-month-old R6/1 mouse. LFS (bar) induced LTD of the EPSP amplitude (filled circles). Inserts are traces of the intracellular EPSP from the points indicated (averages of five sweeps). (B) Dual recording in a slice prepared from an 8-month-old R6/1 mouse. LFS (bar) induced LTD in both the intracellular EPSP amplitude (filled circles) and associated field EPSP slope (open triangles). Inserts are traces of the intracellular EPSP and field EPSP from the points indicated (are averages of five sweeps). The scale bars represent 10 ms and 5 mV in both (A and B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have shown that following an initial period of normal development, progressive synaptic dysfunction occurs several months prior to the manifestation of classical neuropathological and neurological phenotype in R6/1 mice.

Motor dysfunction and a progressive reduction in body mass (characteristics of the clinical phenotype of HD) developed in the R6/1 mice at 20 weeks (5 months), in agreement with previous reports (21Go,30Go). NIIs, which occur both in HD (31Go) and transgenic mouse models (14Go), are observed in brain regions known to be susceptible to neurodegeneration. We observed NIIs in R6/1 hippocampal areas CA1, CA3 and DG, as well as throughout the cortex and striatum at 7 months. However, we also observed region- and age-specific diffuse accumulation of the huntingtin peptide within cell nuclei using an antibody (S830) targeting the N-terminal sequence of mutant huntingtin. Nuclear localization was first detected in hippocampal area CA1 at 1 month and later in area CA3, DG, striatum and cortex. The diffuse cellular/nuclear localization of mutant huntingtin correlates well with the early synaptic dysfunction we report here.

In truncated construct models, the cellular processing of huntingtin believed to be required for neurotoxicity (32Go) and nuclear accumulation (33Go) seen in full-length models is bypassed and is likely to explain the early nuclear localization seen in such models. The pattern of nuclear accumulation observed here is similar to that of inclusion formation in R6/2 mice; inclusions appear first in CA1 (but not in CA3) at 3 weeks of age (19Go,22Go). Although the rate of symptomatic onset in R6/2 is too swift to readily dissect the temporal relationship between behavioural abnormalities, neurophysiological alterations and inclusion formation (19Go), the current study demonstrates that inclusion formation is secondary to dysfunction, but correlates with the overt ‘clinical’ phenotype. In full-length and knockin models, behavioural deficits are observed prior to (or in the absence of) inclusion formation (18Go,34Go–36Go), suggesting that diffuse nuclear localization of mutant huntingtin and the presence of protein micro-aggregates within the nucleus and neuropil correlate better with early disease progression than NIIs.

An immediate question arising from this study concerns the defining characteristics of a CA1 pyramidal cell that lead it to first exhibit nuclear huntingtin localization. It is known that normal huntingtin shuttles into and out of the nucleus (33Go). Although the exact mechanism of huntingtin nuclear entry is still unknown, it has been reported that accumulation of mutant huntingtin fragments within the nucleus is associated with the loss of a nuclear export signal within the context of the full-length protein (33Go). It has been proposed that huntingtin might enter the nucleus via interactions with other proteins that do carry a classic nuclear localization sequence (33Go), such as calcium-binding protein (CBP). Huntingtin is known to interact with CBP (37Go), and CBP (expressed at highest levels within the hippocampus) (38Go) has been heavily implicated in hippocampal synaptic plasticity (39Go). It is plausible that the CA1 pyramidal cell, with its high turnover of plasticity-related molecules (such as CBP), is initially more vulnerable to nuclear huntingtin localization due to this and/or other related molecular processes. The interaction between mutant huntingtin and CBP also correlates with altered gene transcription (37Go,40Go) and as such may be an early contributor to abnormal cellular physiology, such as aberrant synaptic plasticity. Indeed, the temporal pattern of nuclear localization of mutant huntingtin in both the hippocampal subfields and the cortex correlates well with altered synaptic plasticity evident in these brain regions.

The most conspicuous alteration in synaptic function reported here is an age-dependent re-emergence of a developmentally regulated form of LTD in slices from R6/1 mice, which was fully apparent prior to the onset of an overt motor phenotype or formation of nuclear inclusions in these animals. In WT mice, this form of LTD is expressed in a highly age-dependent manner at the CA3–CA1 synapse. Crucially, the ability to support LTD is entirely lost at ~1 month of age (23Go). In this study, slices prepared from both WT and R6/1 mice aged <1 month expressed LTD. The ability to support LTD was diminished by 1 month of age, and neither WT nor R6/1 slices exhibited significant mean reductions from baseline following LFS conditioning. By 2 months of age, the result of LFS was significantly different in R6/1 slices from that observed in WT, and by 3–4 months of age, the pattern of plasticity had markedly diverged between littermate controls and transgenic animals, with LTD being a defining feature of hippocampal plasticity in R6/1 mice.

In young WT mice, LTD induction is dependent upon the activation of NMDA receptors (23Go). The induction of LTD in adult R6/1 animals was also shown to be entirely dependent upon the activation of NMDA receptors. Recently, it has been reported that LTD induction crucially requires the involvement of a class of NMDA receptors that contain the NR2B subunit (41Go). Moreover, it has been suggested that the decline in the ability of young animals to support LTD is attributable to a developmental decrease in the expression of these subunits (42Go,43Go). In this study, we show that ifenprodil, a selective NR2B subtype antagonist, reduced the magnitude of LTD in adult R6/1 slices by >40%, strongly suggesting that NR2B-containing NMDA receptors contribute to the aberrant plasticity observed in these mice. The concentration of ifenprodil used here is reported to block completely currents generated by NR1/NR2B receptor complexes but is only partly effective against NR1/NR2A/NR2B triheteromers (26Go); it is therefore possible that the residual LTD seen in R6/1 ifenprodil-treated slices is attributable to the latter.

The view that abnormalities in NMDA receptor signalling might underlie altered synaptic plasticity in the R6/1 hippocampus is supported by several observations. First, NMDA-induced whole cell currents in NMDA receptor transfected cell lines (specifically NR2B subunit-containing receptors) are increased by the presence of mutant huntingtin (44Go) and secondly, NR2B-mediated currents are enhanced in both cultured striatal neurones (45Go) and at cortico-striatal synapses in acute slices (46Go) from YAC transgenic HD mice. Furthermore, medium spiny neurones in striatal slices from YAC mice, transgenic N-terminal fragment-expressing HD mice (including R6/2) and knockin HD mice have been shown to exhibit enhanced responsiveness to NMDA application both after and prior to the onset of overt symptoms (47Go,48Go). This study is the first detailed description of a progressive derailment of a functional neural correlate of cognitive processing and one that is likely to be associated with altered NMDA receptor function in HD. Importantly, the re-emergence of aberrant LTD reported here coincides with the appearance of altered cognition in R6/1 mice reported elsewhere (49Go,50Go). Significantly, changes in LTD expression in R6/1 mice were fully manifest prior to the deterioration in the intrinsic electrophysiological properties of CA1 pyramidal cells, confirming that the functional deficit underlying augmented LTD is synaptic in origin.

Interestingly, the re-emergence of LTD may contribute, in an accumulative manner, to the loss of cell capacitance seen in CA1 neurones in slices prepared from symptomatic animals. Cell capacitance is an indirect measure of plasma membrane surface area and a decrease in capacitance is indicative of a loss of surface membrane (51Go). It has been reported that the induction of LTD is associated with a reduction in the size of dendritic spines (52Go), and hence, it is conceivable that the aberrant LTD reported here may, in part, contribute to a progressive and accumulative loss of cell membrane. Indeed, it has been recently reported that there is a decrease in spine density and dendritic spine length in both striatal medium spiny neurones and cortical pyramidal neurones in symptomatic R6/1 mice (53Go).

In summary, the central theme reported here is that the hippocampus in R6/1 mice progressively acquires a tendency towards synaptic depression after a period of near-normal developmental plasticity that is similar to that of WT littermates. Thus, unlike the R6/2 line, the R6/1 mouse exhibits a delayed synaptic phenotype (see schematic timeline in Fig. 8) and is therefore a more appropriate model of the human condition. Furthermore, the late onset of LTD in the R6/1 mouse suggests that it is a more fitting model in which to study the earliest indicators of disease onset, progression and the efficacy of therapeutic agents. Our results suggest that therapeutic strategies aimed at the intervention of aggregate formation may be ineffective as therapies in the treatment of early cognitive decline in HD. We suggest that strategies aimed at moderating NMDA receptor activity (and specifically directed at the NR2B subunit) could help to maintain normal synaptic plasticity and prevent or lessen cognitive decline in HD.


Figure 0928
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  		Figure 8. Comparison of R6/2 with R6/1 HD mice. A model describing the development of hippocampal aberrant synaptic plasticity, behavioural phenotype progression (points below bar) and biochemical/cellular alterations (points above bar) in R6 mice. Aberrant plasticity (LTD) seen in the R6/2 was expressed at the youngest age studied (5 weeks) (19Go). Weight loss and clasping occurred near to death (21Go), motor dysfunction (58Go), cognitive dysfunction (59Go) and NII formation (22Go) are recorded at similar ages. In R6/1 mice, decreased D1 receptor labelling is apparent at 1 month (Milnerwood et al., unpublished data), an age at which activity levels have been shown to be altered (60Go). Cognitive dysfunction has been reported at 10 weeks (49Go,50Go) and motor dysfunction at 14 weeks (54Go). At this age D1 and D2 receptor labelling is increased (within the hippocampus; Milnerwood et al., unpublished data) and LTD is expressed in ~50% of experiments, and by 16 weeks, CA1 cell properties are altered. At 20 weeks clasping and weight loss are evident, NII were detected at 28 weeks (7 months).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Transgenic mice
The founders of the Open University R6/1 colony were a gift from the University Laboratory of Physiology, Oxford University, UK, courtesy of Dr Anthony Hannan and Dr Anton van Dellen. Hemizygotic R6/1 males (21Go) were mated with CBAxC57BL/6 females and housed in an enriched environment (54Go). At weaning (3 weeks), all mice were given identity marks and tail-tip samples were taken for DNA extraction and genotyping by transgene PCR (21Go) and were analysed for CAG length (all mice used in this study were confirmed to have a CAG length of ~116Q) (Vatsavayai et al., submitted for publication). For experiments using slices from animals aged <3 weeks, DNA sample were taken postmortem.

Assessment of the onset of clinical phenotype
Mice were periodically weighed and the onset of motor disturbance was assessed by tail suspension. Mice were suspended for 15 s per trial and considered positive for clasping if they demonstrated a hind limb clasp in three or more of 10 consecutive trials.

Immunohistochemistry to detect mutant huntingtin
R6/1 and age-matched WT littermate mice (three animals of each genotype at three age points, total 18) were sacrificed by cervical dislocation and immediate decapitation in accordance with the UK legislation [Animal (Scientific Procedures) Act 1986]. Brains were rapidly removed and 400 µm coronal slices were prepared on a vibratome (Campden Instruments Inc., USA). Slices were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich Company Ltd, UK), then in 2% PFA overnight before transfer to 0.1M phosphate-buffered saline (PBS) (pH 7.4) and stored at 4°C. Slices were temporarily mounted in 5% agar and re-sectioned at 50 µm thickness before re-immersion in PBS, in preparation for immunohistochemistry. Free-floating sections were blocked/permeabilized (2% Fish gelatine, 0.01% sodium azide, 0.1% Triton X-100 in PBS), and endogenous peroxidase activity was quenched (3% H2O2 30 min) prior to further rinsing. Sections were then treated with primary antibody (1:2000 dilution of S830 sheep polyclonal antibody) raised against the product of the N-terminal region to 53 glutamine residues of exon 1 of the human gene (a gift from Professor Gillian Bates, Department of Medical and Molecular Genetics, GKT School of Medicine, King's College London, UK), and left to incubate overnight under constant agitation at 4°C. Slices were again washed, then exposed to secondary antibody (1:500 dilution of biotinylated anti-sheep IgG, Vector Laboratories, UK) and left to incubate for 1 h at room temperature. The immunoperoxidase reaction was performed using avidin–biotin complex (Vectastrain Elite kit, Vector Laboratories Ltd, UK) and diaminobenzidene (0.05% in TBS+10 µl 30% H2O2) as chromogen. Nuclei were counterstained with Methyl green or Harris' haematoxylin. Sections were dehydrated in a graded series of alcohols, cleared in xylene and cover slipped using DPX as mountant. Negative control sections were included, where the primary antibody was omitted.

Quantitative image analysis
Image analysis was performed in order to assess the relative levels of immunolabelled mutant huntingtin with progressive age in R6/1 animals. The profile of immunoreactivity (mean percentage of immunoreactive product per defined area) was assessed in accordance with established protocols (55Go,56Go). Analysis was performed blind with respect to age and genotype on consecutive sections of the hippocampus for each animal. A survey of the immunoreacted tissue sections was performed by two independent operators (Austen J. Milnerwood, Payam Rezaie) to verify specific immunoreactivity in duplicate sections subsequently processed to quantitative image analysis. Briefly, non-overlapping RGB images were digitally captured at random within the defined areas, providing a systematic survey throughout each region for each genotype at the three ages examined. Images (each field measuring 150 µm wide, with a height of 112 µm) were captured via a digital camera (JVC KY-F75V, JVC Professional Europe Ltd, London, UK) mounted onto a Nikon Microphot-FX microscope (Nikon UK Ltd, UK) using a 40x objective and neutralizing grey filters. All parameters including the lamp intensity, digital camera set-up and microscope calibration were held constant. Optimal segmentation of immunoreactive profiles was analysed using the Image-Pro Plus® program (Version 5.0, Media Cybernetics, UK) according to the previously described semi-automated thresholding method based on the optical density of the immunoreactive product (55Go–57Go). Macros were subsequently recorded to transfer the data to a spreadsheet. These were plotted as the mean percentage area of immunoreactivity per field±SD for each group and the significance was determined by Student's t-test.

Electrophysiology
Brains were isolated from R6/1 and WT littermates. Briefly, transverse hippocampal slices (400 µm) were prepared, area CA3 was excised and slices were transferred to an interface recording chamber (Scientific Systems Design Inc., USA) maintained at 28°C and constantly perfused with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) (containing in mM: 120 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 1.2 NaH2PO4, 23 NaHCO3, 11 glucose) and left to incubate for a minimum of 1.5 h prior to experimentation.

Hippocampal CA1 field potentials were evoked by constant current stimuli (40 µs) applied via monopolar stimulating electrodes (impedance 5 M{Omega}; AM Systems, USA) to distinct CA3 Schaffer-collateral commissural projections. Field potentials were recorded via extracellular glass microelectrodes (impedance 5–8 M{Omega}, filled with 1 M NaCl and 2% Pontamine blue) placed in the stratum radiatum of CA1 using either a Neurolog AC-preamp or an Axoclamp 2B amplifier (Digitimer, UK; Axon Instruments Inc., USA, respectively). High-frequency tetani consisted of three trains of 100 shocks at 100 Hz with an inter-train interval of 10 s. For the purposes of assessing the probability of the induction of LTD, it was defined as a stable reduction (>10%) of the fEPSP (field EPSP) slope 1 h post-conditioning. Analysis of synaptic responses was by manual cursor placement, set to measure the fEPSP initial linear slope at a fixed latency or population spike amplitude (software: A/Dvance 3.6). Stimulus intensity was set to produce a response which was just below the threshold for population spike activity detected in the fEPSP, and evoked at 0.033 Hz for at least 20 min, to ensure a stable baseline prior to conditioning. All drugs (purchased from Tocris Cookson Ltd, UK and Sigma-Aldrich Company Ltd) were diluted in ACSF and perfused into the recording chamber for a minimum of 20 minutes prior to experimentation. The NMDA-sensitive glutamate receptor antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 µM except where stated) and the NR2B NMDA receptor subtype-specific antagonist {alpha}-(4-hydroxyphenyl)-ß-methyl-4-benzyl-1-piperidineethanol tartrate salt (ifenprodil; 10 µM) were used to investigate NMDA receptor-mediated activity.

For all statistical analyses, data obtained from each in vitro slice preparation was used to calculate the sample mean, number and standard error or deviation, and the number of animals used to generate slices for each series of experiments is indicated in brackets. For each data set, to avoid potential bias, the contribution of each animal was equally weighted. Data are expressed as the percent change in fEPSP slope or population spike amplitude±SEM. Statistical analysis was performed by unpaired and paired Student's t-test where appropriate; the latter test was always performed using absolute values. LTP, LTD and depotentiation values for each experiment were calculated as the change in mean fEPSP slope between the 5 min period prior to conditioning and 55–60 min period post-conditioning, unless stated otherwise.

Intracellular recording electrodes (impedance 60–150 M{Omega} filled with 3 M KAc, connected to an Axoclamp-2B amplifier in bridge mode) were used to impale CA1 hippocampal pyramidal cells. Passive membrane properties were recorded by stepwise current injection (–0.8 to +0.6 nA) to generate a current–voltage (I/V) relationship, including positive pulses to assess threshold and AP characteristics. AP threshold potential, AP amplitude and AP half-width (ms at 50%) were determined. Membrane resistance (Rin) was calculated by the slope of a line of best-fit through the linear portion of the I/V plot (R2 always >0.98) and the membrane time constant ({tau}m) from the 0.4 nA deflection, by determining the time (ms) taken to achieve (1–1/ex100)% of the maximal and steady-state potential. Membrane capacitance (Cm) was calculated from Rin and {tau}m using the relationship Cm={tau}m/Rin (51Go). Synaptic responses were generated and analysed in the same manner as that for field potentials.


   ACKNOWLEDGEMENTS
 
We would like to thank Mr Steve Walters, Mrs Dawn Sadler and Mrs Karen Evans of the Open University for their excellent technical assistance, Drs Tony Hannan and Anton van Dellen (Oxford, UK) for their help in establishing our R6/1 colony and Dr Ole Paulsen (Oxford, UK) for allowing us to conduct pilot experiments in his laboratory. We would also like to thank Professor Gillian Bates (London, UK) for the generous gift of the S830 antibody. This work was funded by the Open University Research Development Committee and the Royal Society.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Department of Psychiatry and Brain Research Centre, University of British Columbia, Vancouver, Canada. Back

{ddagger} Present address: Mental Retardation Research Center, University of California at Los Angeles, Los Angeles, CA, USA. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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