Behavioural, physiological and morphological analysis of a line of apolipoprotein E knockout mouse

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Neuroscience Vol. 85, No. 1, pp. 93–110, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00598-8

BEHAVIOURAL, PHYSIOLOGICAL AND MORPHOLOGICAL ANALYSIS OF A LINE OF APOLIPOPROTEIN E KNOCKOUT MOUSE R. ANDERSON,* J. C. BARNES,* T. V. P. BLISS,† D. P. CAIN,‡ K. CAMBON,§ H. A. DAVIES,§ M. L. ERRINGTON,† L. A. FELLOWS,* R. A. GRAY,* T. HOH,‡ M. STEWART,§ C. H. LARGE* and G. A. HIGGINS*¶ *Neuroscience Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, U.K. †Department of Neurophysiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K. ‡Department of Psychology, University of W. Ontario, London, Ontario, Canada, N6A 5C2 §Department of Biology, The Open University, Walton Hall, Milton Keynes MK7 6AA, U.K. Abstract––Using apolipoprotein E knockout mice derived from the Maeda source [Piedrahita J. A. et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, 4471–4475], we have studied the influence of apolipoprotein E gene deletion on normal CNS function by neurological tests and water maze learning, hippocampal ultrastructure assessed by quantitative immunocytochemistry and electron microscopy, CNS plasticity, i.e. hippocampal long-term potentiation and amygdaloid kindling, and CNS repair, i.e. synaptic recovery in the hippocampus following deafferentation. In each study there was little difference between the apolipoprotein E knockout mice and wild-type controls of similar age and genetic background. Apolipoprotein E knockout mice aged eight months demonstrated accurate spatial learning and normal neurological function. Synaptophysin and microtubule-associated protein 2 immunohistochemistry and electron microscopic analysis of these animals revealed that the hippocampal synaptic and dendritic densities were similar between genotypes. The induction and maintenance of kindled seizures and hippocampal long-term potentiation were indistinguishable between groups. Finally, unilateral entorhinal cortex lesions produced a marked loss of hippocampal synaptophysin immunoreactivity in both groups and a marked up-regulation of apolipoprotein E in the wild-type group. Both apolipoprotein E knockout and wild-type groups showed immunohistochemical evidence of reactive synaptogenesis, although the apolipoprotein E knockout group may have initially shown greater synaptic loss. It is suggested that either apolipoprotein E is of no importance in the maintenance of synaptic integrity and in processes of CNS plasticity and repair, or more likely, alternative (apolipo)proteins may compensate for the loss of apolipoprotein E in the knockout animals.  1998 IBRO. Published by Elsevier Science Ltd. Key words: apolipoprotein E, Alzheimer’s disease, plasticity, repair, LTP, cognition.

amino acids at positions 112 and 158.37,80 Recently, genetic association studies have identified the ApoE gene as a major risk factor for late-onset Alzheimer’s disease, such that the age of onset is modified in an isoform-dependent manner.10,22,69,72,77 People carrying one or more ApoE4 alleles show an earlier age of onset than those with an ApoE3 allele, who, in turn, tend to have an earlier age of onset than those with one or more ApoE2 allele. Despite extensive research into the role of ApoE in the periphery, very little is known of its role in the CNS. An important approach to understanding the function of ApoE in human disease is the study of transgenic animals expressing the different human isoforms.81 A desirable precursor to such studies is the characterization of animals in which the rodent protein has been removed; these studies may in themselves provide clues to the function of the protein. Transgenic mice in which the ApoE gene has

Apolipoprotein E (ApoE) is a 34000 mol. wt member of a family of lipoproteins, whose role in the sequestration and trafficking of cholesterol in the periphery has been studied extensively.5,22,25,37 In humans, the protein exists in three common isoforms, termed E2, E3 and E4, which differ in two single ¶To whom correspondence should be addressed at: Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd, Basel, Switzerland. Abbreviations: AD, afterdischarge; Apo, apolipoprotein; BSA, bovine serum albumin; DG, dentate gyrus; ECL, entorhinal cortex lesion; EDTA, ethylenediaminetetraacetate; EPSP, excitatory postsynaptic potential; GAP43, growth-associated protein 43; GFAP, glial fibrillary acidic protein; HFS, high-frequency stimulation; LDL, low density lipoprotein; LRP, low density lipoprotein receptor-related protein; LTP, long-term potentiation; MAP-2, microtubule-associated protein 2; PBS, phosphate-buffered saline; PCR, polymerase chain reaction. 93

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Fig. 1. Western blot of whole cortex probed with an antibody to ApoE (Chemicon, AB947, dilution 1:8000). The subjects used were four ApoE knockout mice and four wild-type age-matched controls. Note the complete absence of a band at mol. wt 34000 in the knockout group. A second minor band was evident at around mol. wt 46000; this was seen in animals from both groups. ko, knockout.

been deleted have been produced by various laboratories.58,59,79 Consistent findings in these animals are raised plasma cholesterol and marked atherosclerosis with age.59,73,79,82 The present series of experiments has been designed to characterize ApoE-deficient mice of between four and 12 months of age in a variety of models. Given the well-documented role of ApoE in lipid transport,37 neurite outgrowth23,49,50,64 and CNS remodelling,2,60,61 we were interested in studying processes of cognition, synaptic plasticity and repair in these animals. Specifically, we have studied these mice in a well-validated cognitive task, the Morris water maze,47,48 two models of synaptic plasticity, long-term potentiation (LTP) and kindling,4,6,7,32,68 and synaptic recovery following unilateral entorhinal cortex lesions.20,36,40,41,71,75 Furthermore, following the work of Masliah et al.,43 showing disruption to the neuronal cytoskeleton and synaptic loss in ApoE knockout mice beyond four months of age, we were interested in examining the hippocampal structure of the ApoE knockout mouse using electron microscopy and immunocytochemistry. EXPERIMENTAL PROCEDURES

Animals and housing ApoE transgenic mice were obtained from a colony produced by Dr N. Maeda and colleagues (University of North Carolina, Chapel Hill, NC, U.S.A.). The ApoE knockouts were generated from germline transmitting chimeras, which were a mixture of C57BL/6J blastocysts and 129J modified embryonic stem cells.58 These offspring were then backcrossed six times with C57BL/6J animals before transfer to the U.K. Test groups comprised male ApoE knockout and wild-type littermate controls at four to 12 months of age which were produced by selective matings between heterozygous ApoE knockout breeding pairs. All comparisons between ApoE knockout and wild types are in mice of equivalent age. Mouse genotype was determined by polymerase chain reaction (PCR) analysis (see below).

Following these procedures, all mice were transferred to the relevant laboratories for the appropriate tests at the following sites [i.e. behaviour, entorhinal cortex lesions, LTP (12 months of age), immunohistochemistry: GW Stevenage, U.K.; electron microscopy analysis: Open University, U.K.; kindling: University of Western Ontario, Canada; LTP (six months of age): NIMR, Mill Hill, U.K.]. Genotyping of transgenics The genotype of the mice was confirmed by extraction of DNA from tail biopsies using Strataclean Purification Resin (Stratagene). DNA (1 µl; 300–1000 ng) was analysed using the ApoE-specific primers 5 -CTTACTCTACACAGGAT GCCTAGCC-3 and 5 -TTCCCAGAAGTTGAGAAGCT GCGG-3 , and the neomycin-specific primer 5 -AATG GGCTGACCGCTTCCTCGTG-3 . Standard 25-µl PCR reactions (Promega) were set up (94C for 40 s, 70C for 1 min, 72C for 2 min: 30 cycles) and the amplified products analysed on a 2% agarose gel. The presence of a 221-base pair amplified fusion product and lack of a 467-base pair wild-type product confirmed the genotype as homozygote ApoE knockout. Conversely, the presence of the 467-base pair product and absence of the 221-base pair product confirmed the genotype as wild type. Heterozygous mice were excluded from all studies. In addition to PCR, measurements of plasma lipid levels were used to further confirm the phenotypic status of the mice. The mice were anaesthetized with a mixture of isofluorane (5% in oxygen) and nitrous oxide (ratio 1:3). A whole blood sample (500 µl) was taken via cardiac puncture, added to an EDTA-coated tube (Monoject KE/2.5) and spun at 1400 r.p.m. for 10 min at 4C in an Eppendorf bench centrifuge. A 120-µl aliquot of the supernatant was removed and cholesterol and triglyceride concentrations determined colorimetrically using a Kone Ultra Analyser. In some animals, further characterization of genotype was conducted by western blot analysis of whole forebrain tissue (Fig. 1). Behavioural tests For these studies, a group of 12 male ApoE knockout and 12 wild-type littermate controls aged eight to 10 months were used. Prior to water maze testing, all mice were subjected to a battery of neurological tests as described previously,20 with additional measures of motor coordination, determined by measuring the mean latency of three trials to fall from a mouse rotarod. Locomotor activity was

Characterization of ApoE knockout mice measured by beam breaks over a 1-h test period in activity chambers (35 cm long22 cm wide18 cm deep). Rearing was not measured. The water maze was a white perspex circular pool (100 cm diameter, 30 cm deep) filled with water (261C) to a depth of 25 cm and rendered opaque by the addition of a liquid latex compound (Warner Jenkinson). The maze was isolated with screens and a variety of distal cues placed prominently around the maze. The behaviour of the mice was recorded by an overhead camera and tracking system (HVS Image, Hampton, U.K.). For cued training, the mice were required to locate a visible escape platform (6.5 cm diameter) which was painted black, flagged and protruded 1 cm above the water surface. Each mouse was tested over four sessions (two per day, three trials per session with a 10-min inter-trial interval). In this case, both the starting and platform positions were randomly varied to avoid habituation to a particular quadrant. If a mouse failed to locate the platform within the maximum trial period of 60 s it was led to and placed on the platform for 10 s. Following the cued task, the mice received eight sessions (three trials per session, 10 min inter-trial interval) over four consecutive days with the placed escape platform (6.5 cm diameter) painted white and positioned just below the water surface. The position of the placed platform was fixed for each individual mouse, although the start position was varied. Following the eighth session, the mice underwent a probe trial which involved removal of the placed platform and analysis of the search pattern for 60 s. The time spent searching the quadrant that formerly contained the hidden escape platform was recorded as a percentage and compared with that spent searching in the other quadrants. Data from the neurological tests and water maze probe test were analysed by t-test or one-way ANOVA. The water maze acquisition studies were analysed by two-way mixed factor ANOVA with session as the within-subjects variable and genotype as the between-subjects variable. In all cases following a significant main effect, subsequent post hoc comparisons were made using Dunnett’s t-test. CNS morphology Following water maze testing, a subgroup of mice used in the behavioural studies (n=6 per genotype) were taken, culled by decapitation and the brain hemisected. One side was placed in 2% paraformaldehyde at 4C in phosphatebuffered saline (PBS) for 18 h before wax blocking; 8-µm sections were then taken from the anterior hippocampal and frontoparietal regions. The other hemisphere was homogenized and prepared for western blotting and other biochemical assays (data not shown). Blood samples from these animals were taken for cholesterol analysis. Slides were dewaxed in xylene, rehydrated and incubated for 1 h with (5%) normal swine serum before overnight application of undiluted primary synaptophysin antibody (MCA860, Serotec, U.K.) or microtubule-associated protein 2 (MAP-2) monoclonal antibody [M4528, Sigma; diluted 1:50 in PBS containing 2% normal swine serum, 1% bovine serum albumin (BSA), 0.3% Triton] at 4C. Following the overnight incubation, sections were washed in PBS before incubation for 30 min with biotinylated anti-mouse antibody (Vectastain Elite avidin–biotin–peroxidase complex mouse kit). Following a further wash, the sections were incubated for 30 min in the presence of fluorescein– streptavidin (2% in PBS; Vector Labs) prior to a final wash and mounting with Vectashield mountant medium (Vector Labs). Within 48 h of immunostaining, the slides were examined using a laser scanning confocal microscope (TCS4D; Leica Lasertechnic, Germany) and images captured of the CA1, CA3 and dentate regions. Confocal images were analysed using a Leica Q500MC image analyser (Leica, U.K.; see Ref. 20 for further details).

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The remaining mice (n=6 per genotype) from the same water maze experiment were anaesthetized with sodium pentobarbitone and then perfused transcardially with 15 ml saline solution followed by 150 ml of phosphate-buffered fixative containing 2% glutaraldehyde and 2% paraformaldehyde. Whole brains were removed and placed in fresh fixative overnight at 4C. Frontal slabs of 1 mm thickness across the entire dorsal hippocampus in the right hemisphere were dissected, and trimmed to leave a block containing CA1, CA3 and the dentate gyrus (DG). Hippocampal tissue was prepared for electron microscopy by postfixing in 1% osmium tetroxide for 1 h, dehydrating through graded alcohols and embedding in Epon resin. Blocks were hardened in an oven at 60C for 24–48 h. Ultrathin sections of silver interference colour were cut on an ultramicrotome with a diamond knife and mounted on single-slot grids coated with Pioloform/carbon support film. Pairs of serial sections were mounted on the same slot grid and stained with uranyl acetate and lead citrate. Numerical synapse density was estimated using an unbiased disector routine in the middle part of the molecular layer of the DG, and in the middle part of the basal (in stratum oriens) and apical (in stratum radiatum) dendrites of CA1 cells. For each subregion of the hippocampus, 12 pairs of digital images of the sections were acquired along the axis of the cell layer, from a JEM 1010 electron microscope at a magnification of 12000 using a Kodak Megaplus digital camera. On the digital images, synapses were identified by the presence of both pre- and postsynaptic densities, with an associated vesicle cloud, and they were then classified as shaft or spine depending on the postsynaptic target site. No attempt was made to distinguish between symmetric and asymmetric synapses, though most possessed asymmetrical junctions and were presumed to be excitatory in nature. The mean synaptic numerical density per area was calculated using the disector formula:74 Nv syn=Q
syn/t·A, where Q
syn is the total number of counted synapse profiles that appear only in the nominated section, t is the section thickness determined by the electron scattering method and A is the area of the counting frame. The stereological determination of the granular cell density in the DG was estimated using the same disector method. Pairs of adjacent transverse sections of 2 µm thickness were prepared from the embedded tissue and stained with Toluidine Blue. For each animal, three pairs of digital images along the upper limb of the DG were acquired from a light microscope at a magnification of 80 using a camera connected to a Joyce–Loebl Magiscan MD system. Spatial density in the layer of granular cells in the DG was expressed in terms of the total number of cells contained within a parallelepiped of unit area passing through the cell body layer. In vivo electrophysiology Electrophysiological characterization was carried out on male ApoE knockout and wild-type littermates aged six and 12 months. Mice were anaesthetized with urethane (1.8 g/kg, i.p.) for six-month-old mice and isoflurane (2% in 50% O2/50% N2O) for 12-month-old mice. Mice were positioned in a stereotaxic frame and normal core body temperature monitored constantly using a rectal temperature probe and maintained by a homeothermic heated blanket. The surface of the brain was exposed for electrode placement and a glass recording micropipette was positioned in the hilus of the DG (1.8–2.0 mm posterior to bregma, 1.5–1.6 mm lateral to the midline) for recording field potentials, and a bipolar stimulating electrode positioned in the angular bundle of the medial perforant path (2.9–3.2 mm lateral to lambda). Electrode depth was adjusted to obtain the maximum evoked response from single square-wave test pulses (50 µs, 300 µA). At the start of each experiment, input–output relationships were determined to

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assess the maximal slope of the evoked field potential and identify the stimulation parameters required to produce approximately 50% of the maximal excitatory postsynaptic potential (EPSP) slope and a population spike with an amplitude of approximately 1 mV. These parameters were used for all subsequent test and paired-pulse stimuli. The following electrophysiological characterization was carried out in each mouse sequentially. Paired-pulse facilitation of field EPSP was studied using pulses of amplitude below the threshold for evoking a population spike, delivered at intervals of between 10 and 80 ms in 5-ms steps, each pair separated by 30 s. This was then repeated using pulses of higher amplitude in order to evaluate the effects on the population spike; paired pulses were delivered at intervals of between 10 ms and 1 s. Following this, single test pulses were delivered at 30-s intervals and a stable pretetanus period of 30 min obtained prior to the induction of LTP using using high-frequency stimulation (HFS; six trains of six pulses at 400 Hz, 200 ms inter-train interval). This was repeated six times at 20-s intervals. Recording of test pulses was continued for at least 1 h following the HFS. All data were analysed using Spike 2 software (Cambridge Electronic Design) and the EPSP slope and population spike amplitude of each response were determined. Kindling Subjects were seven ApoE knockout mice, four wild-type littermate controls and five C57/b6 background strain controls, all of which were females. At the time of experimentation all animals were approximately six months of age. Surgical techniques for bilateral implantation of indwelling amygdaloid electrodes involved standard stereotaxic techniques as described previously.8 Electrodes were constructed of twisted Teflon-insulated Nichrome wire 127 µm in diameter soldered to gold-plated connector pins. Implantation was into the basolateral amygdala under 10 mg xylazine and pentobarbital anaesthesia (60–80 mg/kg) at the following coordinates: AP +2.5 mm relative to interaural zero; L3.3 mm from midline; DV-5.3 mm from the skull surface. A miniature connector and leads attached the pins to brain stimulation and recording equipment. Histological examination of approximately half of the subjects in each group, selected at random, confirmed the accuracy of the electrode placements. After 10 days recovery, trains of biphasic square wave pulses (1 ms each, 60 pulse pairs per second, for 1 s) were applied through the electrode at increasing intensity once every 2 min to determine after discharge (AD) threshold. The hemisphere yielding AD (minimum duration 5 s) at the lowest stimulation intensity was selected for kindling. Once daily stimulation at 110% AD threshold was applied beginning 24 h later until three generalized convulsions occurred. Stimulation was applied once daily thereafter between 10.00 and 14.00. Polygraphic records were obtained from both electrodes before and after each stimulation. AD occurred in response to each suprathreshold stimulation. Kindling sessions of the first three mice in each group were videotaped, and the tapes were replayed for scoring the convulsions using Racine’s65 categories, with stage 1 indicating brief behavioural immobility with ear flattening or twitching of the facial musculature and stage 5 indicating generalized convulsions. Convulsions of subsequent mice in each group were scored by direct observation. After three stage 5 convulsions mice were allowed three to four weeks without stimulation and then rekindled with the same stimulation intensity as a measure of retention of kindling. Statistical analysis of the results was by ANOVA. Entorhinal cortex lesions Male mice aged four months were used for this study. The techniques employed were essentially as described in

Ref. 20, except that the lesions were made using a Scouten knife blade, rather than electrolytically. Briefly, mice were anaesthetized with a hypnorm/hypnovel mixture before unilateral transection of the perforant path was made using a knife blade angled 6 from vertical, using the following coordinates (AP +0.5 mm from lambda; L
1.0 mm from midline; DV
4.0 mm from the skull surface; blade extended 2.5 mm for entire DV travel). Following recovery from surgery, both ApoE knockout and wild-type mice were culled at days 6, 14, 28, 70 and 140 post-lesion (n=4 or 5 per genotype at each time-point). Under anaesthesia, all animals received a 20-ml cardiac perfusion of PBS; the brain was then removed and placed for 18 h in PBS containing 2% paraformaldehyde at 5C. After this period, the brains were dehydrated and wax embedded for subsequent coronal sectioning (8 µm thickness). A subgroup of unlesioned mice (n=4 per genotype) were selected randomly for blood cholesterol measurement. Sections from each animal at each time-point were selected based on equivalent (anterior) hippocampal regions, and were immunohistochemically processed in parallel. Three main studies were undertaken: (i) the study of ApoE expression in the wild-type animals; (ii) changes in synaptophysin immunoreactivity in the molecular layer following entorhinal cortex lesion (ECL); and (iii) changes in thickness of the inner molecular layer following ECL using growth-associated protein 43 (GAP-43) immunohistochemistry. The protocol for GAP-43 immunostaining was essentially as described for synaptophysin (see under ‘‘CNS morphology’’), except GAP-43 (5 µg/ml; 1379 011, Boehringer, U.K.) was applied overnight. Confocal analysis and subsequent quantification of the synaptophysin and GAP-43 immunoreactivity was identical to that described previously.20 Sections were processed for ApoE immunoreactivity using an avidin–biotin–peroxidase procedure. Sections were deparaffinized and pretreated with 0.3% H2O2 in PBS for 30 min to eliminate endogenous peroxidase. After a 1-h incubation in 5% normal rabbit serum in PBS to block non-specific immunoglobulin G binding, tissues were incubated overnight at 4C with goat polyclonal ApoE antibody (Chemicon, AB947) diluted 1:10000 in PBS containing 3% normal rabbit serum, 1% BSA and 0.3% Triton X-100. Sections were subsequently incubated in biotinylated anti-goat antibody (Vector anti-goat avidin–biotin– peroxidase complex ‘‘Elite’’ kit) for 30 min at room temperature followed by the avidin–biotin–peroxidase complex for 30 min at room temperature. Peroxidase activity was visualized by reacting the sections with diaminobenzidine (Vector Labs, SK-4100) for 4 min, resulting in a brown reaction product. Each incubation step was preceded by three 5-min washes in PBS. Sections were then rinsed in distilled water, dehydrated and coverslipped with xylene. In control experiments, some sections were incubated with either preabsorbed anti-ApoE antibody or without the anti-ApoE antibody. In these sections, no specific immunoreactivity was observed. The number of ApoEimmunoreactive structures at days 6, 14, 28 and 70 postECL were counted with the aid of a Sight Systems Image Analyser (Sight Systems, Brighton, U.K.). ApoE-positive counts were taken from defined regions of the hippocampus, i.e. the molecular layer, the lacunosum moleculare, the CA3 region, the CA4/hilus region and the CA1/CA2 region. For double immunolabelling sections to ApoE and glial fibrillary acidic protein (GFAP), immunoperoxidase staining for ApoE immunoreactivity was performed using the same procedure as described above. Sections were treated for 30 min with 0.3% H2O2 in PBS following the diaminobenzidine reaction for the first antibody. Sections were then incubated with 5% normal horse serum in PBS for 1 h followed by incubation overnight at 4C in mouse monoclonal anti-GFAP antibody (Serotec, MCA363) diluted 1:5000 in PBS containing 3% normal horse serum, 1% BSA

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Fig. 2. (A) Performance of wild type () and ApoE knockout ( ) mice on water maze learning in both the cued (sessions 1–4) and place (sessions 5–12) tasks. Data shown are for path length, although there were no differences in swim speeds between the groups (see text); n=12 per group. At no time-points were there any group differences. (B) Performance of wild-type and ApoE knockout mice in a 60-s probe test conducted 10 min after the final place training session. Each group is represented by four histobars corresponding to each quadrant of the water maze [adjacent left, island quadrant (filled bar), adjacent right, opposite, respectively]. Note that both groups showed a significant preference for the island quadrant, but there was no significant difference between groups. (C) Path plots for the median wild-type (upper) and ApoE knockout (lower) mouse. ko, knockout. and 0.3% Triton X-100. The sections were subsequently incubated in biotinylated anti-mouse antibody (Vector anti-mouse avidin–biotin–peroxidase complex ‘‘Elite’’ kit) for 30 min at room temperature followed by incubation in the avidin–biotin–peroxidase complex for 30 min at room temperature. The sections were then reacted for 6 min with undiluted True Blue peroxidase substrate (Dynatech Labs, 71-00-64). Each incubation step was preceded by three 5-min washes in PBS. Consequently, ApoE-positive cells were stained brown and GFAP-positive astrocytes were stained blue. Sections were rinsed in distilled water, dehydrated and coverslipped with xylene. RESULTS

Behavioural tests There was no significant difference in neurological measures between ApoE knockout and wild-type littermate control mice at eight to 10 months of age. Similarly, body temperature and rotarod scores were indistinguishable and neither genotype displayed abnormal secretory signs (data not shown). Furthermore, cumulative locomotor activity scores over the 60-min observation period did not differ between genotypes, i.e. total activity scores for wild-type and ApoE knockout mice were 2101167 and 1813172 counts, respectively (t22=0.2, NS). Rearing was not measured. In the water maze experiment, Fig. 2A shows path length to locate the cued platform (sessions 1–4) and path length to locate the placed platform (sessions 5–12). There was no main effect of genotype in either the cued (F1,22=0.9, P=0.4) or the place task (F1,22=0.05, P=0.8). For example, path lengths at the 12th session were 21627 and 21329 cm for wild-type and ApoE knockout mice, respectively. In

addition, there was no significant difference in swim speeds (F1,22=1.6, P=0.7). For example, swim speeds of wild-type and ApoE knockout mice at the 12th session were 19.00.7 and 20.50.6 cm/s, respectively. Probe tests confirmed that ApoE knockout and wild-type animals showed spatial learning, as evidenced by a significant preference for the quadrant that previously contained the island platform (percentage time in island quadrant: wild type, 394%; ApoE knockout, 494%; Fig. 2B). There was no significant difference in the percentage time spent searching in the island quadrant between ApoE knockout and wild-type groups (P=0.09). Median probe data path plots for ApoE knockout and wild-type mice are shown in Fig. 2C. At the completion of this study, plasma cholesterol levels were determined in a subgroup of mice (n=5). All mice genotyped as being ApoE knockout had elevated plasma cholesterol compared to the wildtype group (wild type, 2.30.2 mmol/l; ApoE knockout, 15.01.3 mmol/l; P<0.001). Morphometric studies Hippocampal synaptophysin and microtubuleassociated protein 2 immunohistochemistry. Quantitative analysis of confocal images to synaptophysinimmunostained sections containing the anterior hippocampus failed to reveal any differences between wild-type and ApoE knockout mice (see Fig. 3). There was high immunostaining throughout the molecular region of the DG, as well as the radiatum and oriens subfields of the CA1–CA4 region in both groups. In contrast, the cell body areas of the DG

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Fig. 3.

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Table 1. Mean synapse densityS.E.M. in the dentate gyrus and CA1 region of the hippocampus (expressed as the number of synapses per µm3 of tissue) Shaft synapse

Wild type ApoE knockout

Spine synapse

DG

CA1 basal

CA1 apical

DG

CA1 basal

CA1 apical

DG neuronal density

0.340.12 0.310.05

0.150.03 0.210.04

0.100.01 0.150.04

3.210.2 3.110.26

3.280.17 3.10.3

3.140.19 3.600.21

0.100.02 0.110.01

Neuron density in the DG region is expressed as the total number of neurons within a parallelepiped of 1 µm2 passing through the granular cell layer. On no measure were any significant differences noted between wild-type and ApoE knockout mice (n=6 per group).

and CA fields showed no synaptophysin immunoreactivity. Synaptophysin is a synapse-specific protein, hence the similarity of immunofluorescence for these hippocampal subregions suggests equivalent synaptic densities between wild-type and ApoE knockout mice. Similarly, adjacent sections stained with an antibody to MAP-2 failed to show any qualitative difference between the two groups (Fig. 3). This was confirmed by image analysis, where the area of neuropil occupied by MAP-2 immunoreactivity was identical between groups. Omission of the primary antibody resulted in the complete absence of signal (data not shown). Mean numerical density of synapses and neuronal cell bodies in the dentate gyrus and CA1 region of the hippocampus. Qualitative ultrastructural analysis of the hippocampus revealed no visible alterations in the brain of ApoE knockout mice compared with wildtype mice. A set of electron micrographs from the CA1 apical region and the molecular layer of the DG are shown in Fig. 4 for both wild-type and ApoE knockout mice. Most of the presynaptic terminals make either simple or complex asymmetric contacts on dendritic spines. The terminals appear normal, with no sign of dilatation in either group of animals; they contain abundant synaptic vesicles. No dendritic vacuolization was observed in either group of animals and the dendrites show similar microtubular distribution and mitochondria of normal appearance in both ApoE knockout and wild-type mice. In both ApoE knockout and wild-type mice, mean synaptic densities for shaft and spine synapses are shown in Table 1 for the middle part of both basal and apical dendrites of CA1 pyramidal neurons. No significant differences were noted in the mean values of any of these synaptic categories in either region between the ApoE knockout or wild type. Mean synaptic densities, and separately those for shaft and spine synapses, in the middle part of the

molecular layer of the DG are shown in Table 1, and no significant differences were observed in the mean values for any of these synaptic categories between the ApoE knockout or wild-type mice. The mean neuronal density in the granular cell layer measured on the upper limb of the DG was 0.100.02 neurons/ µm2 per layer of cells for the wild-type mice and 0.110.01 neurons/µm2 per layer of cells for the ApoE knockout mice. There was no statistical difference between the two groups of animals regarding this parameter.

In vivo electrophysiology Blood samples were obtained from 12-month-old ApoE knockout and wild-type mice for determination of plasma cholesterol in order to phenotype the mice used for electrophysiological characterization. The predicted elevated plasma lipid profile was observed in ApoE knockout mice (wild type, 3.00.1 mmol/l; ApoE knockout, 13.90.7 mmol/l; P<0.01; n=4). The degree of LTP was determined using parameters of EPSP slope and population spike amplitude, and comparing the pre- and post-HFS values. At six months, there was no significant difference between ApoE knockout and wild types in the degree of LTP as described by the increase at 60 min post-HFS of both EPSP slope (ApoE knockout, 16.92.9; wild type, 15.84.7) and population spike (ApoE knockout, 39951; wild type, 43590) (Fig. 5A, C). Indeed, this was the case at 12 months (EPSP slope: ApoE knockout, 18.35.5; wild type, 17.59.7; population spike: ApoE knockout, 512124; wild type, 48084; Fig. 5B, D). It was noted that the mean increases in population spike amplitude were higher in wild types than ApoE knockout at both six and 12 months, although statistical significance was not achieved.

Fig. 3. Laser confocal images of hippocampal sections taken from wild-type and ApoE knockout mice following immunostaining with antibodies to synaptophysin (upper panel) and MAP-2 (lower panel). The final column shows the subsequent quantification by image analysis: ( ) wild type; () ApoE knockout mice (n=6 per group). The synaptophysin images were taken from the anterior hippocampal area and show the DG and the medial blade of the CA1 region (magnification: 100). The MAP-2 images were taken from the CA1 region; the pyramidal cell layer is evident at the top of the figure (magnification: 400).

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Fig. 4. Electron micrographs from the hippocampus of eight-month-old wild-type and ApoE knockout mice. A and B are from the molecular layer of the DG, and C and D are from the apical dendritic region of CA1 pyramidal neurons (stratum radiatum). In both wild-type (A–C) and ApoE knockout (B–D) mice, the synaptic ultrastructure is similar. Most synaptic contacts occur on dendritic spines (star, simple synapse; asterisk, perforated synapse). The presynaptic terminals contain abundant vesicles (double arrows). Microtubules in dendrites (d) display a similar distribution in both wild-type and ApoE knockout mice. Magnification: 35000.

There was no significant difference in paired-pulse phenomena of either EPSP slope or population spike amplitude between ApoE knockout and wild-type littermate control mice at either six or 12 months (Fig. 5E–H). Indeed, there does not appear to be a decline in the degree of paired-pulse phenomena or LTP between the ages of six and 12 months in either ApoE knockout or wild-type controls.

Kindling The littermate wild-type and C57/BL6 control groups did not differ on any measure, and therefore were combined into a pooled control group for graphing and statistical analysis purposes. AD was easily evoked from both groups at low stimulation intensities, and AD thresholds did not differ between

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Fig. 5.

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Entorhinal cortex lesions Both ApoE knockout and wild-type mice showed good recovery from the surgery, and there was no obvious difference in terms of weight gain and neurological function assessed one to six days postlesion (data not shown). A subgroup of mice from this colony were killed for cholesterol determination, and found to give the predicted plasma lipid profile (wild type, 1.70.3 mmol/l; ApoE knockout, 5.71.0 mmol/l; P=0.02; n=4 per group).

Fig. 6. (A) Convulsion stage across sessions during kindling (left) and rekindling (right). Convulsion stages are from Racine.65 For purposes of graphic comparison between groups, mice that completed a third stage 5 convulsion before session 12 were assigned their last AD duration and convulsion stage values thereafter. (B) AD duration (s) across sessions during kindling (left) and rekindling (right). () Wild-type/C57/Bl6; ( ) ApoE knockout mice. *P<0.05 vs wild-type/C57/Bl6 controls.

the groups (ApoE knockout, 92.527.8 µA; pooled controls, 72.818.1 µA; P>0.05; Fig. 6A). All mice kindled rapidly and displayed a typical murine stage 5 generalized convulsion by session 10 (ApoE knockout, 5.61.0 sessions to first stage 5; pooled controls, 5.71.0 sessions; P>0.05; Fig. 6B). Mice in both groups displayed comparable increases in convulsion severity, although a significant genotypetrial interaction emerged on the measure of AD duration (F7,98=2.4, P<0.03). Thus, from trial 6, the ApoE knockout group showed higher AD durations relative to controls. During rekindling performed three to four weeks after initial kindling, the groups displayed comparable AD durations and all mice displayed a robust generalized stage 5 convulsion on the first or second rekindling stimulation (Fig. 6A, B). A small difference was found in the number of sessions required to rekindle the two groups (ApoE knockout group, 1.40.2 sessions; pooled control group, 1.00.0 sessions; F1,14=5.9, P<0.03; Fig. 6A).

Change in synaptophysin immunoreactivity after entorhinal cortex lesions. In sham-lesioned animals, there was a high level of synaptophysin immunofluorescence throughout the molecular layer of the DG, which did not differ between the groups (e.g., see Fig. 3). Six days following ECL, however, there was a highly significant reduction in the middle and outer molecular layer, which gradually returned to sham levels by day 70 (Fig. 7). These changes were seen in both the wild-type and ApoE knockout mice, although at each time-point the ApoE knockout mice showed a slightly lower level of synaptophysin immunoreactivity (Fig. 7). Overall, ANOVA revealed a main effect of genotype of borderline significance (middle layer: F1,35=3.9, P=0.05; outer layer: F1,35=5.5, P<0.05) and a main effect of time (both layers: F6,35 >19, P<0.01), but no genotypetime interaction (middle layer: F6,35=0.4, NS; outer layer: F6,35=0.6, NS). Therefore, synaptic recovery as measured by synaptophysin immunoreactivity was evident in ApoE knockout and wild-type mice, and the rate of this recovery was similar, although ApoE knockout mice showed a slightly greater loss of synaptophysin immunoreactivity post-lesion. Change in growth-associated protein 43 immunoreactivity after entorhinal cortex lesions. In sham animals from both wild-type and ApoE knockout mice, a similar pattern of GAP-43 immunoreactivity was noted, with the highest level of staining seen in the inner molecular zone. After ECL, there was a clear expansion of GAP-43 immunoreactivity within this region, as shown by a main effect of time (F5,30=13.4, P<0.01; see Fig. 7). However, these changes were seen in both ApoE knockout and wild-type mice, and no main effect of genotype (F1,30=0.7, NS) or genotypetime interaction (F5,30=0.7, NS) were found.

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Fig. 7. (A–C) Quantification of changes in synaptophysin immunoreactivity in wild-type () and ApoE knockout mice ( ) in the inner, middle and outer regions of the molecular layer following ECL. At each time-point, the level of immunofluorescence was determined for each animal on the lesioned side and calculated as a percentage compared to the corresponding sham side. ‘‘Pre’’ refers to a sham lesion compared to unoperated side; n=4 or 5 at each time-point, per genotype. Note that pre-lesion levels of synaptophysin immunoreactivity did not differ between groups (see Fig. 3). (D) Change in thickness in the inner molecular layer following ECL as assessed by GAP-43 immunohistochemistry. *P<0.05 vs ‘‘Pre’’. At no time-point was a significant difference noted between wild-type and ApoE knockout mice in either parameter.

Change in apolipoprotein E expression after entorhinal cortex lesions in wild-type mice. On the shamlesioned side of wild-type mice, there were very low levels of ApoE immunoreactivity largely confined to the lacunosum moleculare and the neuropil of the DG (molecular layer) and CA field. No staining was evident in the granule or pyramidal cell layers. In contrast, on the lesion side at day 6, there was a marked up-regulation of ApoE immunoreactivity, particularly in the molecular layer, CA3 and CA4 hilus regions (see Fig. 8A, B). Quantification confirmed the robustness of these changes and analysis at various time-points revealed that ApoE expression peaked at day 6, and declined steadily thereafter such that by day 28 little change could be detected between the sham and lesioned sides (see Table 2). Double staining with GFAP revealed that, at each timepoint, 75–90% of ApoE-positive structures co-stained for GFAP (Fig. 8C); occasional ApoE immunoreactivity independent of GFAP was noted (Fig. 8D).

ApoE western blot analysis using pooled tissue from two wild-type mice at day 6 post-lesion showed an increase in a 34000 mol. wt band on the lesioned side compared to the sham side (Fig. 8E). In addition, a second band at approximately 46000 mol. wt was noted, but in this instance only on the lesioned side. DISCUSSION

In the present series of experiments we have studied whether ApoE-deficient mice differ from wild-type controls in terms of cognitive ability, CNS structure (primarily hippocampal morphology), neuronal plasticity, assessed by the development and maintenance of amygdala kindled seizures or hippocampal LTP, and mechanisms of CNS repair, assessed by studying the synaptic remodelling that follows entorhinal cortex lesions. The results from each of these studies will now be considered in turn.

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Fig. 8. ApoE immunohistochemistry of the anterior hippocampus of a wild-type mouse on the lesioned (A) and sham-lesioned (B) sides at day 6 post-ECL. Note the clusters of immunoreactivity in the neuropil, particularly the molecular region of the DG on the lesioned side. There is negligible staining on the sham-lesioned side. In the majority of cases (C), ApoE immunoreactivity (brown) was associated with GFAP (blue), although occasionally some ApoE staining independent of GFAP was seen (D), as indicated by the arrow. (E) Western blot of hippocampal tissue from two wild-type mice, pooled according to sham (A) or lesioned (B) side. Tissue was probed with an ApoE antibody. Note the increase in immunoblot at mol. wt 34000 on the lesioned side. Also note the presence of a band at around mol. wt 46000 on the lesioned side only. Table 2. Changes in hippocampal apolipoprotein E expression at various time-points following entorhinal cortex lesions in wild-type mice Day 6

DG molecular layer Lacunosum moleculare CA3 region CA4/hilus region CA1/CA2 region

Day 14

Day 28

Sham

Lesion

Sham

Lesion

Sham

Lesion

2813 7931 3727 6139 93

30133* 1275 14874* 17427* 2715

43 87 22 0 22

15199* 10860* 13040* 17370* 249*

0 1611 0 22 0

5133* 5635 2920* 1414* 44

Data presented are the actual number of stuctures per unit area (µm210) positively stained with a polyclonal antibody to ApoE (Chemicon, AB947), as measured by image analysis. *P<0.05 vs sham-lesioned side (paired t-test).

Cognitive and neurological function In agreement with our previously published findings in female ApoE knockout mice of equivalent age,1,22 we could find no evidence of impaired water

maze learning in male ApoE knockout mice. Thus, rates of acquisition of cued and spatial (place) learning were indistinguishable between groups, and subsequent probe tests confirmed the use of a spatial strategy to perform the task. Furthermore, similarity

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of swim speeds suggested no obvious neurological difference between the groups, a finding supported by more formal neurological tests. Finally, analysis of plasma cholesterol levels confirmed the nature of ApoE gene deletion.59,79,82 These results differ from some recent studies, which have suggested cognitive impairments in ApoE knockout mice.19,44,52 In particular, our findings differ from those of Oitzl et al.,52 who reported negligible learning during the water maze trials and an unusual repetitive behaviour classified as ‘‘wall bumping’’. We have no immediate explanation for these differences beyond the source of these transgenic lines (see next section). However, the present results are consistent with the absence of electrophysiological or structural abnormalities in the hippocampus of our ApoE knockout mice. We are currently developing other cognitive models to extend this characterization of ApoE knockout mice. CNS morphology Gross inspection of CNS morphology using synaptophysin immunohistochemistry failed to reveal any major structural differences between ApoE knockout and wild-type mice (e.g., see Fig. 3A for the hippocampus). Comparison between these mice in terms of computer-aided quantitative analysis of MAP-2 and synaptophysin immunoreactivity failed to show differences in dendritic and presynaptic terminal density. Since these studies were conducted in mice aged eight to 10 months, the data contrast with those of Masliah et al.,43 who reported decreases in both markers within the molecular layer region of the DG and frontal cortex from mice of similar age. Although not formally presented in the current report, we have also failed to observe differences in the frontal cortex of ApoE knockout mice from our colony. Electron microscopy analysis of synapse density in CA1 and the DG confirmed the lack of difference between wild-type and ApoE knockout mice. Furthermore, stereological assessment of the granule cell density showed that deletion of the ApoE gene had no effect on the development or maintenance of neurons in the hippocampus. Electron microscopy analysis failed to detect the dendritic vacuolization reported previously.43 Clearly, methodological differences such as holding conditions, nature of the transgenic line, tissue preparation and quantification techniques may account for these disparate results. Regarding the

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nature of the transgenic line, differences in the transgene construct, embryonic stem cell line, host blastocyst strain, background strain, number of backcrossings and nature of the control line may all contribute to the observed phenotype. For example, variable susceptibility to transgenes,3,24 excitotoxins70 and cognitive/neurological differences11,18,33,54 have been reported between mouse strains. The animals analysed in the study by Masliah et al.43 were derived from the Rockerfeller University59 and UCSD line, whereas our mice were bred from the colony derived at the University of North Carolina, Chapel Hill.58 However, it should be noted that if a phenotype remains consistent between lines despite these caveats then it is probably significant. For instance, elevated plasma cholesterol and atherosclerosis are commonly reported for ApoE knockout mice, irrespective of source.59,79,82 It is interesting to note that two other preliminary studies, in addition to the present work, have failed to detect major structural abnormalities within the CNS of ApoE knockout mice up to one year of age.16,55 However, we are allowing an additional colony of ApoE knockout and wild-type littermate controls to age further, to see if differences emerge. Synaptic plasticity and kindling studies Mechanisms of plasticity such as those underlying LTP or kindling require the complex interaction of synaptic machinery,4,38 postsynaptic protein synthesis15,53 and structural modification,13 as well as modulatory influences from axon collaterals or afferent inputs.78 Consequently, studies of synaptic plasticity might reveal subtle abnormalities of CNS function or development which are not discernible from behavioural studies, or which are not manifest as a structural abnormality. However, it would be interesting to characterize synaptic plasticity in animals which do show structural deficits, such as those reported by Masliah et al.43 Animals at six and 12 months of age showed no deficits in any of the paired-pulse phenomena. Facilitation of the synaptic response (EPSP) is characteristic of normal presynaptic function;27 inhibition of an evoked population spike at short inter-pulse intervals is dependent on the integrity of hilar inhibitory circuits, and facilitation of the population spike at longer inter-pulse intervals may suggest normal function of extrahippocampal feedback circuits.66 The induction of LTP in ApoE knockout animals was also comparable to that in wild-type mice, and

Fig. 5. LTP and paired-pulse phenomena in ApoE knockout () and wild-type littermates ( ) at six and 12 months of age. (A, B) LTP of field EPSP slope. (C, D) Population spike amplitude. (E, F) Paired-pulse facilitation of EPSP. (G, H) Inhibition and facilitation of population spike. Individual points represent the meanS.E.M. EPSP slope or population spike amplitude of responses from six ApoE knockout and wild-type mice. The high-frequency train was delivered at t=0. Data are expressed as percentage of pre-HFS levels for LTP and percentage change for paired-pulse studies. There were no significant differences between wild-type and ApoE knockout mice in any of the parameters measured.

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potentiation was maintained for the duration of these acute experiments (1 h post-tetanus). Thus, there was no obvious deficit in mechanisms underlying induction or early maintenance of LTP. These results suggest that the absence of the ApoE gene had no effect on these complex mechanisms within the hippocampus, or on the development of the infrastructure necessary for their expression. It should be noted that the present experiments were limited to the DG; future studies should also include investigation of synaptic plasticity in CA3 or CA1, since normal LTP in one subfield of the hippocampus is no guarantee of normality in another.51 Six- to eight-month-old ApoE knockout and wild-type mice showed similar sensitivity to kindling stimulation, and each animal kindled equally rapidly and exhibited behavioural manifestations that were comparable across groups, and typical of murine amygdaloid kindling.7,8 However, a difference emerged in that the ApoE knockout group showed longer AD duration from trial 6, and this difference was maintained for the remainder of the trial period. We have no immediate explanation for this finding, except to note that the sudden increase in AD duration as the final stages of kindling are approached is typical of control groups from previous experiments (e.g., see Refs 7, 8). Thus, it could be argued that the control group in the present study showed an atypical AD response. At the completion of stage 5 kindling, the animals were left for four weeks before undergoing further kindling. Again, both groups rekindled rapidly, with all mice displaying stage 5 generalized convulsions on the first or second rekindling stimulation. Although the ApoE knockout group took slightly, but significantly, longer to rekindle to stage 5, the difference in rekindling rate was small and isolated, in that the groups did not differ in AD strength during rekindling, or in the characteristics of the convulsions themselves. The degree of temporary fall back in convulsion expression by the ApoE knockout group was comparable to that seen in normal rats kindled in the amygdala.12 In summary, the groups were comparable in seizure sensitivity, initial kindling rate and kindling permanence. In conclusion, these electrophysiological investigations suggest that the absence of ApoE does not compromise the normal development of the CNS or the expression of plasticity. There is currently no evidence for a role of ApoE in the mechanisms underlying synaptic plasticity, although ApoE’s putative involvement in microtubule stabilization,50,76 lipid transport37,60 and neurite outgrowth23,49,50,64 could indicate that the protein has a part to play in structural modifications accompanying neural plasticity. Since these changes may not occur for some hours after the induction of LTP, it would be prudent to study the progress of potentiation to at least this point in time. To this end, we are developing methods for recording from freely

moving mice.14 The study of kindling, however, did follow the expression of the phenomenon for weeks, during which structural alterations are likely to have occurred. Entorhinal cortex lesions The main findings from this study was that ApoE knockout mice clearly retained the ability for reactive synaptogenesis following ECL, although there may have been subtle differences between the test groups. Thus, following ECL there was an approximately 40% decrease in synaptophysin immunoreactivity in the middle and outer molecular layer at day 6 postlesion; however, by day 140 post-lesion this decrease had virtually disappeared in both groups. At each earlier time-point, however, the level of synaptophysin immunoreactivity was slightly lower in the ApoE knockout group, even at day 6. This may suggest a greater extent of damage following the lesion, a finding which may be consistent with some emerging data suggestive of ApoE knockout mice being more susceptible to acute CNS trauma.9,21,31,34,42 For instance, ECL is associated with microglial and astrocytic activation in terminal areas.62,71,75 Glial activation is associated with the release of reactive oxygen species and toxic cytokines, e.g., interleukin-1, interleukin-6 and tumour necrosis factor á, which may facilitate the phagocytic clearance of cellular debris by microglia prior to the generation of new synapses.17,56,57,71 Recent evidence suggests that ApoE may have a neuroprotective role, e.g., as an immunomodulator or antioxidant;9,21,30,31,34,46 this may preserve some synapses which might otherwise be lost by the gliosis which accompanies ECL. Similar to the synaptophysin result, changes in GAP-43 expansion within the inner molecular layer suggest that neuronal sprouting from the commisural/associational system can occur in ApoE knockout and wild-type mice.35,40 Again, no significant differences between these groups were noted, although the ApoE knockout group seemed to show a delayed response. Further studies of ApoE expression in the wild-type animals were consistent with the work of Poirier and co-workers. Thus, ApoE protein at day 6 was mainly confined to the neuropil and co-localized with GFAP, consistent with the synthesis of this protein in reactive astrocytes.62 Temporal expression of ApoE peaked at day 6, and by day 28 was returning to pre-lesion levels. Since these changes preceded the increase in GAP-43 and synaptophysin expression, this would be consistent with the proposed role for ApoE as a chaperone to carry lipid between astrocytes to neurons undergoing resprouting via uptake through the LDL/LRP receptor system.60,61 However, given the findings of Poirier and coworkers, and the changes in ApoE expression seen in wild-type mice in the present study, it is interesting to

Characterization of ApoE knockout mice

note that processes of reactive synaptogenesis can still be maintained in ApoE knockout mice. Popko et al.63 have shown that, following sciatic nerve crush, regenerating neurons in ApoE knockout mice were morphologically similar to those in control animals, despite a recognized role for ApoE in the redistribution of lipid necessary for sciatic nerve regeneration.5,25,37 Popko et al.63 concluded that, in the ApoE knockout line, other apolipoproteins may compensate for ApoE, e.g., ApoD, ApoA-I, ApoA-IV or ApoJ (clusterin, SGP-2).26 Indeed, ApoJ may be synthesized by astrocytes, can cross the blood–brain barrier39 and, like ApoE, its expression has been shown to increase in rat brain following ECL, kainate injection or cerebral ischaemia.28,29,45 Thus, it is feasible that ApoJ may compensate for loss of ApoE, for a lipid trafficking role for ApoJ has been described.26 Considering the subtle changes between the wild-type and ApoE knockout mice, however, it would be of interest to further explore these differences in other models of neurodegeneration and repair. These experiments are presently ongoing.21 Final comments The present studies have shown that ApoE gene deletion in mice does not have any marked effect on water maze learning, hippocampal structure or in

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processes of hippocampal/amygdaloid plasticity and repair. It should be noted that this does not preclude changes in other regions of the brain which were not studied in the present report. The null results might imply that ApoE is not involved in processes of cognition, CNS maintenance, plasticity and repair. However, a more likely explanation is that, in the knockout mice, alternative factors compensate for the absence of ApoE.67 In this respect, it is important to note that other apolipoproteins may be present in the CNS; one of these, ApoJ, may functionally substitute for ApoE. With respect to understanding the role of ApoE in Alzheimer’s disease, mice expressing human ApoE isoforms on a murine ApoE knockout background may provide a more valuable tool81 for future studies.

Acknowledgements—We would like to thank F. Boon (University of Western Ontario, Canada), for technical assistance; D.P.C. was supported by a grant from NSERC, Canada. Part of this work was conducted as an MRC LINK award between Glaxo Wellcome, The Open University and the National Institute for Medical Research (principal investigators: Charles Large, Tim Bliss, Steven Rose, Mike Stewart). Further thanks are due to David Bemister and Sarah Prior for maintaining the transgenic facility, Scott Poynter for mouse genotyping and Michael Evans for arranging the transfer of animals across sites.

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