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β-Amyloid pathology in the entorhinal cortex of rats induces memory deficits: Implications for Alzheimer’s disease
Neuroscience 147 (2007) 28 –36
-AMYLOID PATHOLOGY IN THE ENTORHINAL CORTEX OF RATS INDUCES MEMORY DEFICITS: IMPLICATIONS FOR ALZHEIMER’S DISEASE E. SIPOS,a* A. KURUNCZI,b Á. KASZA,a J. HORVÁTH,a K. FELSZEGHY,c S. LAROCHE,d J. TOLDI,e Á. PÁRDUCZ,b B. PENKEa AND Z. PENKEa,d
mer’s disease and for screening drug candidates designed against A pathology. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
a
University of Szeged, Institute of Medical Chemistry, Dóm tér 8., H-6720 Szeged, Hungary
Key words: amyloid beta, Alzheimer’s disease, animal model, learning, memory.
b
Biological Research Center of Szeged, Temesvári krt. 62., H-6701 Szeged, Hungary
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive deterioration and memory loss. In the brain, neuropathological hallmarks of AD lesions include diffuse and neuritic extracellular amyloid plaques— consisting of fibrils formed from the amyloid- peptide (A) generated by endoproteolytic cleavage of the amyloid precursor protein (APP), reactive microglial cells, dystrophic neurites and bundles of astrocytic processes—and intracellular neurofibrillary tangles (Kar et al., 2004; Lahiri and Greig, 2004; Tran et al., 2002). The amyloid cascade hypothesis, which postulates a primary role of A in AD, is supported by several lines of experimental evidence (Golde et al., 2006; http://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asp). All four genes now known to be linked to familial AD or be risk factors for the sporadic forms of the disease have been shown to increase A production (APP, PS1, PS2) or A deposition in the brain (apoE; Selkoe and Podlisny, 2002). Injection of A into the brain can rapidly induce morphological degeneration, including neurite dystrophy, loss of synaptic integrity, microglia activation, reactive astrocytosis and neuron death (Kar et al., 2004; Nagele et al., 2004; Tran et al., 2002). It is becoming increasingly clear that the sequence and aggregation state of A has a major impact on its toxicity and mechanisms of action (Golde et al., 2006; Haass and Selkoe, 2007; Stephan and Phillips, 2005). For better designing therapeutic interventions targeting the A cascade, it is necessary to gain a better appraisal of the molecular and cognitive effects of different A forms. The most widely used models of AD, transgenic mouse lines, do not provide direct means to test the effects of different A forms on the development of the symptoms. This is why it is important to establish animal models where symptoms mimicking AD develop after injection of a known A form into the brain. Amyloid plaques appear in an early stage of AD in the temporal neocortex, the entorhinal cortex (EC) and the hippocampus (Braak and Braak, 1991; Duyckaerts, 2004; Thal et al., 2000, 2002). Neurodegeneration of the EC likely plays a major part in the memory dysfunction observed at early stages of AD (DeToledo-Morrell et al.,
c
Research Group for Psychopharmacology, Hungarian Academy of Sciences–Semmelweis University, Hûvösvölgyi u. 116., H-1021 Budapest, Hungary
d
Neurobiology of Learning, Memory and Communication, CNRS, Univ Paris Sud, UMR 8620, 91405 Orsay, France
e
University of Szeged, Department of Comparative Physiology, Közép fasor 52., H-6726 Szeged, Hungary
Abstract—Alzheimer’s disease is characterized by the presence of senile plaques in the brain, composed mainly of aggregated amyloid- peptide (A), which plays a central role in the pathogenesis of Alzheimer’s disease and is a potential target for therapeutic intervention. Amyloid plaques occur in an increasing number of brain structures during the progression of the disease, with a heavy load in regions of the temporal cortex in the early phases. Here, we investigated the cognitive deficits specifically associated with amyloid pathology in the entorhinal cortex. The amyloid peptide A1– 42 was injected bilaterally into the entorhinal cortex of rats and behavioral performance was assessed between 10 and 17 days after injection. We found that parameters of motor behavior in an open-field as well as spatial working memory tested in an alternation task were normal. In contrast, compared with naive rats or control rats injected with saline, rats injected with A1– 42 showed impaired recognition memory in an object recognition task and delayed acquisition in a spatial reference memory task in a water-maze, despite improved performance with training in this task and normal spatial memory in a probe test given 24 h after training. This profile of behavioral deficits after injection of A1– 42 into the entorhinal cortex was similar to that observed in another group of rats injected with the excitotoxic drug, N-methyl-D-aspartate. Immunohistochemical analysis after behavioral testing revealed that A1– 42 injection induced a reactive astroglial response and plaque-like deposits in the entorhinal cortex. These results show that experimentally-induced amyloid pathology in the entorhinal cortex induces selective cognitive deficits, resembling those observed in early phases of Alzheimer’s disease. Therefore, injection of protofibrillar-fibrillar A1– 42 into the entorhinal cortex constitutes a promising animal model for investigating selective aspects of Alzhei*Corresponding author. Tel: ⫹36-62-546-806; fax: ⫹36-62-546-806. E-mail address: [email protected] (E. Sipos). Abbreviations: A, amyloid- peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; EC, entorhinal cortex; GFAP, glial fibrillary acidic protein; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline solution.
0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.011
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2004; Stoub et al., 2006). The EC relays multimodal processed information from the sensory cortical areas to the hippocampus, as well as information processed by the hippocampus to permanent storage sites in the neocortex (Heinemann et al., 2000; Poucet et al., 2003), and as such is assumed to play an important role in memory processes. For example, lesion of the EC impairs various types of memory, including spatial and recognition memory and forms of operant learning (Davis et al., 2001; Eijkenboom et al., 2000; Kopniczky et al., 2006; Miwa and Ueki, 1996; Parron et al., 2004; Parron and Save, 2004; Ramirez et al., 1988; Ueki et al., 1994). In this study, our aim was to establish whether injection of a defined A form in the EC of rats induces histopathological alterations and cognitive deficits resembling those observed in human AD. To this end, we injected protofibrillar-fibrillar A1– 42 into the EC of rats, and examined the development of amyloid plaques and reactive astrocytosis in this structure as well as behavioral performance in spatial working and reference memory tasks and in a nonspatial, recognition memory task classically associated with temporal lobe function.
EXPERIMENTAL PROCEDURES Subjects and housing Adult male Wistar rats (n⫽54; Charles River, Isazeg, Hungary, bred at the University facility), weighing 300 –350 g and aged 8 –9 weeks at the beginning of the experiment were used as subjects. After arrival in the laboratory, they were housed by groups of four under constant temperature and lighting conditions (23 °C, 12-h light/dark cycle, lights on at 07:00). Rat chow and tap water were provided ad libitum. All efforts were made to minimize the number of animals and their suffering throughout the experiments. Experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Formal approval to conduct the experiments described has been obtained from the Animal Experimentation Committee of the University of Szeged.
Peptide synthesis and structure analysis A1– 42 was synthesized in our laboratory by a solid-phase procedure involving the use of Wang-resin and Fmoc chemistry (Zarandi et al., 2007). The synthetic peptides were purified on a C-4 RP-HPLC column with an acetonitrile gradient; pure fractions were pooled and lyophilized. A1– 42 was repeatedly aggregated and lyophilized from aqueous solution resulting in protofibrils (diameter 4 –5 nm, length 150 –200 nm) and fibrils (diameter 7–10 nm, length 300 nm–1 m) as observed using transmission electron microscopy (see Datki et al., 2004). Purity control and proof of structure were achieved by amino acid analysis and mass spectrometry (ESI MS, FinniganMat TSQ 7000).
Surgery, solutions Rats were anesthetized by i.p. injection with a mixture of ketamine (10.0 mg/0.1 kg) and xylazine (0.8 mg/0.1 kg). They were then placed in a stereotaxic frame, a midline sagittal incision was made in the scalp, and holes were drilled on the skull. The solution was injected into the EC at three bilateral sites (2.5 l per site) with a Hamilton syringe in a total of 60 min. The coordinates were (from bregma): AP: ⫺6.4, ⫺6.8, ⫺7.8; ML: ⫾4.0, ⫾3.6, ⫾3.0; DV: ⫺8.2, ⫺7.8, ⫺7.4 (Paxinos and Watson, 1982). Control animals were
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injected with sterile physiological saline. Amyloid-treated rats (A group) were injected with a 10⫺4 M A1– 42 solution in distilled water prepared immediately before injection. It has been shown that this solution contains mostly protofibrillar and fibrillar form of A1– 42 (Zarandi et al., 2007). N-methyl-D-aspartate (NMDA)treated rats (NMDA group) were injected with a 0.04 M NMDA (Sigma-Aldrich, Budapest, Hungary) solution in distilled water. Rats were treated with antibiotics and analgesics for 3 days after the surgery.
Behavioral testing Animals (nine intact, 17 control, 18 A, 10 NMDA rats) were allowed to recover for 10 days before the beginning of the behavioral tests. They were handled and weighed each day after the operation. All the behavioral experiments were carried out in the same room, illuminated by a halogen lamp giving diffused and dim light. A video camera was mounted on the ceiling directly above the test apparatus and images were relayed to a video tracking system. The behavior of the animals was automatically and/or manually recorded with the software EthoVision 2.3 (Noldus Information Technology, Wageningen, the Netherlands, 2002).
Open field test To assess the consequences of injections on explorative behavior, a 5-min open field test was performed on day 10 after A injection. The open field consisted of a black plastic rectangular arena (50⫻70⫻30 cm), the floor of which was covered with wood chips. At the beginning of the test, the animals were placed in the center of the arena. The following parameters were determined automatically: (1) total traveled distance, (2) percentage of time spent in the peripheral zone (thigmotaxis, Simon et al., 1994). The behavior was also scored by an observer who was unaware of the treatment of individual rats to record rearing, grooming and defecation behaviors.
Object recognition The object recognition test was used to assess whether recognition memory is impaired by A treatment. This test was carried out in the open field apparatus, on the day following the open field test, which therefore served also as a habituation session to the environment of the object recognition test. The test consisted of a single acquisition session (sampling phase), followed by a retention test 2 h later. Two types of objects were used, different in color, shape and material. For acquisition, each rat was placed in the middle of the arena containing two identical objects, and was left free to explore them for a total of 30 s of object exploration. Exploration of an object was defined as directing the nose to the object at a distance of less than 2 cm or touching it with the nose. Turning around or sitting on the object was not considered as object exploration. The animal was then placed back in the home cage for 2 h. Object recognition was tested in a 5-min session, during which one object used during the acquisition phase was replaced by a novel object. The nature and the spatial position of the objects were counterbalanced within each group in order to avoid any bias due to a preference that rats may have for a given object or its position in the arena. During the retention phase, the duration of exploration of each object was recorded, and the ratio of novel object exploration/total exploration was expressed as a percentage.
Spatial navigation in the water maze Spatial learning and memory were assessed in a Morris water maze on days 12–16 post-surgery (eight intact, nine control, eight A, nine NMDA rats). The maze consisted of a circular tank made of blue plastic (diameter: 130 cm, height: 80 cm) filled to a depth
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of 45 cm with water of 21⫾1 °C. The water was rendered opaque by the addition of milk. The tank was divided into four virtual quadrants. A white, hidden escape platform (diameter: 10 cm) was submerged in the middle of one of the quadrants (training quadrant), 30 cm from the rim of the pool and 2 cm below the water surface. The platform was not visible at water level. The rats had to learn to find the hidden platform on the basis of several constant spatial cues around the pool. During the training phase, five blocks of three trials were conducted between 09:00 and 13:00 h (on days 1, 2 and 3 of the test), and between 14:00 and 18:00 h (on days 1 and 2 of the test). Four different starting positions were used at the limits of the quadrants around the perimeter of the tank. A trial began by placing the rat into the water facing the wall of the pool; the starting position was varied pseudo-randomly over the trials. Rats were given a maximum of 90 s to find the platform and were then allowed to stay on it for 10 s. If a rat failed to escape from the water within 90 s, it was gently guided to the platform, and stayed there for 10 s. After the acquisition phase, retention was assessed in a 60-s probe trial (trial with the platform removed), on the day following the last training block. The data recorded by video-tracking were used to calculate the time to reach the platform, swim speed and swim paths lengths (distance) during acquisition trials as well as percentage of time spent in each of the four virtual quadrants and crossings over the platform’s position during the probe test. The means of the data from each block of trials were used for statistics.
Spontaneous alternation in a Y-maze In order to assess short-term memory, a Y-maze test was carried out on day 17 post-surgery. The apparatus was made of wood painted in black, and had three identical arms (each 14 cm wide, 19 cm high and 51 cm long), positioned in an equal angle. The floor of the maze was covered by wood chips. At the beginning of the test, each rat was placed in the center of the arena facing one arm. During the 10-min test period, the animal was allowed to move freely throughout the maze. The sequence of arm entries was recorded manually. Spontaneous alternation behavior was defined as successive entries into the three arms, on overlapping triplet sets (Yamada et al., 1998). Percentage of spontaneous alternation behavior was calculated as the ratio of actual to possible alternations, the latter defined as (total number of arm entries⫺2).
Statistics The results of different groups in the open field, object recognition, water-maze probe test and Y-maze tests were compared with one-way analysis of variance, followed by Fisher’s LSD post hoc tests for multiple comparison when appropriate. In the Morris water maze test, the swimming distance and latency data did not have a normal distribution, they were therefore analyzed with Kruskal-Wallis nonparametric test, followed by Mann-Whitney U tests for multiple comparison. Mean performance for individual groups was compared with chance level by one-sample Student’s t-test. The exploration durations of the two objects during the acquisition phase, as well as the time spent in the target and other quadrants in the Morris water maze were compared by paired Student’s t-test. In the description of results, F values (analysis of variance) and t values (Student’s t-test) and H values (KruskalWallis test) are given. The degrees of freedom (groups, subjects) for each test are shown as subscripts following the F, t and H. Statistical significance was set at Pⱕ0.05.
phosphate-buffered saline solution (PBS; room temperature, pH 7.4), followed by 300 ml of ice-cold paraformaldehyde solution (4% in phosphate buffer, pH 7.4). The brains were removed, post-fixed in the same fixative for 2 h, cryoprotected in sucrose solution (30% in PBS) for at least 36 h at 4 °C, and cut on a cryostat in 30 m coronal sections. Brain slices were collected and stored at ⫹4 °C in PBS for free-floating immunohistochemistry or Thioflavin T staining.
Immunohistochemistry and Thioflavin T staining Glial fibrillary acidic protein (GFAP) immunoreactivity, indicating reactive astrocytosis, was visualized by immunostaining with rabbit antibody (DakoCytomation, Glostrup, Denmark) used at a 1:20,000 dilution in PBS (pH 7.4). For A immunoreactivity, a mouse monoclonal antibody (4G8; AbCam, Cambridge, UK) was used at a 1:500 dilution. After quenching of endogenous peroxidase activity and a blocking step, the sections were incubated overnight at 4 °C with the primary antibody in the presence of 20% goat serum and Triton X-100 0.2%. On the following day, the sections were washed in PBS and incubated 1 h at room temperature with the secondary biotinylated mouse anti-rabbit antibody for GFAP (Sigma, 1:400), goat anti-mouse for 4G8 (Vector Laboratories, 1:500). The peroxidase reaction was carried out using the Vectastain Elite ABC Kit system (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine as the substrate and NiCl2 as an intensifier. After immunostaining and washing, all sections were mounted on gelatin-coated slides, air-dried, dehydrated and coverslipped with DPX mountant for histology (Fluka BioChemika, Buchs, Switzerland). For fibrillar amyloid-specific Thioflavin T staining (e.g. Kayed and Glabe, 2006), one series of sections was mounted on gelatincoated slides and dried overnight at room temperature. The sections were then washed in distilled water, incubated with a 0.5% Thioflavin T (Sigma-Aldrich, St. Louis, MO, USA) solution for 30 min, dehydrated and coverslipped. At least six sections per brain, (every twelfth section) from the dorsal hippocampus to the EC, from nine control and nine amyloid-treated rats were immunostained. The sections were processed at the same time and using the same solutions to reduce variability in immunostaining. Digital photographs were taken using a light microscope (Olympus Vanox-T AH-2; Olympus, Tokyo, Japan) and a CCD camera (Spot RT, Diagnostic Instruments, Sterling Heights, MI, USA). The observer of the sections was unaware of the treatment of the brains during the qualitative analysis, which was done with a computerized image analysis system (ImagePro Plus, Media Cybernetics, Bethesda, MD, USA).
RESULTS Open field In the open field test, parameters of exploratory behavior (total distance traveled, rearings) and anxiety (% time spent on periphery, grooming, defecation) were measured 10 days after the injections. Results showed that there was no significant difference among the groups in any of these parameters (total distance traveled: F3,50⫽0.14, P⫽0.93; rearings: F3,50⫽0.93, P⫽0.43; % time spent on periphery: F3,50⫽1.73, P⫽0.17; grooming: F3,50⫽0.13, P⫽0.94; defecation: F3,50⫽2.13, P⫽0.11).
Histology
Object recognition
After the behavioral tests, rats were killed by transcardial perfusion between days 20 –23 post-surgery. They were deeply anesthetized and perfused through the ascending aorta with 100 ml
The object recognition task was conducted on the day following the open field test, in the same apparatus. The two objects were explored equally during the acquisition
E. Sipos et al. / Neuroscience 147 (2007) 28 –36
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impaired in this task (Fig. 2B, C). All groups spent more time in the training quadrant than in the others (compared with the mean of the time spent in the other quadrants; intact t7⫽5.88, P⫽0.001; control t8⫽3.31, P⫽0.01; A t7⫽10.4, P⬍0.001; NMDA t8⫽4.80, P⫽0.001). The four groups spent a similar amount of time in the training quadrant (F3,31⫽0.29, P⫽0.83). Moreover, the number of crossings over the platform was also similar between the groups
Fig. 1. A1– 42 or NMDA injection into the EC impairs object recognition memory. Retention performance is expressed as the percentage of time spent exploring the novel object over the total time of object exploration (mean⫾S.E.M.). Intact and control animals explored preferentially the novel object while performance of animals injected with A1– 42 (A) or NMDA did not differ from chance level. * Different from chance level at P⬍0.05.
phase (t⫽0.28, P⫽0.78). During the retention phase, significant between-group differences in performance were observed (F3,50⫽4.41, P⫽0.007). Specifically, both intact and control rats explored preferentially the novel object (time spent exploring the novel object significantly different from chance level; t18⫽4.72, P⫽0.0001 and t8⫽3.04, P⫽0.01, respectively; Fig. 1), while rats injected with A1– 42 or with NMDA showed a similar level of exploration of the novel and familiar objects (t17⫽1.06, P⫽0.3 and t9⫽0.78, P⫽0.45, respectively; Fig. 1). Between-group comparisons showed that intact and control rats had similar recognition memory performance and performed better than the A1– 42-treated or NMDA-treated rats (A-control: P⫽0.009; A-intact: P⫽0.006; NMDA-control: P⫽0.038; NMDA-intact: P⫽0.02). All the groups had a similar overall duration of object exploration during the retention phase (F3,50⫽0.98, P⫽0.43). Spatial learning in the water maze The Morris water maze test was used to assess spatial learning and memory on days 12–16 after the injections. The results showed that although performance of each group improved significantly across blocks of trials, learning was slower in the A- and the NMDA-treated groups, compared with control rats (Fig. 2A). Specifically, in the third block, A- and NMDA-treated rats swam longer distances to find the platform than control rats (H3,31⫽7.63, P⫽0.05; control-A: P⫽0.027, control-NMDA: P⫽0.1). In the fourth block, intact rats were quicker to find the platform than all the other groups (H3,31⫽10.57, P⫽0.01; intact-control: P⫽0.02; intact-A: P⫽0.007; intact-NMDA: P⫽0.006). No differences were observed between NMDA- and A-treated groups in the third or the fourth block (P⫽0.847 and P⫽0.700, respectively). There were no significant differences among the groups during the other blocks (first block: H3,31⫽3.3, P⫽0.34; second block: H3,31⫽4.18, P⫽0.24; fifth block: H3,31⫽6.86, P⫽0.07). Despite slower learning in the A1– 42-treated and NMDA-treated rats, performance at the probe test given 24 h after learning showed that spatial memory was not
Fig. 2. Injection of A1– 42 or NMDA in the EC resulted in slower spatial learning in the water-maze. (A) Performance during acquisition is expressed as the mean (⫾S.E.M.) swim paths lengths (distance) during the five training blocks. In block 3, A and NMDA rats swam a longer distance than controls to find the platform. By block 5, however, there was no significant difference between groups. (B) Injection of A1– 42 or NMDA did not affect performance in the probe trial 24 h after training. All groups spent more time in the training quadrant than in the other three quadrants, with no significant difference between groups (mean⫾S.E.M. is shown). (C) During the probe test, there was no significant difference between groups in the number of crossings over the place where the platform was during acquisition (mean⫾S.E.M. is shown).
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intact: t8⫽3.27, P⫽0.011; control: t18⫽6.79, P⬍0.0001; A: t17⫽7.17, P⬍0.0001; NMDA: t9⫽5.63, P⫽0.0003; Fig. 3). The performance of the groups did not differ from each other (F3,50⫽0.058, P⫽0.98). Moreover, the total number of arm entries was comparable in the different groups (F3,50⫽1.95, P⫽0.13), suggesting a similar level of motivation for exploration. Histology Fig. 3. Injection of A1– 42 or NMDA did not alter spontaneous alternation behavior in a Y-maze. Histograms show the percentage of alternation (consecutive entries into three different arms) over the total number of arm entries for each group (mean⫾S.E.M.). * P⬍0.05; *** P⬍0.001, compared with chance.
(F3,31⫽0.93, P⫽0.43). Finally, there were no significant differences in swim speed among the groups at any stage of behavioral testing. Spontaneous alternation The effect of A1– 42 on spontaneous alternation was examined on day 17 post-injection in a Y-maze. All groups showed alternation performance above chance level (50%,
After behavioral testing, ⬃3 weeks after A1– 42 injections, histological examination was performed on the brains of amyloid-treated and control rats to assess inflammatory reaction and the presence of aggregated material in the EC. First, GFAP staining was used to detect reactive astrogliosis as an indication of an inflammatory reaction. In contrast to control animals in which slight astrogliosis was found restricted to the injection tracts (Fig. 4A, C), likely caused by mechanical damage, astrocyte proliferation was apparent in a much larger area extending farther apart from the amyloid injection sites in the EC (Fig. 4B, D). Moreover, in amyloid-treated animals, astrocyte somata had a larger appearance and GFAP staining was more intense than in controls.
Fig. 4. Representative examples of GFAP staining in the EC of a control (A, high magnification: C) and an A1– 42-injected (B, high magnification: D) rat. In control animals, only some reactive astrocytes along the injection track can be observed among normal glial elements. In the amyloid-treated group, numerous reactive astrocytes can be seen around the injection sites, extending to the whole EC.
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Fig. 5. Representative examples of sections showing A1– 42 staining (4G8 antibody) in the EC of a control (A) and an amyloid-injected (B, C) rat. In the amyloid-treated group, aggregates with apparent filaments can be seen around the injection sites (B, high magnification: C). The fibrillar nature of at least some amyloid aggregates is confirmed by positive Thioflavin T staining (light patch on D).
Second, to assess whether injection of A1– 42 peptides led to amyloid deposits in the EC, specific amyloid immunostaining (4G8) was used. Examination of the EC revealed plaque-like depositions in A1– 42-injected animals (Fig. 5B, C), but only non-specific staining in controls (Fig. 5A). The presence of amyloid depositions was the highest around the injection sites, with only a few deposits observed in other parts of EC. The diffuse plaque-like structures were diverse in size, but they were all composed of many fibril-like structures. The presence of fibrillar A was confirmed by the strong Thioflavin T positive staining observed in the EC (Fig. 5D), as this colorant is specific for A of fibrillar structure (Kayed and Glabe, 2006). The extent of A immunoreactivity and of astrogliosis in the EC and adjacent structures was analyzed on serial brain sections. A schematic representation of the smallest and largest extent of the area covered by amyloid deposits and reactive astroglia is shown in Fig. 6. In each individual case, good overlap was observed between the areas of astrocytic proliferation and A deposits (see details in figure legend).
DISCUSSION The purpose of the present study was to examine the specific contribution of amyloid pathology in the EC to the cognitive deficits associated with AD. To this end, we tested learning and memory ability of rats injected with A1– 42 into the EC in different tasks believed to depend on the integrity of medial temporal lobe structures. Our results show for the first time that injection of A into the EC of rats results in selective impairments in memory functions, char-
acterized by spatial learning retardation and severe deficits in object recognition memory. Recognition memory is dependent on the integrity of structures of the medial temporal lobe, including the hippocampus and, possibly to an even greater extent, the surrounding parahippocampal areas (see review in Mumby, 2001; Squire et al., 2004). Previous studies have shown that excitotoxic lesions of the EC impair recognition memory in rats (Galani et al., 1998; Mumby and Pinel, 1994; Parron and Save, 2004). Here, we confirm the importance of the EC in object recognition memory, as NMDA injections severely impair performance in the novel object recognition task, and we show that injection of A1– 42 also results in a similar recognition memory deficit in this task. The deficit cannot be accounted for by an alteration in object exploration, as all rats explored the two novel objects for an identical time during the acquisition phase and had a similar amount of total object exploration during the test phase. Spatial reference memory measured in the Morris water maze is largely dependent on an intact hippocampus, and can also be affected by lesions of certain parahippocampal areas (reviewed in Aggleton et al., 2000). EC lesions have been reported to impair place navigation (Aggleton et al., 2000; Eijkenboom et al., 2000; Kopniczky et al., 2006; Parron et al., 2004; Spowart-Manning and van der Staay, 2005). However, some studies failed to find an effect of EC lesions (e.g. Galani et al., 1998), probably due to the use of proximal rather than distal landmarks (Burwell et al., 2004; Parron et al., 2004), or to other differences in experimental paradigms affecting the choice of different navigational strategies in the water maze (Aggleton et al.,
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Fig. 6. Schematic drawings of the region lesioned by A injection on three different coronal sections at AP ⫺6.3, ⫺6.8 and ⫺7.8 from bregma (Paxinos and Watson, 1982). We found intense amyloid and GFAP staining (dark gray) in the vicinity of the injection sites. The largest extension of A immunoreactivity and astrocytic proliferation extending over these areas is shown in light gray. CA, CA1 region of the hippocampus; DG, dentate gyrus of the hippocampus; Prh, perirhinal cortex.
2000). In our experimental conditions, NMDA lesion of the EC slowed down the rate of spatial learning, without otherwise affecting the rats’ ability to learn with repeated training or the expression of good spatial memory performance 24 h after training. With injections of A1– 42, again a similar profile of impairment was observed. In contrast, in our experimental conditions we found no evidence for a working memory impairment in the spontaneous alternation task after either NMDA or A1– 42 injection. Although there is some evidence that the EC may be involved in this task, at least at some point of ontogeny (Blozovski and Hess, 1989; Degroot and Parent, 2000; Gibbs et al., 1987), spatial working memory deficits are more consistently found after hippocampal lesions (reviewed in Lalonde, 2002) and, interestingly, working memory deficits have been clearly identified after A1– 42 injection into the hippocampus (O’Hare et al., 1999; Stephan et al., 2001). Thus, our study reveals that injection of A1– 42 inducing the formation of stable A aggregates in the EC results in a profile of cognitive impairments similar to that observed after NMDA injection, suggesting that injected A induces functional alteration of the EC. Bilateral excitotoxic lesion of the EC in rats has been suggested as a model for the early stages of human AD, because they share some similarities in their pathological and cognitive characteristics (Spowart-Manning and van der Staay, 2005). Moreover, cholinergic drugs used in the treatment of AD have been reported to improve recovery from the behavioral deficit induced by EC lesion (Spowart-Manning and van der Staay, 2005). However, injection of excitotoxic molecules is likely to induce neuron loss or lesions in the EC that have a neuropathological origin different from the lesions observed in AD. Our model therefore seems more suitable in the context of understanding the etiology of AD
or for testing therapeutic candidate molecules designed to protect against A pathology. Direct injection of A peptides into specific brain areas has been a widely used strategy to test animal models of AD. In these models, the nature and degree of cognitive impairments observed after A injection depend largely on the targeted structure, the A sequence length and aggregation state, the solvent, the time elapsed after the injection and the behavioral tasks used (Chacon et al., 2004; De Ferrari et al., 2003; Shen et al., 2001; Stephan et al., 2001; for a review, see Stephan and Phillips, 2005). It has been suggested that injection of soluble (monomeric or oligomeric) forms of A induces an immediate, but transient impairment of memory because of their detrimental effects on synaptic plasticity (Cleary et al., 2005; Stephan et al., 2001; Stephan and Phillips, 2005). On the other hand, the later-onset and longer-lasting effects on memory induced by injections of aggregated insoluble forms of A (or by the injection of the soluble but highly fibrillogenic A1– 42) are probably related to inflammatory reactions induced by fibrillar A (Stephan et al., 2003; Stephan and Phillips, 2005). Our histological analysis showed that the injected soluble A aggregated into plaque-like structures in the EC and we also confirmed previous data showing signs of intense inflammatory reaction characterized by activation of astrocytes and microglial cells surrounding and infiltrating the deposits formed after the injection of aggregated A, regardless of the target structure in the CNS (e.g. Giovannelli et al., 1998; Stephan and Phillips, 2005). Accumulation of astrocytes has also been observed around senile plaques in human AD, and their peripheral distribution around A deposits suggests a role in containing or circumscribing the abnormal protein, a well-known function
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of astrocytes in response to injury (Vehmas et al., 2003). Moreover, a recent study in a triple transgenic mouse model of AD reported that the inflammatory reaction that occurs in this model appears earlier in the EC than in the hippocampus (Janelsins et al., 2005). Although we cannot dissociate the specific contribution of aggregated amyloid peptide and inflammation in the memory deficits observed here after injection in the EC, it is possible that cognitive dysfunction induced by fibrillar A into the EC can be at least in part due to the associated extensive inflammatory reaction (Stephan et al., 2003; reviewed in Sastre et al., 2006).
CONCLUSION In conclusion, our results suggest that injection of protofibrillar-fibrillar A1– 42 into the EC of rats constitutes a suitable experimental model for some aspects of the early stages of AD. This in vivo model could be valuable for investigating specifically in the EC the differential pathological and cognitive consequences of different sequences and different aggregation states of A peptides, and for screening drug candidates designed to combat the deleterious effects of fibrillar A. Acknowledgments—This work was supported by grants from NKTH-RET 08/2004 DNT, OTKA TS 049817, ETT 476/2006 and NKFP1/A/005/2004. The authors thank the chemists of the Institute of Medical Chemistry for A synthesis and Professor Csaba Nyakas for helpful discussion.
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(Accepted 5 April 2007) (Available online 17 May 2007)
-AMYLOID PATHOLOGY IN THE ENTORHINAL CORTEX OF RATS INDUCES MEMORY DEFICITS: IMPLICATIONS FOR ALZHEIMER’S DISEASE E. SIPOS,a* A. KURUNCZI,b Á. KASZA,a J. HORVÁTH,a K. FELSZEGHY,c S. LAROCHE,d J. TOLDI,e Á. PÁRDUCZ,b B. PENKEa AND Z. PENKEa,d
mer’s disease and for screening drug candidates designed against A pathology. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
a
University of Szeged, Institute of Medical Chemistry, Dóm tér 8., H-6720 Szeged, Hungary
Key words: amyloid beta, Alzheimer’s disease, animal model, learning, memory.
b
Biological Research Center of Szeged, Temesvári krt. 62., H-6701 Szeged, Hungary
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive deterioration and memory loss. In the brain, neuropathological hallmarks of AD lesions include diffuse and neuritic extracellular amyloid plaques— consisting of fibrils formed from the amyloid- peptide (A) generated by endoproteolytic cleavage of the amyloid precursor protein (APP), reactive microglial cells, dystrophic neurites and bundles of astrocytic processes—and intracellular neurofibrillary tangles (Kar et al., 2004; Lahiri and Greig, 2004; Tran et al., 2002). The amyloid cascade hypothesis, which postulates a primary role of A in AD, is supported by several lines of experimental evidence (Golde et al., 2006; http://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asp). All four genes now known to be linked to familial AD or be risk factors for the sporadic forms of the disease have been shown to increase A production (APP, PS1, PS2) or A deposition in the brain (apoE; Selkoe and Podlisny, 2002). Injection of A into the brain can rapidly induce morphological degeneration, including neurite dystrophy, loss of synaptic integrity, microglia activation, reactive astrocytosis and neuron death (Kar et al., 2004; Nagele et al., 2004; Tran et al., 2002). It is becoming increasingly clear that the sequence and aggregation state of A has a major impact on its toxicity and mechanisms of action (Golde et al., 2006; Haass and Selkoe, 2007; Stephan and Phillips, 2005). For better designing therapeutic interventions targeting the A cascade, it is necessary to gain a better appraisal of the molecular and cognitive effects of different A forms. The most widely used models of AD, transgenic mouse lines, do not provide direct means to test the effects of different A forms on the development of the symptoms. This is why it is important to establish animal models where symptoms mimicking AD develop after injection of a known A form into the brain. Amyloid plaques appear in an early stage of AD in the temporal neocortex, the entorhinal cortex (EC) and the hippocampus (Braak and Braak, 1991; Duyckaerts, 2004; Thal et al., 2000, 2002). Neurodegeneration of the EC likely plays a major part in the memory dysfunction observed at early stages of AD (DeToledo-Morrell et al.,
c
Research Group for Psychopharmacology, Hungarian Academy of Sciences–Semmelweis University, Hûvösvölgyi u. 116., H-1021 Budapest, Hungary
d
Neurobiology of Learning, Memory and Communication, CNRS, Univ Paris Sud, UMR 8620, 91405 Orsay, France
e
University of Szeged, Department of Comparative Physiology, Közép fasor 52., H-6726 Szeged, Hungary
Abstract—Alzheimer’s disease is characterized by the presence of senile plaques in the brain, composed mainly of aggregated amyloid- peptide (A), which plays a central role in the pathogenesis of Alzheimer’s disease and is a potential target for therapeutic intervention. Amyloid plaques occur in an increasing number of brain structures during the progression of the disease, with a heavy load in regions of the temporal cortex in the early phases. Here, we investigated the cognitive deficits specifically associated with amyloid pathology in the entorhinal cortex. The amyloid peptide A1– 42 was injected bilaterally into the entorhinal cortex of rats and behavioral performance was assessed between 10 and 17 days after injection. We found that parameters of motor behavior in an open-field as well as spatial working memory tested in an alternation task were normal. In contrast, compared with naive rats or control rats injected with saline, rats injected with A1– 42 showed impaired recognition memory in an object recognition task and delayed acquisition in a spatial reference memory task in a water-maze, despite improved performance with training in this task and normal spatial memory in a probe test given 24 h after training. This profile of behavioral deficits after injection of A1– 42 into the entorhinal cortex was similar to that observed in another group of rats injected with the excitotoxic drug, N-methyl-D-aspartate. Immunohistochemical analysis after behavioral testing revealed that A1– 42 injection induced a reactive astroglial response and plaque-like deposits in the entorhinal cortex. These results show that experimentally-induced amyloid pathology in the entorhinal cortex induces selective cognitive deficits, resembling those observed in early phases of Alzheimer’s disease. Therefore, injection of protofibrillar-fibrillar A1– 42 into the entorhinal cortex constitutes a promising animal model for investigating selective aspects of Alzhei*Corresponding author. Tel: ⫹36-62-546-806; fax: ⫹36-62-546-806. E-mail address: [email protected] (E. Sipos). Abbreviations: A, amyloid- peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; EC, entorhinal cortex; GFAP, glial fibrillary acidic protein; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline solution.
0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.011
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2004; Stoub et al., 2006). The EC relays multimodal processed information from the sensory cortical areas to the hippocampus, as well as information processed by the hippocampus to permanent storage sites in the neocortex (Heinemann et al., 2000; Poucet et al., 2003), and as such is assumed to play an important role in memory processes. For example, lesion of the EC impairs various types of memory, including spatial and recognition memory and forms of operant learning (Davis et al., 2001; Eijkenboom et al., 2000; Kopniczky et al., 2006; Miwa and Ueki, 1996; Parron et al., 2004; Parron and Save, 2004; Ramirez et al., 1988; Ueki et al., 1994). In this study, our aim was to establish whether injection of a defined A form in the EC of rats induces histopathological alterations and cognitive deficits resembling those observed in human AD. To this end, we injected protofibrillar-fibrillar A1– 42 into the EC of rats, and examined the development of amyloid plaques and reactive astrocytosis in this structure as well as behavioral performance in spatial working and reference memory tasks and in a nonspatial, recognition memory task classically associated with temporal lobe function.
EXPERIMENTAL PROCEDURES Subjects and housing Adult male Wistar rats (n⫽54; Charles River, Isazeg, Hungary, bred at the University facility), weighing 300 –350 g and aged 8 –9 weeks at the beginning of the experiment were used as subjects. After arrival in the laboratory, they were housed by groups of four under constant temperature and lighting conditions (23 °C, 12-h light/dark cycle, lights on at 07:00). Rat chow and tap water were provided ad libitum. All efforts were made to minimize the number of animals and their suffering throughout the experiments. Experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Formal approval to conduct the experiments described has been obtained from the Animal Experimentation Committee of the University of Szeged.
Peptide synthesis and structure analysis A1– 42 was synthesized in our laboratory by a solid-phase procedure involving the use of Wang-resin and Fmoc chemistry (Zarandi et al., 2007). The synthetic peptides were purified on a C-4 RP-HPLC column with an acetonitrile gradient; pure fractions were pooled and lyophilized. A1– 42 was repeatedly aggregated and lyophilized from aqueous solution resulting in protofibrils (diameter 4 –5 nm, length 150 –200 nm) and fibrils (diameter 7–10 nm, length 300 nm–1 m) as observed using transmission electron microscopy (see Datki et al., 2004). Purity control and proof of structure were achieved by amino acid analysis and mass spectrometry (ESI MS, FinniganMat TSQ 7000).
Surgery, solutions Rats were anesthetized by i.p. injection with a mixture of ketamine (10.0 mg/0.1 kg) and xylazine (0.8 mg/0.1 kg). They were then placed in a stereotaxic frame, a midline sagittal incision was made in the scalp, and holes were drilled on the skull. The solution was injected into the EC at three bilateral sites (2.5 l per site) with a Hamilton syringe in a total of 60 min. The coordinates were (from bregma): AP: ⫺6.4, ⫺6.8, ⫺7.8; ML: ⫾4.0, ⫾3.6, ⫾3.0; DV: ⫺8.2, ⫺7.8, ⫺7.4 (Paxinos and Watson, 1982). Control animals were
29
injected with sterile physiological saline. Amyloid-treated rats (A group) were injected with a 10⫺4 M A1– 42 solution in distilled water prepared immediately before injection. It has been shown that this solution contains mostly protofibrillar and fibrillar form of A1– 42 (Zarandi et al., 2007). N-methyl-D-aspartate (NMDA)treated rats (NMDA group) were injected with a 0.04 M NMDA (Sigma-Aldrich, Budapest, Hungary) solution in distilled water. Rats were treated with antibiotics and analgesics for 3 days after the surgery.
Behavioral testing Animals (nine intact, 17 control, 18 A, 10 NMDA rats) were allowed to recover for 10 days before the beginning of the behavioral tests. They were handled and weighed each day after the operation. All the behavioral experiments were carried out in the same room, illuminated by a halogen lamp giving diffused and dim light. A video camera was mounted on the ceiling directly above the test apparatus and images were relayed to a video tracking system. The behavior of the animals was automatically and/or manually recorded with the software EthoVision 2.3 (Noldus Information Technology, Wageningen, the Netherlands, 2002).
Open field test To assess the consequences of injections on explorative behavior, a 5-min open field test was performed on day 10 after A injection. The open field consisted of a black plastic rectangular arena (50⫻70⫻30 cm), the floor of which was covered with wood chips. At the beginning of the test, the animals were placed in the center of the arena. The following parameters were determined automatically: (1) total traveled distance, (2) percentage of time spent in the peripheral zone (thigmotaxis, Simon et al., 1994). The behavior was also scored by an observer who was unaware of the treatment of individual rats to record rearing, grooming and defecation behaviors.
Object recognition The object recognition test was used to assess whether recognition memory is impaired by A treatment. This test was carried out in the open field apparatus, on the day following the open field test, which therefore served also as a habituation session to the environment of the object recognition test. The test consisted of a single acquisition session (sampling phase), followed by a retention test 2 h later. Two types of objects were used, different in color, shape and material. For acquisition, each rat was placed in the middle of the arena containing two identical objects, and was left free to explore them for a total of 30 s of object exploration. Exploration of an object was defined as directing the nose to the object at a distance of less than 2 cm or touching it with the nose. Turning around or sitting on the object was not considered as object exploration. The animal was then placed back in the home cage for 2 h. Object recognition was tested in a 5-min session, during which one object used during the acquisition phase was replaced by a novel object. The nature and the spatial position of the objects were counterbalanced within each group in order to avoid any bias due to a preference that rats may have for a given object or its position in the arena. During the retention phase, the duration of exploration of each object was recorded, and the ratio of novel object exploration/total exploration was expressed as a percentage.
Spatial navigation in the water maze Spatial learning and memory were assessed in a Morris water maze on days 12–16 post-surgery (eight intact, nine control, eight A, nine NMDA rats). The maze consisted of a circular tank made of blue plastic (diameter: 130 cm, height: 80 cm) filled to a depth
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of 45 cm with water of 21⫾1 °C. The water was rendered opaque by the addition of milk. The tank was divided into four virtual quadrants. A white, hidden escape platform (diameter: 10 cm) was submerged in the middle of one of the quadrants (training quadrant), 30 cm from the rim of the pool and 2 cm below the water surface. The platform was not visible at water level. The rats had to learn to find the hidden platform on the basis of several constant spatial cues around the pool. During the training phase, five blocks of three trials were conducted between 09:00 and 13:00 h (on days 1, 2 and 3 of the test), and between 14:00 and 18:00 h (on days 1 and 2 of the test). Four different starting positions were used at the limits of the quadrants around the perimeter of the tank. A trial began by placing the rat into the water facing the wall of the pool; the starting position was varied pseudo-randomly over the trials. Rats were given a maximum of 90 s to find the platform and were then allowed to stay on it for 10 s. If a rat failed to escape from the water within 90 s, it was gently guided to the platform, and stayed there for 10 s. After the acquisition phase, retention was assessed in a 60-s probe trial (trial with the platform removed), on the day following the last training block. The data recorded by video-tracking were used to calculate the time to reach the platform, swim speed and swim paths lengths (distance) during acquisition trials as well as percentage of time spent in each of the four virtual quadrants and crossings over the platform’s position during the probe test. The means of the data from each block of trials were used for statistics.
Spontaneous alternation in a Y-maze In order to assess short-term memory, a Y-maze test was carried out on day 17 post-surgery. The apparatus was made of wood painted in black, and had three identical arms (each 14 cm wide, 19 cm high and 51 cm long), positioned in an equal angle. The floor of the maze was covered by wood chips. At the beginning of the test, each rat was placed in the center of the arena facing one arm. During the 10-min test period, the animal was allowed to move freely throughout the maze. The sequence of arm entries was recorded manually. Spontaneous alternation behavior was defined as successive entries into the three arms, on overlapping triplet sets (Yamada et al., 1998). Percentage of spontaneous alternation behavior was calculated as the ratio of actual to possible alternations, the latter defined as (total number of arm entries⫺2).
Statistics The results of different groups in the open field, object recognition, water-maze probe test and Y-maze tests were compared with one-way analysis of variance, followed by Fisher’s LSD post hoc tests for multiple comparison when appropriate. In the Morris water maze test, the swimming distance and latency data did not have a normal distribution, they were therefore analyzed with Kruskal-Wallis nonparametric test, followed by Mann-Whitney U tests for multiple comparison. Mean performance for individual groups was compared with chance level by one-sample Student’s t-test. The exploration durations of the two objects during the acquisition phase, as well as the time spent in the target and other quadrants in the Morris water maze were compared by paired Student’s t-test. In the description of results, F values (analysis of variance) and t values (Student’s t-test) and H values (KruskalWallis test) are given. The degrees of freedom (groups, subjects) for each test are shown as subscripts following the F, t and H. Statistical significance was set at Pⱕ0.05.
phosphate-buffered saline solution (PBS; room temperature, pH 7.4), followed by 300 ml of ice-cold paraformaldehyde solution (4% in phosphate buffer, pH 7.4). The brains were removed, post-fixed in the same fixative for 2 h, cryoprotected in sucrose solution (30% in PBS) for at least 36 h at 4 °C, and cut on a cryostat in 30 m coronal sections. Brain slices were collected and stored at ⫹4 °C in PBS for free-floating immunohistochemistry or Thioflavin T staining.
Immunohistochemistry and Thioflavin T staining Glial fibrillary acidic protein (GFAP) immunoreactivity, indicating reactive astrocytosis, was visualized by immunostaining with rabbit antibody (DakoCytomation, Glostrup, Denmark) used at a 1:20,000 dilution in PBS (pH 7.4). For A immunoreactivity, a mouse monoclonal antibody (4G8; AbCam, Cambridge, UK) was used at a 1:500 dilution. After quenching of endogenous peroxidase activity and a blocking step, the sections were incubated overnight at 4 °C with the primary antibody in the presence of 20% goat serum and Triton X-100 0.2%. On the following day, the sections were washed in PBS and incubated 1 h at room temperature with the secondary biotinylated mouse anti-rabbit antibody for GFAP (Sigma, 1:400), goat anti-mouse for 4G8 (Vector Laboratories, 1:500). The peroxidase reaction was carried out using the Vectastain Elite ABC Kit system (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine as the substrate and NiCl2 as an intensifier. After immunostaining and washing, all sections were mounted on gelatin-coated slides, air-dried, dehydrated and coverslipped with DPX mountant for histology (Fluka BioChemika, Buchs, Switzerland). For fibrillar amyloid-specific Thioflavin T staining (e.g. Kayed and Glabe, 2006), one series of sections was mounted on gelatincoated slides and dried overnight at room temperature. The sections were then washed in distilled water, incubated with a 0.5% Thioflavin T (Sigma-Aldrich, St. Louis, MO, USA) solution for 30 min, dehydrated and coverslipped. At least six sections per brain, (every twelfth section) from the dorsal hippocampus to the EC, from nine control and nine amyloid-treated rats were immunostained. The sections were processed at the same time and using the same solutions to reduce variability in immunostaining. Digital photographs were taken using a light microscope (Olympus Vanox-T AH-2; Olympus, Tokyo, Japan) and a CCD camera (Spot RT, Diagnostic Instruments, Sterling Heights, MI, USA). The observer of the sections was unaware of the treatment of the brains during the qualitative analysis, which was done with a computerized image analysis system (ImagePro Plus, Media Cybernetics, Bethesda, MD, USA).
RESULTS Open field In the open field test, parameters of exploratory behavior (total distance traveled, rearings) and anxiety (% time spent on periphery, grooming, defecation) were measured 10 days after the injections. Results showed that there was no significant difference among the groups in any of these parameters (total distance traveled: F3,50⫽0.14, P⫽0.93; rearings: F3,50⫽0.93, P⫽0.43; % time spent on periphery: F3,50⫽1.73, P⫽0.17; grooming: F3,50⫽0.13, P⫽0.94; defecation: F3,50⫽2.13, P⫽0.11).
Histology
Object recognition
After the behavioral tests, rats were killed by transcardial perfusion between days 20 –23 post-surgery. They were deeply anesthetized and perfused through the ascending aorta with 100 ml
The object recognition task was conducted on the day following the open field test, in the same apparatus. The two objects were explored equally during the acquisition
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impaired in this task (Fig. 2B, C). All groups spent more time in the training quadrant than in the others (compared with the mean of the time spent in the other quadrants; intact t7⫽5.88, P⫽0.001; control t8⫽3.31, P⫽0.01; A t7⫽10.4, P⬍0.001; NMDA t8⫽4.80, P⫽0.001). The four groups spent a similar amount of time in the training quadrant (F3,31⫽0.29, P⫽0.83). Moreover, the number of crossings over the platform was also similar between the groups
Fig. 1. A1– 42 or NMDA injection into the EC impairs object recognition memory. Retention performance is expressed as the percentage of time spent exploring the novel object over the total time of object exploration (mean⫾S.E.M.). Intact and control animals explored preferentially the novel object while performance of animals injected with A1– 42 (A) or NMDA did not differ from chance level. * Different from chance level at P⬍0.05.
phase (t⫽0.28, P⫽0.78). During the retention phase, significant between-group differences in performance were observed (F3,50⫽4.41, P⫽0.007). Specifically, both intact and control rats explored preferentially the novel object (time spent exploring the novel object significantly different from chance level; t18⫽4.72, P⫽0.0001 and t8⫽3.04, P⫽0.01, respectively; Fig. 1), while rats injected with A1– 42 or with NMDA showed a similar level of exploration of the novel and familiar objects (t17⫽1.06, P⫽0.3 and t9⫽0.78, P⫽0.45, respectively; Fig. 1). Between-group comparisons showed that intact and control rats had similar recognition memory performance and performed better than the A1– 42-treated or NMDA-treated rats (A-control: P⫽0.009; A-intact: P⫽0.006; NMDA-control: P⫽0.038; NMDA-intact: P⫽0.02). All the groups had a similar overall duration of object exploration during the retention phase (F3,50⫽0.98, P⫽0.43). Spatial learning in the water maze The Morris water maze test was used to assess spatial learning and memory on days 12–16 after the injections. The results showed that although performance of each group improved significantly across blocks of trials, learning was slower in the A- and the NMDA-treated groups, compared with control rats (Fig. 2A). Specifically, in the third block, A- and NMDA-treated rats swam longer distances to find the platform than control rats (H3,31⫽7.63, P⫽0.05; control-A: P⫽0.027, control-NMDA: P⫽0.1). In the fourth block, intact rats were quicker to find the platform than all the other groups (H3,31⫽10.57, P⫽0.01; intact-control: P⫽0.02; intact-A: P⫽0.007; intact-NMDA: P⫽0.006). No differences were observed between NMDA- and A-treated groups in the third or the fourth block (P⫽0.847 and P⫽0.700, respectively). There were no significant differences among the groups during the other blocks (first block: H3,31⫽3.3, P⫽0.34; second block: H3,31⫽4.18, P⫽0.24; fifth block: H3,31⫽6.86, P⫽0.07). Despite slower learning in the A1– 42-treated and NMDA-treated rats, performance at the probe test given 24 h after learning showed that spatial memory was not
Fig. 2. Injection of A1– 42 or NMDA in the EC resulted in slower spatial learning in the water-maze. (A) Performance during acquisition is expressed as the mean (⫾S.E.M.) swim paths lengths (distance) during the five training blocks. In block 3, A and NMDA rats swam a longer distance than controls to find the platform. By block 5, however, there was no significant difference between groups. (B) Injection of A1– 42 or NMDA did not affect performance in the probe trial 24 h after training. All groups spent more time in the training quadrant than in the other three quadrants, with no significant difference between groups (mean⫾S.E.M. is shown). (C) During the probe test, there was no significant difference between groups in the number of crossings over the place where the platform was during acquisition (mean⫾S.E.M. is shown).
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intact: t8⫽3.27, P⫽0.011; control: t18⫽6.79, P⬍0.0001; A: t17⫽7.17, P⬍0.0001; NMDA: t9⫽5.63, P⫽0.0003; Fig. 3). The performance of the groups did not differ from each other (F3,50⫽0.058, P⫽0.98). Moreover, the total number of arm entries was comparable in the different groups (F3,50⫽1.95, P⫽0.13), suggesting a similar level of motivation for exploration. Histology Fig. 3. Injection of A1– 42 or NMDA did not alter spontaneous alternation behavior in a Y-maze. Histograms show the percentage of alternation (consecutive entries into three different arms) over the total number of arm entries for each group (mean⫾S.E.M.). * P⬍0.05; *** P⬍0.001, compared with chance.
(F3,31⫽0.93, P⫽0.43). Finally, there were no significant differences in swim speed among the groups at any stage of behavioral testing. Spontaneous alternation The effect of A1– 42 on spontaneous alternation was examined on day 17 post-injection in a Y-maze. All groups showed alternation performance above chance level (50%,
After behavioral testing, ⬃3 weeks after A1– 42 injections, histological examination was performed on the brains of amyloid-treated and control rats to assess inflammatory reaction and the presence of aggregated material in the EC. First, GFAP staining was used to detect reactive astrogliosis as an indication of an inflammatory reaction. In contrast to control animals in which slight astrogliosis was found restricted to the injection tracts (Fig. 4A, C), likely caused by mechanical damage, astrocyte proliferation was apparent in a much larger area extending farther apart from the amyloid injection sites in the EC (Fig. 4B, D). Moreover, in amyloid-treated animals, astrocyte somata had a larger appearance and GFAP staining was more intense than in controls.
Fig. 4. Representative examples of GFAP staining in the EC of a control (A, high magnification: C) and an A1– 42-injected (B, high magnification: D) rat. In control animals, only some reactive astrocytes along the injection track can be observed among normal glial elements. In the amyloid-treated group, numerous reactive astrocytes can be seen around the injection sites, extending to the whole EC.
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Fig. 5. Representative examples of sections showing A1– 42 staining (4G8 antibody) in the EC of a control (A) and an amyloid-injected (B, C) rat. In the amyloid-treated group, aggregates with apparent filaments can be seen around the injection sites (B, high magnification: C). The fibrillar nature of at least some amyloid aggregates is confirmed by positive Thioflavin T staining (light patch on D).
Second, to assess whether injection of A1– 42 peptides led to amyloid deposits in the EC, specific amyloid immunostaining (4G8) was used. Examination of the EC revealed plaque-like depositions in A1– 42-injected animals (Fig. 5B, C), but only non-specific staining in controls (Fig. 5A). The presence of amyloid depositions was the highest around the injection sites, with only a few deposits observed in other parts of EC. The diffuse plaque-like structures were diverse in size, but they were all composed of many fibril-like structures. The presence of fibrillar A was confirmed by the strong Thioflavin T positive staining observed in the EC (Fig. 5D), as this colorant is specific for A of fibrillar structure (Kayed and Glabe, 2006). The extent of A immunoreactivity and of astrogliosis in the EC and adjacent structures was analyzed on serial brain sections. A schematic representation of the smallest and largest extent of the area covered by amyloid deposits and reactive astroglia is shown in Fig. 6. In each individual case, good overlap was observed between the areas of astrocytic proliferation and A deposits (see details in figure legend).
DISCUSSION The purpose of the present study was to examine the specific contribution of amyloid pathology in the EC to the cognitive deficits associated with AD. To this end, we tested learning and memory ability of rats injected with A1– 42 into the EC in different tasks believed to depend on the integrity of medial temporal lobe structures. Our results show for the first time that injection of A into the EC of rats results in selective impairments in memory functions, char-
acterized by spatial learning retardation and severe deficits in object recognition memory. Recognition memory is dependent on the integrity of structures of the medial temporal lobe, including the hippocampus and, possibly to an even greater extent, the surrounding parahippocampal areas (see review in Mumby, 2001; Squire et al., 2004). Previous studies have shown that excitotoxic lesions of the EC impair recognition memory in rats (Galani et al., 1998; Mumby and Pinel, 1994; Parron and Save, 2004). Here, we confirm the importance of the EC in object recognition memory, as NMDA injections severely impair performance in the novel object recognition task, and we show that injection of A1– 42 also results in a similar recognition memory deficit in this task. The deficit cannot be accounted for by an alteration in object exploration, as all rats explored the two novel objects for an identical time during the acquisition phase and had a similar amount of total object exploration during the test phase. Spatial reference memory measured in the Morris water maze is largely dependent on an intact hippocampus, and can also be affected by lesions of certain parahippocampal areas (reviewed in Aggleton et al., 2000). EC lesions have been reported to impair place navigation (Aggleton et al., 2000; Eijkenboom et al., 2000; Kopniczky et al., 2006; Parron et al., 2004; Spowart-Manning and van der Staay, 2005). However, some studies failed to find an effect of EC lesions (e.g. Galani et al., 1998), probably due to the use of proximal rather than distal landmarks (Burwell et al., 2004; Parron et al., 2004), or to other differences in experimental paradigms affecting the choice of different navigational strategies in the water maze (Aggleton et al.,
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Fig. 6. Schematic drawings of the region lesioned by A injection on three different coronal sections at AP ⫺6.3, ⫺6.8 and ⫺7.8 from bregma (Paxinos and Watson, 1982). We found intense amyloid and GFAP staining (dark gray) in the vicinity of the injection sites. The largest extension of A immunoreactivity and astrocytic proliferation extending over these areas is shown in light gray. CA, CA1 region of the hippocampus; DG, dentate gyrus of the hippocampus; Prh, perirhinal cortex.
2000). In our experimental conditions, NMDA lesion of the EC slowed down the rate of spatial learning, without otherwise affecting the rats’ ability to learn with repeated training or the expression of good spatial memory performance 24 h after training. With injections of A1– 42, again a similar profile of impairment was observed. In contrast, in our experimental conditions we found no evidence for a working memory impairment in the spontaneous alternation task after either NMDA or A1– 42 injection. Although there is some evidence that the EC may be involved in this task, at least at some point of ontogeny (Blozovski and Hess, 1989; Degroot and Parent, 2000; Gibbs et al., 1987), spatial working memory deficits are more consistently found after hippocampal lesions (reviewed in Lalonde, 2002) and, interestingly, working memory deficits have been clearly identified after A1– 42 injection into the hippocampus (O’Hare et al., 1999; Stephan et al., 2001). Thus, our study reveals that injection of A1– 42 inducing the formation of stable A aggregates in the EC results in a profile of cognitive impairments similar to that observed after NMDA injection, suggesting that injected A induces functional alteration of the EC. Bilateral excitotoxic lesion of the EC in rats has been suggested as a model for the early stages of human AD, because they share some similarities in their pathological and cognitive characteristics (Spowart-Manning and van der Staay, 2005). Moreover, cholinergic drugs used in the treatment of AD have been reported to improve recovery from the behavioral deficit induced by EC lesion (Spowart-Manning and van der Staay, 2005). However, injection of excitotoxic molecules is likely to induce neuron loss or lesions in the EC that have a neuropathological origin different from the lesions observed in AD. Our model therefore seems more suitable in the context of understanding the etiology of AD
or for testing therapeutic candidate molecules designed to protect against A pathology. Direct injection of A peptides into specific brain areas has been a widely used strategy to test animal models of AD. In these models, the nature and degree of cognitive impairments observed after A injection depend largely on the targeted structure, the A sequence length and aggregation state, the solvent, the time elapsed after the injection and the behavioral tasks used (Chacon et al., 2004; De Ferrari et al., 2003; Shen et al., 2001; Stephan et al., 2001; for a review, see Stephan and Phillips, 2005). It has been suggested that injection of soluble (monomeric or oligomeric) forms of A induces an immediate, but transient impairment of memory because of their detrimental effects on synaptic plasticity (Cleary et al., 2005; Stephan et al., 2001; Stephan and Phillips, 2005). On the other hand, the later-onset and longer-lasting effects on memory induced by injections of aggregated insoluble forms of A (or by the injection of the soluble but highly fibrillogenic A1– 42) are probably related to inflammatory reactions induced by fibrillar A (Stephan et al., 2003; Stephan and Phillips, 2005). Our histological analysis showed that the injected soluble A aggregated into plaque-like structures in the EC and we also confirmed previous data showing signs of intense inflammatory reaction characterized by activation of astrocytes and microglial cells surrounding and infiltrating the deposits formed after the injection of aggregated A, regardless of the target structure in the CNS (e.g. Giovannelli et al., 1998; Stephan and Phillips, 2005). Accumulation of astrocytes has also been observed around senile plaques in human AD, and their peripheral distribution around A deposits suggests a role in containing or circumscribing the abnormal protein, a well-known function
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of astrocytes in response to injury (Vehmas et al., 2003). Moreover, a recent study in a triple transgenic mouse model of AD reported that the inflammatory reaction that occurs in this model appears earlier in the EC than in the hippocampus (Janelsins et al., 2005). Although we cannot dissociate the specific contribution of aggregated amyloid peptide and inflammation in the memory deficits observed here after injection in the EC, it is possible that cognitive dysfunction induced by fibrillar A into the EC can be at least in part due to the associated extensive inflammatory reaction (Stephan et al., 2003; reviewed in Sastre et al., 2006).
CONCLUSION In conclusion, our results suggest that injection of protofibrillar-fibrillar A1– 42 into the EC of rats constitutes a suitable experimental model for some aspects of the early stages of AD. This in vivo model could be valuable for investigating specifically in the EC the differential pathological and cognitive consequences of different sequences and different aggregation states of A peptides, and for screening drug candidates designed to combat the deleterious effects of fibrillar A. Acknowledgments—This work was supported by grants from NKTH-RET 08/2004 DNT, OTKA TS 049817, ETT 476/2006 and NKFP1/A/005/2004. The authors thank the chemists of the Institute of Medical Chemistry for A synthesis and Professor Csaba Nyakas for helpful discussion.
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(Accepted 5 April 2007) (Available online 17 May 2007)