RAPID COMMUNICATION

Layer-Specific Alterations to CA1 Dendritic Spines in aMouse Model of Alzheimer’s Disease

P. Merino-Serrais,1 S. Knafo,1,2* L. Alonso-Nanclares,1 I. Fernaud-Espinosa,1 and J. DeFelipe1

ABSTRACT: Why memory is a particular target for the pathologicalchanges in Alzheimer’s Disease (AD) has long been a fundamental ques-tion when considering the mechanisms underlying this disease. It hasbeen established from numerous biochemical and morphological studiesthat AD is, at least initially, a consequence of synaptic malfunctionprovoked by Amyloid b (Ab) peptide. APP/PS1 transgenic mice accumu-late Ab throughout the brain, and they have therefore been employed toinvestigate the effects of Ab overproduction on brain circuitry andcognition. Previous studies show that Ab overproduction affects spinemorphology in the hippocampus and amygdala, both within and outsideplaques (Knafo et al., (2009) Cereb Cortex 19:586-592; Knafo et al.,(in press) J Pathol). Hence, we conducted a detailed analysis of dendri-tic spines located in the stratum oriens and stratum radiatum of theCA1 hippocampal subfield of APP/PS1 mice. Three-dimensional analysisof 18,313 individual dendritic spines revealed a substantial layer-specificdecrease in spine neck length and an increase in the frequency of spineswith a small head volume. Since dendritic spines bear most of theexcitatory synapses in the brain, changes in spine morphology may beone of the factors contributing to the cognitive impairments observed inthis AD model. VVC 2010 Wiley-Liss, Inc.

KEY WORDS: morphology; confocal microscopy; amyloid beta;unbiased stereology; amyloid precursore protein

INTRODUCTION

The presence of Ab plaques is one of the pathological hallmarks ofAD, and they have been associated with changes in neurite morphologyand dendritic spine density (Tsai et al., 2004; Spires et al., 2005; Knafo

et al., 2009a, in press). Apart from amyloid plaques,the Ab peptide accumulates in different forms in AD:intracellular Ab and oligomeric Ab. Indeed, it hasbeen demonstrated that spine heads are targets ofoligomeric Ab (Lacor et al., 2007), and it has beensuggested that targeting and functional disruption ofparticular synapses by Ab oligomers may provide amolecular basis for the specific memory loss in AD(Lacor et al., 2007). Nevertheless, previous studiesindicate that there is only a weak correlation betweenplaque load and cognitive functions (Terry et al.,1991). In fact, plaques are sometimes detected even innondemented patients (Price et al., 2009) and cogni-tive decline is better reflected by the level of solubleAb oligomers, distributed diffusely outside the plaques(Selkoe, 2002). Moreover, at least in aged micebearing AD mutations, plaques occupy a negligiblefraction of the neuropil, less than 5% (Cohen et al.,2009;Knafo et al., 2009a, in press), and therefore, itis unlikely that cognitive impairment in these micearises solely from changes in synapses within theplaques. Hence, AD neuropathological research isincreasingly focusing on the changes in plaque-freeregions of the neuropil and in AD-like pathologyprior to plaque appearance. Here we have analyzeddendritic spines in the CA1 subfield, an area criticalfor spatial orientation and learning (Andersen et al.,2006), in order to determine the microstructural basisof the hippocampal-dependent cognitive impairmentin APP/PS1 mice (Malm et al., 2007). Dendriticspines represent the major postsynaptic elements ofexcitatory synapses in the cerebral cortex (Gray, 1959)and they are fundamental to memory, learning andcognition (Lamprecht and LeDoux, 2004). Dendriticspines undergo remarkable activity-dependent struc-tural changes (Lang et al., 2004; Tsai et al., 2004)and they are targets of oligomeric Ab (Lacor et al.,2007). Therefore, spine morphology may be associ-ated with Ab pathology and synaptic malfunction. Weshow that in APP/PS1 mice, CA1 spines necks are sig-nificantly shorter in the stratum oriens. In addition,the frequency of spines with a small head augments inthe same stratum radiatum of the same subfield. Thesefindings indicate that circuits in the stratum radiatumand the stratum oriens might be affected differently byAD-related mutations. In addition, this study suggests

1 Instituto Cajal (CSIC), Madrid, Spain and Laboratorio de Circuitos Cor-ticales, Centro de Tecnologıa Biomedica, Universidad Politecnica deMadrid, Madrid, Spain; 2Centro de Biologıa Molecular ‘‘Severo Ochoa’’,Consejo Superior de Investigaciones Cientıficas (CSIC)-UniversidadAutonoma de Madrid, Madrid, SpainAdditional Supporting Information may be found in the online version ofthis article.P. Merino-Serrais and S. Knafo contributed equally to this work.Grant sponsor: CIBERNED; Grant number: CB06/05/0,066; Grant spon-sor: EU 6th Framework Program; Grant number: PROMEMORIA LSHM-CT-2,005–512012; Grant sponsor: Spanish Ministerio de Educacion Cien-cia e Innovacion; Grant numbers: BFU2006–13395, SAF2009–09394,BES-2,007–16542; Grant sponsors: Fundacion CIEN (Financiacion deProyectos de Investigacion de Enfermedad de Alzheimer y enfermedadesrelacionadas 2008), Ministry of Science and Technology.*Correspondence to: Dr S. Knafo, Centro de Biologıa Molecular ‘‘SeveroOchoa’’, Consejo Superior de Investigaciones Cientıficas (CSIC)-Universi-dad Autonoma de Madrid, Madrid, Spain. E-mail: sknafo@cbm.uam.esAccepted for publication 25 June 2010DOI 10.1002/hipo.20861Published online in Wiley Online Library (wileyonlinelibrary.com).

HIPPOCAMPUS 00:000–000 (2010)

VVC 2010 WILEY-LISS, INC.

that dendritic spine morphology reflects the synaptic malfunc-tion arising from Ab overexpression.

We used a transgenic mouse line (12 to 14-month-old malemice) expressing a Mo/Hu APP695swe construct in conjunc-tion with the exon 9 deleted variant of human presenilin 1(PS1-dE9: (Scheuner et al., 1996). Age-matched littermatesserved as controls (Tg-). The mice were perfused with 4%paraformaldehyde and coronal sections of the fixed brain wereobtained. A total of 270 pyramidal neurons from Tg2 miceand 262 neurons from APP/PS1 mice were microinjectedindividually with Alexa594 (Invitrogen, Eugene, OR,Figs. 1a,b), and plaques were counterstained with thioflavin-safter injection (Figs. 1a–c). The plaques and dendrites in thestratum oriens (corresponding to basal dendrites) and in thestratum radiatum (collateral apical dendrites) were scanned witha Leica laser scanning multispectral confocal microscope (TCSSP5) using 488 and 594 nm laser lines. Image stacks (Physicalsize 76.9 3 76.9 lm, logical size 1,024 3 1,024 pixels) con-sisted of 100–350 image planes. A 633 Glycerol-immersionlens (NA, 1.3; working distance, 280 lm; refraction index,1.45) was used with a calculated optimal zoom factor of 3.2and a z-step of 0.14 lm (voxel size, 75.1 3 75.1 3 136.4nm). These settings and optics represent the highest resolutioncurrently possible with confocal microscopy. After acquisition,the stacks were processed over 10 iterations with a three-dimen-sional blind deconvolution algorithm (Autodeblur; Autoquant,Media Cybernetics) to reduce the out-of-focus light, andthereby removing the haze and the blur, restoring vital detailsto the datasets (Supporting Information Fig. 1b). The stackswere then opened with Imaris 6.0 (Bitplane AG, Zurich, Swit-zerland), a three-dimensional image processing software. Instacks containing images of Ab plaques (green), the green chan-nel was deleted and the stacks were coded (the codes were notbroken until the quantitative analysis had been completed).Spine density measurements and their morphology wereassessed by another investigator using only the red channel toassure impartiality (Figs. 1f,i). For spine density measurements,image stacks were viewed with a computerized data collectionsystem (Neurolucida 7.1 Confocal module; MicroBrightfield,Inc., Williston, VT), the image of the acquired dendrites wastraced in three-dimensions and the spines were marked during

tracing. To assess the morphology of spines, a solid surface thatexactly matched the contours of the head was constructed foreach spine using Imaris (Fig. 1i), and the length of the spineneck was measured manually in three-dimensions using thesame software (Isosurface module, see detailed methods). Toestimate the density of plaques, plaques were immunostainedwith an anti-Ab antibody in serial sections taken from thesame mice, and unbiased stereology rules were applied usingoptical fractionation and the Nucleator probe (Moller et al.,1990). For all the morphological parameters measured, the val-ues were averaged to give a neuron mean, and neurons fromeach animal were averaged for the animal mean. Normality wastested using the Kolmogorov-Smirnov test and a two-tailedunpaired t-test was used to test for the overall effect. Whenmore than two groups were compared, a one-way ANOVA wasused, followed by Tukey’s Multiple Comparison post hoc test.Data are presented as the mean 6 SEM.

We examined 1,475 amyloid plaques and 532 injectedpyramidal neurons by confocal microscopy (Fig. 1a). Weencountered only five dendrites that passed within plaques inthe stratum oriens (basal dendrites) and no such dendrites inthe stratum radiatum (apical dendrites). Typical plaques thatwere positive for thioflavin-s consisted of a core surrounded bya diffuse less dense ring. The dendrites passing through the pla-ques were located in the diffuse peripheral ring, as describedpreviously (Cruz et al., 1997; Knafo et al., 2009a, in press).Dendrites were categorized according to their location withrespect to the Ab plaques, as: (1) dendrites from transgene-negative (control) mice (Tg-); (2) dendrites located in aplaque-free area (Plaque-free); (3) segments of dendrites withina plaque (Plaque).

Spines Have a Shorter Neck in Plaque-FreeRegions of the Stratum Oriens of APP/PS1 Mice

The spine density in the stratum oriens was significantlydifferent among the three categories of dendrites (P 5 0.006,one-way ANOVA, Fig. 1d). Accordingly, the spine density wassignificantly lower within plaques than in other categories ofdendrites (0.85 6 0.17 spines/lm, N 5 4). However, spinedensity for Plaque-free dendrites (1.33 6 0.054, N 5 7) did

FIGURE 1. Spines are shorter in the stratum oriens of APP/PS1 mice. (a) A panoramic view of neurons injected with Alexa594 (red) and thioflavin-s positive plaques (green) in the hippo-campus (203, oil). (b) Representative projection images (403, oil)of injected neurons and plaques in Tg- (left) and APP/PS1 mice(right). The plaques seen in the stratum radiatum (apicaldendrites) are located close to labeled dendrites but they do notcontain dendrites, as determined by three-dimensional analysis.(c) An example of a dendrite located in the stratum oriens passingthrough a plaque and showing a decrease in spine density withinthe plaque. (d) Spine density is decreased significantly within theplaques. Note that the spine density is similar in Tg- mice and inthe plaque-free areas of APP/PS1 mice. (e) The spine density as afunction of the distance from the soma (Sholl Analysis) is similar

in Tg- and APP/PS1 mice in plaque-free regions. Note the similar-ity in spine density along the length of the dendrite. (f ) Represen-tative projection images of dendrites from Tg- and APP/PS1 mice(633, glycerol). Necks are marked as was done for measurements(g) Decreased average neck length for spines in APP/PS1 miceoutside plaques. (h) Cumulative frequency plots showing the dis-tribution of spine neck length, indicating a shift towards lowervalues in the entire spine population. (i) Representative imagesof dendritic segments with the contours of the spine heads ofAPP/PS1 mice constructed for each spine (see Supporting Infor-mation Fig. 1 for details). (j-k) The head volume is similar for Tg-and APP/PS1 mice. *P < 0.05, Tukey’s Multiple Comparison posthoc test. Scale bar, (a) 250 lm, (b) 25 lm, (c) 5 lm (f) 0.6 lm (i)0.8 lm.

2 MERINO-SERRAIS ET AL.

Hippocampus

FIGURE 1

ALTERED SPINES IN APP/PS1 MICE 3

Hippocampus

not differ significantly from that of control (Tg-) dendrites(1.348 6 0.070, N 5 6). Moreover, a Sholl analysis of thespine density at different distances from the soma revealed thatthe spine density for Plaque-free dendrites was similar to thatof control dendrites over their entire length (Fig. 1e).

Spine neck length and head volume were measured in threedimensions in confocal image stacks. Because of the extensiveloss of spines within plaques, the spines within plaques (31spines) were not included in the analysis of head and neckdimensions. Significant differences in the average spine necklength were found between the two dendritic categories (P 50.018, t-test, Figs. 1g,h). Spines in the APP/PS1 mice had asignificantly shorter neck (26%) than spines in Tg- mice spines(0.548 6 0.040). Head volume was not significantly different

among the two categories of dendrites (P 5 0.88, t-test,Figs. J,k), and was 0.056 6 0.003 lm3, for Tg- dendrites (N 56, 5,709 spines) and 0.055 6 0.003 lm3 for plaque-freedendrites (N 5 7, 5,145 spines). Thus, in the stratum oriensof APP/PS1 mice, spine density is decreased within plaques andspines are shorter outside plaques.

Small-Headed Spines Are More Frequentin Plaque-Free Regions of the StratumRadiatum of APP/PS1 Mice

Spine density and morphology were examined in apicalbranches protruding from the main apical trunk. These den-drites were located up to 300 lm from the stratum pyramidale

FIGURE 2. Increased frequency of spines with a small head inthe stratum radiatum of APP/PS1 mice. (a) Representative projectionimages of dendrites from Tg- and APP/PS1 mice (633, glycerol). (b)The spine density is similar in Tg- mice and in the plaque-free areasof APP/PS1 mice. (c) Spine density as a function of distance fromapical trunk is similar in Tg- mice and APP/PS1 mice in plaque-freeregions. (d) Constant average neck length for spines in APP/PS1mice. (e) Cumulative frequency plots showing the distribution of

spine neck length, indicating a similar distribution for both spinepopulations. (f) The frequency of small spines increases substantiallyin plaque-free areas of APP/PS1 mice. (g) Cumulative frequencycurves showing a shift towards smaller head volumes in APP/PS1mice. (h) A bar graph depicting a significant increase in the frequencyof small spines (volume < 0.03 lm3) in the stratum radiatum ofAPP/PS1 mice. Scale bar, 0.6 lm.

4 MERINO-SERRAIS ET AL.

Hippocampus

(cell body layer). Dendrites located in the stratum lacunosum-moleculare were not included in the analysis. The average spinedensity along the apical dendrites in the stratum radiatum didnot differ significantly in APP/PS1 mice when compared withthe control mice (1.71 6 0.08 for Tg- mice and 1.83 6 0.08for APP/PS1 mice: P 5 0.62, student t-test; Figs. 2a,b). ASholl analysis revealed that the density of spines on Plaque-freedendrites was not significantly different to that of control (Tg-)dendrites over their entire length (Fig. 2c). By contrast tothe stratum oriens, the neck length was similar in both groups(P 5 0.43, t-test, Figs. 2d,e), as it was 0.519 6 0.017 lm forTg- dendrites and 0.494 6 0.024 lm in APP/PS1 mice. Theaverage head volume in this layer did not differ significantlybetween Tg- dendrites (0.038 6 0.003 lm3: N 5 6; 4,437spines) and plaque-free regions (0.033 6 0.003 lm3: N 5 7;6,044 spines. P 5 0.29, t-test, Fig. 2f ). Nevertheless, thecumulative frequency curves clearly indicated a distinct distri-bution of head volumes in the smaller values (Fig. 2g) andindeed, a significant increase (29%, P 5 0.01, t-test) in thefrequency of small-headed spines (head volume < 0.03 lm3)was evident in the plaque-free areas of APP/PS1 mice(Fig. 2h). Thus, we found an increase in the frequency ofsmall-headed spines in the stratum radiatum of APP/PS1 mice.

Amyloid Plaques Occupy a SmallFraction of the CA1

We have described changes in dendritic spines within andoutside of plaques that can affect local synaptic circuits. Toquantitatively determine the impact of plaques on CA1 connec-tivity, we immunocytochemically stained Ab plaques in serialsections of APP/PS1 brains (Supporting Information Fig. 2).Using unbiased stereology, we then determined the totalnumber of plaques and their volume in the stratum oriens andstratum radiatum, from which we could calculate the totalvolume occupied by the plaques. The estimated total numberof CA1 plaques per mouse in one hemisphere was 1,551 6272.6 (range, 1,045–2,081 plaques/mouse; N 5 4: Table 1) inthe stratum oriens and 1985 6 183.8 (range 1,533–2,628plaques/mouse, N 5 5) in the stratum radiatum. The densityof plaques in the stratum oriens was 1,138 6 214.4 plaques/mm3, while it was 1,174 6 113.0 plaques/mm3 in the stratumradiatum (Table 1). The average plaque volume was 0.019 60.002 mm3 in the stratum oriens and 0.029 6 0.0,029 mm3 inthe stratum radiatum (P 5 0.04, t-test). The estimated volumeoccupied by Ab plaques was 1.421 6 0.201% in the stratumoriens and 1.554 6 0.195% in the stratum radiatum (Table 1).These results suggest that under our experimental conditions, Abplaques occupy a relatively small fraction of the CA1 neuropil.

This study shows that dendritic spines in the CA1 subfield aresignificantly affected by Ab, both within plaques and in plaque-free regions. Importantly, some changes in spine morphology inthis region were not evident in the dentate gyrus (Knafo et al.,2009a) or the amygdala (Knafo et al., 2009b) when studiedwith the same tools. For example, no significant differenceswere found in the length of spine neck in these regions,

whereas the neck was significantly shorter in the stratum ori-ens of APP/PS1 mice. Moreover, spines in the stratum oriensare affected differently to spines in stratum radiatum, evenwithin the CA1. These layer-specific morphological altera-tions are underscored by the fact that Ab plaques occupy asimilar total volume of both layers (Table 1), implying a sim-ilar Ab load. Thus, we conclude that AD-related mutationshave distinct effects on spines depending on their location.We also show here that dendrites within plaques are deficientin spines, in accordance with observations in other brainregions (Tsai et al., 2004; Knafo et al., 2009a, in press).Spine loss within Ab plaques can affect local synaptic cir-cuits. However, since plaques occupy a minor fraction of theCA1 (below 2%), the morphological changes observed out-side plaques are more likely to contribute to the synaptic andcognitive impairments found in APP/PS1 mice (Malm et al.,2007). We also found that spine density outside plaques isunchanged in APP/PS1 mice, in accordance to our previousstudies into the dentate gyrus and amygdala (Knafo et al.,2009a, in press). These findings imply that cognitive impair-ment in these mice (Malm et al., 2007) does not arise fromchanges in spine density in plaque-free areas. Rather, it islikely that changes in spine morphology outside plaques con-tribute to these cognitive deficits.

The data presented here shows that the average spine necklength is shorter in the stratum oriens of APP/PS1 mice, bothwithin and outside of the plaques. The morphology of thespine neck fulfils a key role in controlling the time windowcompartmentalization of calcium and other second messengersin spines (Yuste et al., 2000). The shortening of the spine neckin APP/PS1 mice may increase the diffusion between thespine and the dendrite. Spines with a fast diffusion equili-bration along the spine neck may be unable to retain secondmessengers or activate proteins upon the LTP inducing stimu-lus (Bloodgood and Sabatini, 2005). This altered plasticitymay eventually contribute to the cognitive impairment seen inAPP/PS1 mice (Malm et al., 2007).

We also found that APP/PS1 mice have a higher proportionof spines with a small head volume in the stratum radiatum.Importantly, small spines are more abundant after processesof long-term depression (LTD), a form of synaptic plasticitysignificantly enhanced in many models of AD (Shankar et al.,2008). Spine head size determines the size and duration ofsynaptic Ca21 transients (Majewska et al., 2000) and it is

TABLE 1.

Plaque Volume and Density

Parameter

S. Oriens

(N 5 4)

S. Radiatum

(N 5 5)

Estimated total number of plaques 1551 6 272.6 1985 6 183.8

Plaque density (plaque/mm3) 1138 6 214.4 1174 6 113.0

Individual plaque volume (lm3) 0.019 6 0.002 0.029 6 0.003

Volume occupied by plaques (%) 1.421 6 0.201 1.554 6 0.195

ALTERED SPINES IN APP/PS1 MICE 5

Hippocampus

therefore correlated with the magnitude of signals transmittedto the dendritic shaft (Harris and Stevens, 1989; Murthy et al.,2000). Spines with smaller heads have smaller postsynapticdensities (Harris and Stevens, 1989) and contain less AMPAreceptors on their heads when compared to spines with largerheads (Kharazia and Weinberg, 1999), resulting in less sensitiv-ity to glutamate at these spines (Matsuzaki et al., 2001). It istherefore possible that the increase in the frequency of small-headed spines reflects long-term synaptic depression, therebycontributing to the cognitive impairment seen in this ADmodel. Moreover, it is possible that the greater frequency ofspines with large heads in this layer reflects the loss of LTPin APP/PS1 mice (Trinchese et al., 2004). Therefore, thelaminar specific changes observed in spine length might havean important functional consequence in certain hippocampalcircuits. In summary, we show here that spines in the CA1 aremorphologically modified, which may reflect the functionalalterations at synapses induced by Ab overexpression in APP/PS1 mice.

DETAILED METHODS

Intracellular Injections With Alexa 594

Mice were anesthetized with pentobarbital (0.04 mg/kg)and transcardially perfused with 20 ml phosphate buffer (PB)followed by 100 ml of 4% paraformaldehyde (pH 7.4)prepared in the same buffer. The brains were postfixed in thesame solution for 24 h, and coronal sections (150 lm)were obtained on a vibratome and labeled with 10–5 M 4,6-diamidino-2-phenylindole (DAPI, Sigma D9542). Pyramidalneurons in CA1 were injected individually with Alexa594(Invitrogen, Eugene, OR) by passing a steady hyper-polarizing current through the electrode (0.5 to 21.0 nA,Figs. 1a,b). The current was applied until the distal tips ofeach neuron fluoresced brightly.

Morphology

Confocal microscopy

For each pyramidal neuron (5–7 neurons from each mouse,6–7 mice per group), 1–5 randomly selected dendrites werescanned from the soma (basal dendrites) or apical trunk (apicaldendrites) to the tip (125 dendrites total). In APP/PS1 mice,dendrites located within the plaques were also scanned. Foreach stack, the laser intensity and detector sensitivity were setso that the fluorescence signal from the spines occupied the fulldynamic range of the detector. Therefore, while scanning, somepixels were saturated in the dendritic shaft but no pixelswere saturated in the spines. In stacks containing images of Abplaques (green) the green channel was deleted. The stacks werecoded and the codes were not broken until the quantitativeanalysis had been completed.

Spine density

Dendritic spine density was determined by tracing theimage of the acquired dendrites in three dimensions (withNeurolucida). Spines were marked during tracing and all pro-trusions were considered as spines, applying no correction fac-tors to the spine counts. After tracing all the dendrites, each2-channel stack (containing the green channel with amyloidplaques) was viewed with Imaris and we determined whethera dendrite entered a plaque (Knafo et al., 2009a). The traceddendrites were viewed with Neurolucida and the correspond-ing stack with the green channel was opened. The traced den-drites were categorized as dendrites that passed through anamyloid plaque, or dendrites whose entire length was in a pla-que-free area. The reconstructed data were exported to Neuro-lucida Explorer (MicroBrightField Inc., Williston, VT) forquantitative analysis. Spine density was calculated for eachdendrite by dividing the dendritic length by the number ofspines. Spine density was also analyzed as a function of its dis-tance from its origin (Sholl analysis), dividing the length ofthe dendritic segment by the number of spines in each 10 lmstretch from the origin.

Head volume measurement

Intensity thresholds were applied to each dendritic segmentto generate a model of the data that was visualized as a solidsurface (the Spot module, Imaris). With this module, volumebased measurements are added to the volume rendering. TheSpots module models point-like structures in the data (e.g.,dendritic spines) providing a procedure to automatically detectsuch structures, an editor to manually correct any errorsdetected, a viewer to visualize the point-like structures asspheres, and a statistics output that includes the volume of thespine heads (Supporting Information Fig. 1b). A solid surfacethat exactly matched the contours of the head was created foreach dendritic spine (Fig. 1i). The image of each dendrite wasthen rotated in three dimensions and examined to ensure thatthe solid surface created for each spine head was correct. Head-less spines were extremely rare and they were not included inthe analysis. Having taken the measurements, the 2-channelsstacks were opened to view the plaques and the spines werecategorized according to their location (within or outside ofplaques). As described previously in an electron microscopystudy of hippocampal dendritic spines (Trommald and Hulle-berg, 1997), we did not observe a multimodal distribution forneck length or for head volume (Fig. 1–2). Consequently, wewere unable to identify spine groups such as thin, stubby ormushroom (Harris et al., 1992). Therefore, we chose todescribe spine morphology using measured dimensions ratherthan shape categories.

Neck length measurement

To measure the neck length, each dendrite was visualizedwith the Volume mode of Imaris. Individual spine necks weremeasured manually in three dimensions from the interface of

6 MERINO-SERRAIS ET AL.

Hippocampus

the spine neck with the dendritic stalk to the beginning of thespine head (MeasurementPro module) while rotating thedendritic image (Knafo et al., 2009a). If the spine head wasconnected directly to the dendritic stalk, the neck length wasrecorded as zero. As the resolution in the z axis is significantlylower than in the x-y axes, spines protruding perpendicular tothe dendritic stalk in the z axis were not measured. Thus,�60% of spines were measured.

Estimation of the Volume andNumber of Plaques

Immunocytochemistry

In slices taken from brains of the same mice used for themorphological studies, amyloid plaques were stained with anantihuman Ab antibody in systematically sampled coronalsections (50 lm thick, every sixth section in the hippocampus).A pilot study confirmed that this antibody exclusively marksthioflavin-s positive plaques (Knafo et al., 2009a). Free-floatingsections were pretreated with 55% formic acid (r-Aldrich,ACS) and washed with 0.1 M PB. Sections were treated with1% H202 for 30 min to deplete the endogenous peroxidaseactivity, and they were then submerged for 1 h in PB with0.25% Triton-X and 3% horse serum (Vector laboratories Inc.,Burlingame, CA). Sections were incubated overnight at 48Cwith a mouse antihuman Ab antibody (1:50; clone 6F/3D,Dako Glostrup, Denmark) and on the following day, thesections were rinsed and incubated for 2 h with a biotinylatedhorse antimouse antibody (1:200; BA-1,000; Vector). Thesections were then incubated for 1 h in an avidin-biotin per-oxidase complex (Vectastain ABC Elite PK6100, Vector) andfinally, the staining was visualized with the chromogen 3, 30

diaminobenzidine tetrahydrochloride (DAB; r-Aldrich, St Louis,MO). After staining, sections were dehydrated, cleared withxylene and cover-slipped. As a control, some sections wereprocessed as above but without the primary antibody, whichproduced no significant staining. Slices were then counterstainedby the Nissl technique to visualize the hippocampal strata.

Unbiased stereology

Unbiased stereology was used to quantify and measure pla-ques since counting spherical plaques in two-dimensional crosssections provides an imprecise measure of the amount of Ab(Stark et al., 2005). Using a stereotaxic atlas (Paxinos andFranklin, 2001) as a reference, eight slices at 21.1 to 23.8mm from bregma were analyzed in each brain. The Stereo In-vestigator software (Microbrightfield, Colchester, VT) was usedto drive a motorized stage (Prior Scientific, Houston, TX) on adual optical head microscope (Oltmpus BX 51) and to markand measure plaques at 40x (NA, 0.85) under brightfieldoptics. The software sequentially chose random counting frames(100 3 100 lm) in the xyz axes, moving the motorized stageautomatically within the previously delimited zones in the stra-tum oriens and the stratum radiatum.

To estimate the plaque volume after each amyloid plaquewas marked, the edges the plaque were marked with the Nucle-ator probe (Moller et al., 1990). The number of labeledplaques was estimated using the optical fractionator method inStereo Investigator. Plaques were marked only if their edges laywithin the dissector area and they did not intersect forbiddenlines, and if they came into the focus as the optical planemoved through the height of the dissector (20 lm). The guardzone thickness was set to 2 lm. The sum areas of the samplingsite represented around 15% of the total area of the slice. Thissampling method and the section interval was tested in a pilotexperiment to ensure that the estimation of the number ofplaques was representative of the total number.

Acknowledgments

The authors thank C. Hernandez and B. Garcıa for assis-tance with the confocal microscopy, and Dr I. Ferrer (InstitutNeuropatologia, Servei Anatomia Patologica, IDIBELL-Hospi-tal Universitari de Bellvitge, Universitat de Barcelona, Hospi-talet de Llobregat, Spain) for supplying the animals.

REFERENCES

Andersen P, Morris RG, Amaral D, Bliss T, O’Keefe J. 2006. TheHippocampus Book. USA: Oxford University Press.

Bloodgood BL, Sabatini BL. 2005. Neuronal activity regulates diffu-sion across the neck of dendritic spines. Science 310:866–869.

Cohen E, Paulsson JF, Blinder P, Burstyn-Cohen T, Du D, Estepa G,Adame A, Pham HM, Holzenberger M, Kelly JW, Masliah E, Dil-lin A. 2009. Reduced IGF-1 signaling delays age-associated proteo-toxicity in mice. Cell 139:1157–1169.

Cruz L, Urbanc B, Buldyrev SV, Christie R, Gomez-Isla T, Havlin S,McNamara M, Stanley HE, Hyman BT. 1997. Aggregation anddisaggregation of senile plaques in Alzheimer disease. Proc NatlAcad Sci USA 94:7612–7616.

Gray EG. 1959. Electron microscopy of synaptic contacts on dendritespines of the cerebral cortex. Nature 183:1592–1593.

Harris KM, Jensen FE, Tsao B. 1992. Three-dimensional structure ofdendritic spines and synapses in rat hippocampus (CA1) at post-natal day 15 and adult ages: Implications for the maturation ofsynaptic physiology and long-term potentiation [published erratumappears in J Neurosci 1992 Aug; 12(8): following table of con-tents]. J Neurosci 12:2685–2705.

Harris KM, Stevens JK. 1989. Dendritic spines of CA 1 pyramidalcells in the rat hippocampus: Serial electron microscopy with refer-ence to their biophysical characteristics. J Neurosci 9:2982–2997.

Kharazia VN, Weinberg RJ. 1999. Immunogold localization of AMPAand NMDA receptors in somatic sensory cortex of albino rat.J Comp Neurol 412:292–302.

Knafo S, Alonso-Nanclares L, Gonzalez-Soriano J, Merino-Serrais P,Fernaud-Espinosa I, Ferrer I, Defelipe J. 2009a. Widespread changesin dendritic spines in a model of Alzheimer’s disease. Cereb Cortex19:586–592.

Knafo S, Venero C, Merino-Serrais P, Gonzalez-Soriano J, Fernaud-Espi-nosa I, Ferrer I, Santpere G, Defelipe J. 2009b. Morphological altera-tions to neurons of the amygdala and impaired fear conditioning in atransgenic mouse model of Alzheimer’s disease. J Pathol 219:41–51.

Lacor PN, Buniel MC, Furlow PW, Sanz Clemente A, Velasco PT,Wood M, Viola KL, Klein WL. 2007. A{beta} Oligomer-induced

ALTERED SPINES IN APP/PS1 MICE 7

Hippocampus

aberrations in synapse composition, shape, and density providea molecular basis for loss of connectivity in Alzheimer’s disease.J Neurosci 27:796–807.

Lamprecht R, LeDoux J. 2004. Structural plasticity and memory. NatRev Neurosci 5:45–54.

Lang C, Barco A, Zablow L, Kandel ER, Siegelbaum SA, ZakharenkoSS. 2004. Transient expansion of synaptically connected dendriticspines upon induction of hippocampal long-term potentiation.Proc Natl Acad Sci USA 101:16665–16670.

Majewska A, Tashiro A, Yuste R. 2000. Regulation of spine calciumdynamics by rapid spine motility. J Neurosci 20:8262–8268.

Malm TM, Iivonen H, Goldsteins G, Keksa-Goldsteine V, AhtoniemiT, Kanninen K, Salminen A, Auriola S, Van Groen T, Tanila H,Koistinaho J. 2007. Pyrrolidine dithiocarbamate activates akt andimproves spatial learning in APP/PS1 mice without affecting{beta}-amyloid burden. J Neurosci 27:3712–3721.

Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, KasaiH. 2001. Dendritic spine geometry is critical for AMPA receptorexpression in hippocampal CA1 pyramidal neurons 1. Nat Neuro-sci 4:1086–1092.

Moller A, Strange P, Gundersen HJ. 1990. Efficient estimation of cellvolume and number using the nucleator and the disector. J Microsc159:61–71.

Murthy VN, Sejnowski TJ, Stevens CF. 2000. Dynamics of dendriticcalcium transients evoked by quantal release at excitatory hippo-campal synapses. Proc Natl Acad Sci USA 97:901–906.

Paxinos G, Franklin KBJ. 2001. The Mouse Brain in StereotaxicCoordinates, Deluxe, 2nd ed. San Diego, California: AcedemicPress.

Price JL, McKeel DW, Jr, Buckles VD, Roe CM, Xiong C, GrundmanM, Hansen LA, Petersen RC, Parisi JE, Dickson DW. 2009. Neuro-pathology of nondemented aging: Presumptive evidence for preclini-cal Alzheimer disease. Neurobiol Aging 30:1026–1036.

Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N,Bird TD, Hardy J, Hutton M, Kukull W. 1996. Secreted amyloid

beta-protein similar to that in the senile plaques of Alzheimer’s dis-ease is increased in vivo by the presenilin 1 and 2 and APP muta-tions linked to familial Alzheimer’s disease. Nat Med 2:864–870.

Selkoe DJ. 2002. Alzheimer’s disease is a synaptic failure. Science 298:789–791.

Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE,Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, ReganCM, Walsh DM, Sabatini BL, Selkoe DJ. 2008. Amyloid-b proteindimers isolated directly from Alzheimer’s brains impair synaptic plas-ticity and memory. Nat Med 14:837–842.

Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J,Nguyen PT, Bacskai BJ, Hyman BT. 2005. Dendritic spine abnor-malities in amyloid precursor protein transgenic mice demonstratedby gene transfer and intravital multiphoton microscopy. J Neurosci25:7278–7287.

Stark AK, Pelvig DP, Jorgensen AM, Andersen BB, Pakkenberg B. 2005.Measuring morphological and cellular changes in Alzheimer’s demen-tia: A review emphasizing stereology. Curr Alzheimer Res 2:449–481.

Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R,Hansen LA, Katzman R. 1991. Physical basis of cognitive altera-tions in Alzheimer’s disease: Synapse loss is the major correlate ofcognitive impairment. Ann Neurol 30:572–580.

Trinchese F, Liu S, Battaglia F, Walter S, Mathews PM, Arancio O.2004. Progressive age-related development of Alzheimer-likepathology in APP/PS1 mice. Ann Neurol 55:801–814.

Trommald M, Hulleberg G. 1997. Dimensions and density of dendri-tic spines from rat dentate granule cells based on reconstructionsfrom serial electron micrographs. J Comp Neurol 377:15–28.

Tsai J, Grutzendler J, Duff K, Gan WB. 2004. Fibrillar amyloid depo-sition leads to local synaptic abnormalities and breakage of neuro-nal branches. Nat Neurosci 7:1181–1183.

Yuste R, Majewska A, Holthoff K. 2000. From form to function: Cal-cium compartmentalization in dendritic spines. Nat Neurosci 3:653–659.

8 MERINO-SERRAIS ET AL.

Hippocampus