Journal of PathologyJ Pathol 2009; 219: 41–51Published online 8 April 2009 in Wiley InterScience(
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Impaired learning and structural alteration in APP/PS1 mice                                                               ...
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Impaired learning and structural alteration in APP/PS1 mice                                                               ...
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Impaired learning and structural alteration in APP/PS1 mice                                                               ...
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Impaired learning and structural alteration in APP/PS1 mice                                                               ...
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Impaired learning and structural alteration in APP/PS1 mice                                                               ...
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Morphological alterations to neurons of the amygdala


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Morphological alterations to neurons of the amygdala

  1. 1. Journal of PathologyJ Pathol 2009; 219: 41–51Published online 8 April 2009 in Wiley InterScience( DOI: 10.1002/path.2565Original PaperMorphological alterations to neurons of the amygdalaand impaired fear conditioning in a transgenic mousemodel of Alzheimer’s diseaseShira Knafo,1 * † Cesar Venero,2† Paula Merino-Serrais,1 Isabel Fernaud-Espinosa,1 Juncal Gonzalez-Soriano,3Isidro Ferrer,4 Gabriel Santpere4 and Javier DeFelipe1 *1 Instituto Cajal (CSIC), Madrid, Spain2 Department of Psychobiology, Universidad Nacional de Educaci´ n a Distancia, Madrid, Spain o3 Department of Anatomy, Faculty of Veterinary Medicine, Complutense University, Madrid, Spain4 Institut Neuropatolog´a, IDIBELL-Hospital Universitari de Bellvitge, Universitat de Barcelona, Hospitalet ı de LLobregat, Barcelona, Spain*Correspondence to: AbstractShira Knafo or Javier DeFelipe,Instituto Cajal (CSIC), Patients with Alzheimer’s disease (AD) suffer from impaired memory and emotionalMadrid, Spain. disturbances, the pathogenesis of which is not entirely clear. In APP/PS1 transgenic mice,E-mail:; a model of AD in which amyloid β (Aβ) accumulates in the brain, we have neurons in the lateral nucleus of the amygdala (LA), a brain region crucial to establish† These cued fear conditioning. We found that although there was no neuronal loss in this region authors contributed and Aβ plaques only occupy less than 1% of its volume, these mice froze for shorter timesequally to this work. after auditory fear conditioning when compared to their non-transgenic littermates. WeThe authors have no conflicts of performed a three-dimensional analysis of projection neurons and of thousands of dendriticinterest to disclose. spines in the LA. We found changes in dendritic tree morphology and a substantial decrease in the frequency of large spines in plaque-free neurons of APP/PS1 mice. We suggest that these morphological changes in the neurons of the LA may contribute to the impaired auditory fear conditioning seen in this AD model. Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Received: 8 January 2009 Wiley & Sons, Ltd. Revised: 25 March 2009 Keywords: Alzheimer’s disease; unbiased stereology; morphology; confocal microscopy; Accepted: 27 March 2009 APP; PS1; amyloid; plaques; cognition; dementia; learning; dendritic spinesIntroduction For example, the lateral nucleus of the amygdala (LA) is a key site of plasticity that underlies fear learn-Alzheimer’s disease (AD) is a progressive neurode- ing [7,8]. The LA receives sensory input from thegenerative disease that causes dementia and emotional thalamus and cerebral cortex, and it generates emo-disturbances [1]. AD is neuropathologically charac- tional responses by activating different subcorticalterized by the accumulation of extracellular fibril- regions [9]. The outputs of the LA arise from pro-lar amyloid beta peptide (Aβ) in amyloid plaques jection neurons [10], which were the subject of this(plaques) and of intraneuronal neurofibrillary tangles study.consisting of aggregated hyperphosphorylated tau, and In mice, the expression of proteins that are impli-by elevated brain levels of soluble Aβ oligomers. cated in familial AD — a chimeric mouse/humanPlaques and neurofibrillary tangles are distributed in amyloid precursor protein (Mo/HuAPP695swe) and athe hippocampus, neocortex, and in subcortical regions mutant human presenilin 1 (PS1-dE9) — leads to thesuch as the amygdala, nucleus basalis, thalamus, locus early appearance of amyloid plaques [11]. We usedcoeruleus, and raphe nuclei [2]. The amygdala plays these double transgenic (APP/PS1) mice to investi-a major role in the processing and memorizing of gate the effects of Aβ overproduction and deposi-emotional reactions [3]. The amygdala of AD patients tion on auditory fear conditioning, and to examineundergoes significant shrinkage, distortion, and loss whether Aβ deposition provokes neuronal loss andof neurons, as well as extensive gliosis [4,5]. More- changes in the morphology of projection neurons inover, the extent of amygdaloid atrophy correlates pos- the LA. Previous studies performed in transgenic miceitively with the degree of emotional memory impair- carrying a single APP transgene, or different com-ment [6]. The different nuclei of the amygdala have binations of the APP and PS1 transgenes, reportedunique connections and they fulfil specific functions. contrasting results after training in the auditory cuedCopyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons,
  2. 2. 42 S Knafo et alfear conditioning paradigm [12–14]. Hence, we eval- conditioning was performed by pairing a tone to auated whether cued fear memory was impaired in foot shock and evaluating freezing during tone pre-12- to 14-month-old APP/PS1 mice. We show that sentation. Pain threshold was evaluated by applyingauditory fear conditioning is severely dampened in an electric current at increasing intensities and deter-APP/PS1 mice and that this cognitive impairment is mining the intensity that provoked discomfort. Detailsnot attributable to neuronal loss in the LA. of the behavioural procedures are described in the Sup- As projection neurons in the LA receive inputs porting information, Supporting material.through asymmetric synapses located mainly on theheads of dendritic spines [15], we examined the mor- Stereology and morphologyphology of the dendritic trees and of individual den-dritic spines in this region. Dendritic spines represent Unbiased stereology was employed to evaluate thethe main postsynaptic elements of excitatory synapses volume occupied by Aβ plaques and to determinein the cerebral cortex [16] and they are fundamen- the neuronal density using Stereo Investigator soft-tal in memory, learning, and cognition [17]. Dendritic ware (MicroBrightfield, Inc, Williston, VT, USA). Thespines undergo significant activity-dependent struc- density of the plaques and neurons in the LA wastural changes [18], which are also influenced by spine estimated using the optical fractionator method. Tohead size [19]. Importantly, recent evidence indicates estimate the plaque volume, the edges of each amy-that spine heads are affected by oligomeric Aβ [20] loid plaque were marked with the Nucleator probeand therefore the morphology of spine heads may link [27]. Dendrites were traced with Neurolucida (Micro-Aβ pathology with synaptic dysfunction. Brightfield) and spine morphology was measured with Previous studies have only identified a weak corre- Imaris software [28]. Details of the histological andlation between the presence of Aβ plaques and AD morphological procedures appear in the Supportingdementia [21], questioning whether plaques contribute information, Supporting dementia [22]. In an attempt to find a structuralbasis for amygdala-dependent cognitive impairment, Statisticswe used advanced imaging and measurement tech-niques to examine the morphology of the dendritic For the behavioural study, the results were analysedtrees and of individual spines. We measured the head using a two-tailed unpaired t-test or repeated measurevolume and neck length of thousands of dendritic ANOVA with the percentage of freezing for eachspines in the LA, both within and outside plaques. minute of the test as the repeated measure. ForWe found that the morphology of the dendritic tree the morphological study, the results were analysedof projection neurons that do not interact directly using a two-tailed unpaired t-test to test for thewith plaques is modified in the amygdala of APP/PS1 overall effect. When more than two groups weremice and that there is a significant decrease in large compared (for analyses of dendrites and spines), one-spines on these neurons. This is the first morphological way ANOVA was used, followed by Newman–Keulsdescription of dendrites and spines in the amygdala of multiple comparison post-hoc tests. Comparisons ofan AD model, although similar studies have been per- Sholl analysis plots were performed with two-wayformed previously in the hippocampus and neocortex ANOVA, followed by the Bonferroni post-hoc test.[23,24]. Comparisons between cumulative distributions were made according to two-sample Kolmogorov–Smirnov tests [29]. The significance of the results was acceptedMaterials and methods at p < 0.05 and the data are presented as mean ± SE.All experimental procedures were carried out in accor-dance with the guidelines set out in the European ResultsCommunity Council Directives (86/609/EEC). Normal anxiety-related behaviourMice We first determined, using an elevated plus-maze,The APP/PS1 mouse line used in this study (age whether APP/PS1 mice differ in their levels of anx-12–14 months, males) expressed a Mo/Hu APP695- iety, a factor that may influence the results of fearswe construct in conjunction with the exon-9-deleted conditioning [30] (see the Supporting information,variant of human presenilin 1 (PS1-dE9) [25]. The Supporting material). APP/PS1 mice displayed normalspecific strain was B6C3-Tg (APPswe, PSEN1dE9) anxiety-related behaviour (Figures 1a and 1b), imply-85Dbo/J. Age-matched littermates without the trans- ing that any change in fear conditioning cannot begene (Tg− ) served as controls. explained by different levels of anxiety.Behavioural procedures Impaired auditory fear conditioningAnxiety-related behaviour was evaluated in an ele- Auditory fear conditioning is a model for emotionalvated plus-maze as described previously [26]. Fear learning in animals and it is a response that dependsJ Pathol 2009; 219: 41–51 DOI: 10.1002/pathCopyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  3. 3. Impaired learning and structural alteration in APP/PS1 mice 43Figure 1. Normal anxiety-like behaviour and impaired auditory fear conditioning in APP/PS1 mice. (a, b) Exploration in theelevated plus-maze was considered a measure of anxiety. The total distance travelled in the maze (a) and the time spent inthe closed and open arms (b) were similar in APP/PS1 and Tg− mice. (c) Average percentage freezing before, during, and aftertone-shock pairings. (d) Freezing responses on the testing day before and during the presentation of the tone. Note that thepercentage of freezing is lower for APP/PS1 mice. ∗ p < 0.05; ∗∗∗ p < 0.001on the amygdala [31,32]. In this learning paradigm, not show alterations in their sensitivity to pain [35]an emotionally neutral auditory conditioned stimulus (see the Supporting information, Supporting material),elicits fear after it is paired with an aversive uncon- implying that conditioning was not affected by painditioned stimulus [33]. We tested the auditory fear perception.conditioning in these mice using immobility (freez-ing) as an index of fear learning [34]. During con-ditioning training, baseline freezing behaviour wasextremely low in both groups of mice before the Amyloid plaques occupy a small fraction of the LApresentation of the tone (Figure 1c). When freezingacross conditioning trials was analysed, it was evi- In an attempt to determine the extent to which audi-dent that both groups acquired fear responses, as tory fear conditioning might be affected by the Aβimplied from the increased freezing during the post- plaques in these animals, we first quantified the vol-shock periods (F2,38 = 18.78, p = 0.0001, repeated ume fraction that they occupied in the LA. We chosemeasures ANOVA). Statistical analyses of the freez- to examine this area because of the key role that iting behaviour between groups indicated no signifi- plays in the acquisition and expression of fear-relatedcant differences when animals were presented with the behaviour [36], and thus we immunocytochemicallytone (t11 = 1.30, p = 0.218) or immediately after foot stained Aβ plaques in sections of APP/PS1 brainsshock (t11 = 1.14, p = 0.277). (Figure 2a). Since counting spherical plaques in two- On the day of testing, both genotypes showed a dimensional cross-sections provides an imprecise mea-low amount of freezing during the baseline period sure of the amount of Aβ, missing small and het-on the test day (t11 = 1.77, p = 0.21, Figure 1d). erogeneous assemblies of Aβ [37], we used unbiasedHowever, following the presentation of the tone, stereology to count plaques and determine their vol-the freezing behaviour increased immediately in the ume. The density of plaques in the LA was 4.10 ± 0.45Tg− mice, while it remained relatively low in the plaques/mm3 (N = 4) and the average plaque volumeAPP/PS1 mice (t11 = 4.70, p = 0.0007). Indeed, the was 1496 ± 317.7 µm3 . The estimated volume occu-averaged freezing response over the 3 min of the pied by the Aβ plaques was only 0.60 ± 0.11%. Thesememory test showed a 42% reduction in APP/PS1 results suggest that Aβ plaques occupy a small volumemice compared with the Tg− animals, indicating fraction of the LA of aged APP/PS1 mice, in accor-that auditory fear conditioning memory is impaired dance with a previous study in which relatively fewin APP/PS1 mice. Importantly, APP/PS1 mice did Aβ plaques were found in the LA of AD patients [38]. J Pathol 2009; 219: 41–51 DOI: 10.1002/path Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  4. 4. 44 S Knafo et alFigure 2. Coronal sections through the amygdala and adjacent regions showing the pattern of distribution of plaques. (a) Anexample of the amygdaloid region as it appears in a section stained with the anti-Aβ antibody and Nissl, as used for plaquequantification. (b, c) Coronal sections from Tg− and APP/PS1 mice stained for anti-NeuN. (d) Double staining for anti-β amyloidand NeuN. (e) Neuron density for Tg− and APP/PS1 mice. BLA = basolateral nucleus of the amygdala; EC = external capsule;LA = lateral nucleus of the amygdala; PIR = piriform cortex. Scale bars: (a) 350 µm; (b–d) 120 µmNeuron density is conserved in LA significantly different between these three groups of neurons (Figures 3d and 3f, and Supporting informa-We determined whether neuronal loss in the LA tion, Supporting Table 1). However, a detailed anal-of APP/PS1 mice might explain the dampening of ysis of the length as a function of the distancethe auditory fear conditioning by assessing neuronal from the soma (Sholl analysis analysed with two-waydensity using unbiased stereology in sections stained ANOVA, p < 0.0001) revealed that neurons belongingwith antibodies for NeuN, a neuron-specific nuclear to APP/PS1 mice had a significantly smaller dendriticprotein [39] (Figures 2b–2d). No neuronal loss wasfound in the LA of APP/PS1 mice (Figure 2e) in line length 30–40 µm from the soma (p < 0.05, Bonfer-with previous studies performed in aged transgenic roni post-hoc test, Figure 3e). Also, there were fewermice with mutant amyloid precursor protein (APP), intersections at 30–40 µm from the soma (p < 0.05,where there was no neuronal loss in cortical areas [11]. Bonferroni post-hoc test, Figure 3g). These findings raised the possibility that the ram- ification of the dendritic tree differed in APP/PS1Altered dendritic structure neurons. Indeed, we found that the three neuronalSince auditory fear conditioning is thought to be medi- types differed significantly in the total number ofated by synaptic changes in the LA [40], and given that dendritic branches per neuron (p = 0.016, one-waymost excitatory synaptic connections occur on den- ANOVA, Figure 3h). Moreover, a post-hoc analysisdritic spines [41], we examined whether dendrites and revealed a significant (p < 0.05, Newman–Keuls mul-dendritic spines were altered in the LA of APP/PS1 tiple comparison test) decrease in this parameter formice. We traced projection neurons in the LA and then PFNs when compared with Tg− neurons (Supportingdivided them into three categories, according to their information, Supporting Table 1). Quantification of thelocation with respect to Aβ plaques (Figures 3a–3c): numbers of each dendritic branch order per neuron(1) neurons from control mice (Tg− ); (2) neurons revealed a change in the number of second-, third-,with no dendrite that enters a plaque (plaque-free and fourth-order dendrites per neuron in PFNs (p <neurons, PFNs); and (3) neurons with at least one 0.001, p < 0.05, and p < 0.01, respectively, two-branch of a dendrite passing through or entering a way ANOVA, Figure 3i, Supporting information, Sup-plaque (plaque neurons, PNs). The total dendritic porting Table 1). Moreover, a post-hoc analysis alsolength and the total number of intersections were not revealed a significant difference between the PNs andJ Pathol 2009; 219: 41–51 DOI: 10.1002/pathCopyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  5. 5. Impaired learning and structural alteration in APP/PS1 mice 45Figure 3. Intracellular injections and morphometric analysis. (a) Panoramic confocal (10×) views of the LA showingAlexa594-injected neurons and thioflavin-s-positive plaques in a Tg− mouse (left) and an APP/PS1 mouse (right). (b) Representativeimages of projection neurons from a Tg− mouse (left) and an APP/PS1 mouse (right). (c) The method used to distinguish dendritesand spines within and outside plaques. Left: a plaque suspected of containing a dendrite due to the rotation of its three-dimensionalimage. Centre: the plaque surface is marked with the aid of the IsoSurface tool of Imaris software. Right: the voxels outside thesurface are set to zero, leaving only the dendritic segment within the plaque. This process was performed after blind morphologicalmeasurements were made. (d) The total dendritic length of the different neuronal categories. (e) Sholl analysis showing thedendritic length as a function of the distance from the soma. Red asterisks represent a significant difference between Tg− and PFN;blue asterisks represent a significant difference between Tg− and PN; black asterisks represent a significant difference between PNand PFN. Inset: diagram showing a traced LA neuron and a series of concentric circles representing the Sholl analysis. This analysisis performed with concentric spheres around the soma rather than circles, in order to give a three-dimensional result. (f) Totaldendritic number of intersections for the different neuronal categories. (g) Sholl analysis showing the number of intersections asa function of the distance from the soma. The colour of the asterisks follows the scheme indicated in e. (h) Total number ofbranches per neuron for the different neuronal categories. (i) Quantity of branches per order per neuron. Scale bars: (a) 50 µm;(b) 25 µm; (c) 5 µm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001PFNs for the fourth branch order (p < 0.05, Bon- described for the neocortex [43] and hippocampusferroni post-hoc test, Figure 3i). We did not detect [28], typical thioflavin-s-positive plaques consisted ofany significant differences in the average tortuosity a core surrounded by a diffuse ring of decreasingof the different branch orders (Supporting informa- density. We encountered 20 dendrites that enteredtion, Supporting Table 1). Thus, we concluded that plaques (Figure 3c) and they were always locatedthe PFNs of APP/PS1 mice had a less complex den- in the diffuse peripheral ring (Figure 3c). Thus, likedritic tree. This finding is in line with a previous the classification of neurons, the dendrites were cat-study in which somatosensory cortical neurons were egorized according to their location with respect toless branched in TG2576 mice, another AD model Aβ plaques (Figure 4a): (1) dendrites from Tg− mice;[42]. (2) dendrites belonging to neurons with no dendrites entering a plaque (PFN); (3) segments of dendritesDecreased dendrite diameter and spine density within a plaque (Plaque); and (4) dendrites arisingwithin plaques from neurons of which one of their branches passed into or entered a plaque, in segments outside plaquesWe examined 259 amyloid plaques and 143 APP/PS1 (PN).injected projection neurons in the LA by laser scan- Dendritic shaft diameter differed significantlyning confocal microscopy (Figures 3a and 3b). As among these four categories of dendrites (p = 0.0001, J Pathol 2009; 219: 41–51 DOI: 10.1002/path Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  6. 6. 46 S Knafo et alFigure 4. The decrease in dendritic diameter and dendritic spine density is limited to plaques. (a) Representative projectionimages of dendrites from Tg− and APP/PS1 mice (63×, glycerol). Tg− , a dendrite from a control mouse; PFN, a dendrite belongingto plaque-free neurons of APP/PS1 mouse; PN, a dendrite arising from a neuron contacting a plaque in a plaque-free segment;Plaque, a dendrite that enters a plaque, with and without the green channel that contains the plaque image. (b) Dendrite diameterwas significantly decreased within plaques and (c) the spine density was also significantly lower inside plaques. (d) Spine densityas a function of the distance from the soma (Sholl analysis) is similar in Tg− mice and APP/PS1 mice in plaque-free regions (PNand PFN). (e) For each plaque-related dendritic segment, the distance of the plaque from the soma was measured, and the ratiobetween the spine density for the segment and the average spine density for the same distance in Tg− mice was calculated.(f) Spine density at increasing distances from the plaque edges. Note that outside the plaques, the spine density is conserved. Scalebar = 5 µm. ∗∗ p < 0.01; ∗∗∗ p < 0.001J Pathol 2009; 219: 41–51 DOI: 10.1002/pathCopyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  7. 7. Impaired learning and structural alteration in APP/PS1 mice 47one-way ANOVA, Figure 4b and Supporting informa- Discussiontion, Supporting Table 2). Accordingly, the dendriticshaft diameter inside plaques was significantly smaller In this study, aged APP/PS1 mice showed a damp-(20 dendrites, p < 0.001, Newman–Keuls multiple ening of auditory fear conditioning, a learning taskcomparison test) than in dendrites from PNs (45 that depends on the LA nucleus of the amygdala [36].dendrites), dendrites from PFNs (58 dendrites), and Using a series of experiments, we found that this inhi-Tg− dendrites (66 dendrites). Conversely, no sig- bition did not arise from a change in anxiety or thenificant differences were found between Tg− and animals’ sensitivity to shock. In addition, we show thatPFN, or PN dendrites in segments that did not pass the volume occupied by plaques in LA is less than 1%through a plaque. The spine density was also signif- and that the neuronal density in this nucleus was con-icantly different among the four categories of den- served. However, we did find that plaque-free neuronsdrites (p = 0.002, one-way ANOVA, Figure 4c) and in the LA of APP/PS1 mice have altered dendritic ram-it was significantly lower within plaques (p < 0.05, ifications and fewer large spines. Although previousNewman–Keuls multiple comparison test) than in the studies have shown changes in dendrites and spines inother categories of dendrites (Supporting information, transgenic mouse models of AD, these studies focusedSupporting Table 2). There were no significant dif- on the neocortex [23,42,44] and hippocampus [28].ferences in the spine density between PFN, PN and The results demonstrate for the first time that morpho-Tg− dendrites. logical changes in dendrites also occur in the amygdala Spine density normally changes as a function of the and that these changes may account for the dampeningdistance from the soma and these changes can be esti- in amygdala-dependent learning. APP/PS1 mice servemated using Sholl analysis. This analysis revealed that as a model of Alzheimer’s disease (AD) since theythe spine density for Tg− dendrites was not signifi- express two of the mutations that exist in patients ofcantly different from the density in PNs and PFNs over familial AD. AD patients show a marked impairmenttheir entire length (Figure 4d and Supporting informa- of fear conditioning [45], implying that the inhibitiontion, Supporting Table 2). The spine density for each of non-declarative memory is common to both ADsegment within a plaque was compared with the aver- patients and APP/PS1 mice. Thus, our results representage spine density at the same distance from the soma an additional demonstration that behavioural featuresin Tg− mice; there was a decrease of 33.43 ± 11.05% of this AD model resemble those found in AD [46].in the spine density within plaques (p = 0.0059, We have shown here that the performance int-test, Figure 4e). We then examined the spine den- an amygdala-dependent task is severely impaired insity as a function of the distance from plaques and we APP/PS1 male mice. Our results are in accordancefound that the spine density in the surrounding den- with the recent demonstration that overexpression ofdrites was not significantly different from the values APP in rodents, leading to elevated levels of Aβ damp-in Tg− mice. Thus, in accordance with our previous ens auditory fear conditioning [12,47]. Importantly,study of the dentate gyrus [28], our data show that the other authors have reported reduced contextual, butdecrease in spine density in the LA was restricted to not cued, fear conditioning memory in 5- or 9-month-plaques. old APP/PS1 mice [13]. The discrepancy between our results and these earlier findings may reflect the dif-Decreased frequency of large spines in PFNs ferent combination of APP and PS1 transgenes in theSpine head volume and neck length were measured in mice used in these two studies, which might affectthree dimensions in confocal image stacks (Figures 5a the amount of Aβ in the brains of these mice. Inand 5b) and for each group, the number of spines addition, different strains were employed in these stud-measured was Tg− , 3949; PFN, 3268; PN, 3577; and ies (C57B6/C3 versus C57B6/SJL), which may bePlaque, 63. No significant differences were found in relevant given that strain differences have alreadythe average spine neck length among these dendritic been reported to influence cued fear conditioning [48].categories (p = 0.48, one-way ANOVA, Figure 5c Indeed, SJL mice are more sensitive to cued fear con-and Supporting information, Supporting Table 2) and ditioning than C3 mice [48] and this sensitivity wouldlikewise, the average head volume was not signifi- facilitate the increase in freezing behaviour after cuedcantly different between the four categories of den- fear conditioning observed by Dineley et al [13]. Thisdrites (p = 0.42, one-way ANOVA, Figure 5d and enhanced freezing behaviour may mask the differencesSupporting information, Supporting Table 2). How- between the transgenic and the wild-type mice. More-ever, a cumulative frequency plot analysis revealed over, the training conditions used in our study (threea statistically significant shift in the head volume of foot shocks of 0.75 mA with an inter-trial intervalspines from PFNs when compared with that of spines of 30 s) were different from those used by Dineleybelonging to the other categories (p = 0.012–0.048, et al(two or five foot shocks of 0.5 mA with a 5 minKolmogorov–Smirnov test, Figure 5e). A decrease of or a 40 s interval between each CS–US pairing). Vari-74% was found in the frequency of large spines (head ation in the intensity of the foot shock, as well as involume > 0.18 µm3 ) when compared with Tg− neu- the inter-trial interval and total time spent condition-rons. Thus, PFNs have a lower frequency of large ing, affects the conditioning in response to explicitspines. and contextual stimuli [31,49,50]. Taken together, the J Pathol 2009; 219: 41–51 DOI: 10.1002/path Copyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  8. 8. 48 S Knafo et alFigure 5. Changes in the spine head volume of plaque-free neurons. (a) Maximum projection confocal images (63×, glycerol)of dendrites representative of each different category. (b) An amplified example of the head volume measurement obtained bydetermining the surface of the spine head (blue). (c, d) Bar graphs of the neck length and head volume indicating that no significantdifferences were found for the average values. (e) Left: a cumulative frequency plot showing the distribution of spine head volumes.Note the significant left shift in head volume in PFN (Kolmogorov–Smirnov test). Right: histograms for head volumes. Tg− , adendrite from a no transgene (control) mouse; PFN, a dendrite from a plaque-free neuron; PN, a dendrite from a plaque neuron;Plaque, a dendrite entering a plaque. See text for further details. Scale bar = 3 µmsetting in which training was performed may favour reasonable to hypothesize that the neuronal loss in thethe differences in fear learning between transgenic and amygdala is a late neuropathological feature of AD andcontrol mice. The alterations to conditioned fear are that other, more subtle synaptic changes may occur inunlikely to result from distinct perception and/or pro- early stages of the disease that cause the impaired fearcessing of the foot shock, or from different levels of memory [52]. We therefore examined dendrites andanxiety, because both Tg− and APP/PS1 mice showed dendritic spines, which are the major sites of synapticthe same pain sensitivity to a foot shock of rising inten- contacts in the brain.sity and similar anxiety-related behaviour. Three different observations from our study support We have shown here that in contrast to AD patients, the notion that Aβ plaques are not responsible forwho suffer marked neuronal loss in the amygdala [51], the dampened auditory fear conditioning in APP/PS1there is no neuronal loss in the LA of aged APP/PS1 mice. (1) Aβ plaques in the LA occupy less thanmice. Given the impaired auditory fear learning in 1% of its volume, implying that the changes withinthese mice, this finding raises doubts as to whether the plaques, the decrease in dendritic diameter andthe fear memory disturbances seen in AD patients arise spine density, are restricted to a small fraction offrom neuronal loss in the amygdala. Instead, it seems the neuropil. (2) The decreased dendritic ramificationJ Pathol 2009; 219: 41–51 DOI: 10.1002/pathCopyright  2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  9. 9. Impaired learning and structural alteration in APP/PS1 mice 49in APP/PS1 mice is limited to PFNs, neurons that and that they are therefore less able to generate feardo not contact Aβ plaques. Branching patterns are memories.related to the degree of compartmentalization of theinputs to the cell. Indeed, it has been proposed that Morphological changes within plaques may havea stronger potential for compartmentalization results a minor effect on cognitionin a significant increase in the representational power We have also shown here that dendrites that passand greater learning and memory capacity [53]. Since through a plaque are thinner and suffer from decreasedPFNs are less complex but they have a similar spine spine density. Aβ plaques have previously been asso-density, the fewer branches probably means the loss of ciated with local synaptic abnormalities and with asynapses, in accordance with previous findings in AD smaller diameter of the neuronal processes [23,44].brains [22,54]. Thus, the reduced complexity of the Since the correlation between the plaque load and thePFNs of APP/PS1 mice may contribute to their limited degree of memory impairment in transgenic mice isability to learn the fear response. Nevertheless, the relatively weak [70,71], the relevance of these plaque-possibility that impaired fear learning and the changes related morphological changes to AD pathogenesis isin dendritic ramification are two unrelated findings unclear and even questioned by many investigatorscannot be discarded. (3) The decrease in the frequency [72]. We suggest that the morphological changes insideof large spines in APP/PS1 mice is limited to the PFNs, plaques might affect local synaptic circuits. Neverthe-implying that the decreased frequency of large spines less, plaques only occupy a small volume of the LA,is not related to plaques. Importantly, we recently less than 1%, and thus the alterations to these localreported that plaque-free regions in the dentate gyrus circuits are restricted to only a small portion of theof APP/PS1 mice also have fewer large spines [28]. neuropil. Therefore, it is more likely that the changesSince in both studies the density of spines remained in dendritic complexity and in the frequency of largeunchanged outside the plaques, a decrease of large spines not directly related to the plaques contribute tospines appears to be a general feature of APP/PS1 the cognitive impairment seen in APP/PS1 mice.mice. Further studies in other brain regions will benecessary to confirm this possibility. The spine head volume reflects the size of the post- Acknowledgementssynaptic density [55–57], which correlates with the This work was supported by the following grants: CIBERNED,number of presynaptic vesicles and with the number RETICEF, Fundaci´ n Caixa (BM05-47-0), The EU 6th Frame- oof docked vesicles [55]. The postsynaptic density area work Programme (PROMEMORIA LSHM-CT-2005-512012),is proportional to the number of postsynaptic receptors and the Spanish Ministry of Science and Technology (grants[58], whereas the number of docked vesicles is propor- BFU2006-13395 to JD and BFU2006-01050 to CV). SK was the recipient of postdoctoral fellowships from the Ram´ n y otional to the ready releasable pool of transmitter [59]. Cajal Programme of the Ministry of Science and Technology.Therefore, the volume of the spine head is likely to We thank Ana Martinez and Gerard Muntane for genotypingbe directly proportional to the average reliability and the mice, and Luis Carrillo for technical assistance.strength of its synapse, and thus it is an important mor-phometric parameter reflecting its activity [55–59]. Ithas been reported that large spines in the LA are post- Supporting informationsynaptic to thalamic afferents and that they harbourR-type voltage-dependent Ca2+ channels (VDCCs), Supporting information may be found in the onlinethereby providing the synapses on their heads with version of this article.the capacity to express associative long-term modifi-cations of synaptic strength [60]. Given that long-termsynaptic plasticity in the LA is required for memory Referencesconsolidation of fear conditioning [3], a decrease inthe frequency of large spines may contribute to the 1. 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