- Email: [email protected]
Behavioral and EEG changes in male 5xFAD mice
PHB-10456; No of Pages 9 Physiology & Behavior xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Physiology & Behavior
1
Behavioral and EEG changes in male 5xFAD mice
2Q1
F. Schneider a, K. Baldauf a,⁎, W. Wetzel b, K.G. Reymann a,b
3 4 5
a
6
H I G H L I G H T S
1 5 1 4 16 17 18 19 20
a r t i c l e
i n f o
21 22 23 24 25 26
Keywords: 5xFAD mice Cognitive deficits Non-cognitive impairments EEG Sleep recording
Transgenic animal models of Alzheimer's disease (AD) are widely used to investigate mechanisms of pathophysiology and cognitive dysfunctions. A model with a very early development of parenchymal plaque load at the age of 2 months is the 5xFAD mouse (Tg6799, Oakley et al. 2006). These 5xFAD mice over-express both human amyloid precursor protein (APP) and human presenilin 1 (PS1). Mice from this line have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of amyloid-β (Aβ). The aim of this study was the behavioral and functional investigations of 5xFAD males because in most studies females of this strain were characterized. In comparison to literature of transgenic 5xFAD females, transgenic 5xFAD males showed decreased anxiety in the elevated plus maze, reduced locomotion and exploration in the open field and disturbances in learning performance in the Morris water maze starting at 9 months of age. Electroencephalogram (EEG) recordings on 6 month old transgenic mice revealed a decrease of delta, theta, alpha, beta and gamma frequency bands whereas the subdelta frequency was increased. EEG recordings during sleep showed a reduction of rapid eye movement sleep in relation to the amount of total sleep. Thus, 5xFAD males develop early functional disturbances and subsequently behavioral deficits and therefore they are a good mouse model for studying Alzheimer's disease. © 2014 Published by Elsevier Inc.
27 28 29 30 31 32 33 34 35 36 37 38 39 40
protein (APP) due to the enzymes β- and γ-secretase [23] and its subsequent aggregation to oligomers, fibrils and extracellular plaques [41]. The cleavage results in a number of isoforms that are between 38 and 43 residues in length. Aβ40 is the most common form, but Aβ42 has a higher tendency to aggregate and is mostly relevant for the pathology of AD [23]. According to the amyloid-cascade-hypothesis by Hardy and Selkoe [16] a point mutation in the APP-, presenilin 1 (PS1)- or presenilin 2 (PS2)- gene causes the accumulation of oligomer Aβpeptides. The imbalance between Aβ production and Aβ clearance leads to hyperphosphorylation of tau and to the formation of intracellular fibrils, which results in synaptic and neuronal loss [16]. In recent years several animal models were generated to represent pathological conditions of AD [27]. One model is the 5xFAD mouse (Tg6799) in which the parenchymal plaque load develops at only 2 months of age [29]. These mice have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of Aβ [32]. The 5xFAD mouse shows hippocampus dependent memory deficits and plaque pathology similar to AD [9,19,29].
51
C
T
E
Article history: Received 17 February 2014 Received in revised form 23 April 2014 Accepted 28 May 2014 Available online xxxx
R O
O
a b s t r a c t
P
Transgenic 5xFAD males showed non-cognitive impairments at 9 months of age. Transgenic 5xFAD males had cognitive impairments at 9 months of age. All EEG bands shifted to a lower band, except the subdelta band at 6 months of age. REM sleep was decreased in 9 month old transgenic 5xFAD males.
D
• • • •
German Centre for Neurodegenerative Diseases (DZNE), D-39120 Magdeburg, Germany Leibniz Institute for Neurobiology, Brenneckestr. 6, D-39118 Magdeburg, Germany
R
R
E
7 8 9 10 11 12 13
b
F
journal homepage: www.elsevier.com/locate/phb
41
47 48 49 50
1. Introduction
Alzheimer's disease (AD) is characterized by an impairment of memory, cognition and spatial orientation [17]. The deposition of amyloid-β (Aβ) peptides as plaques is a hallmark of this disease [16]. Aβ is generated during endoproteolysis of the amyloid precursor
U
44 46
N C O
45 43 42
Abbreviations: Aβ, amyloid-β; AD, Alzheimer's disease; APP, amyloid precursor protein; EEG, electroencephalogramm; EMG, electromyogram; FAD, familiar Alzheimer's disease; PS, presenilin; REM, rapid eye movement sleep; SWS, slow-wave sleep; TS, total sleep; W, waking. ⁎ Corresponding author at: German Centre for Neurodegenerative Diseases (DZNE)-Site Magdeburg, Group Pathophysiology of Dementia, c/o Universitätsklinikum Magdeburg, Leipziger Straße 44, Haus 64, 39120 Magdeburg, Germany. Tel.: +49 391 6724516; fax: +49 391 6724536. E-mail addresses: [email protected] (F. Schneider), [email protected] (K. Baldauf), [email protected] (W. Wetzel), [email protected], [email protected] (K.G. Reymann).
http://dx.doi.org/10.1016/j.physbeh.2014.05.041 0031-9384/© 2014 Published by Elsevier Inc.
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
84
2. Methods
85
2.1. Animals
86
100 101
The 5xFAD mouse model (Tg6799, Jackson Laboratory, Stock 6554) was developed by Oakley et al. [29]. This double transgenic mouse overexpresses APP carrying the Swedish (K670N, M671L), Florida (I716V) and London mutation (V717I) as well as the human PS1 carrying the M14 6 L and L28 6 V mutations. Mutations are expressed under the control of the murine Thy-1-promotor. Mice from this line on the B6SJLF1/J background have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of Aβ. Only males (n = 187) were used in this study and all mice were handled according to German guidelines for animal care. Animals were bred and kept in a temperature controlled room at 20 °C ± 2 °C and a 12/12 h light–dark cycle (light on at 6 a.m.). Food and water were available ad libitum. All animal procedures have been approved by the ethics committee of the German federal state of Sachsen-Anhalt, and are in accordance with the European Communities Council Directive (86/609/EEC).
102
2.2. Phenotypical observations
103
110 111
Transgenic 5xFAD mice and non-transgenic littermates were examined phenotypically for characterization of the 5xFAD strain. Habitus, motor activity, agility, typical behavior and falling reflexes were investigated. To this purpose mice were held on their tail: stretching of paws indicated healthy reflexes, while clasping indicated motoric disturbances. Furthermore nest building was analyzed analogous to Filali et al. [14] by a five point scale: 1 = not noticeably touched, 2 = partially torn up, 3 = mostly shredded but often no identifiable site, 4 = identifiable but flat, 5 = perfect or nearly so.
112
2.3. Behavioral testing
113 114
Mice underwent a consecutive battery of behavioral tests. We started with the elevated plus maze. In the same week the open field test was done. In the second week mice were trained and tested in the Morris water maze. This procedure was maintained for all animals.
95 96 97 98 99
104 105 106 107 108 109
115 116 117 118 119 120 121 122 123 124 125 126
129 130
2.3.3. Morris water maze In a pool (120 cm in diameter, 60 cm deep) filled with opaque water, mice learned to localize a non-visible platform located 0.5 cm under the water surface. Four visible distal landmarks were used as spatial navigation cues. The water temperature was 20 ± 1 °C avoiding hypo- or hyperthermia. In the acquisition phase mice were trained for four days. Four trials were performed each day with an inter trial interval of 30 min (trial 1–16). The starting point was selected randomly for each trial. If a mouse did not reach the platform within 120 s it was placed on the platform for 10 s. On day 5 the probe trial was performed. Thereto the platform was removed and the swim pattern of the mice was recorded for 120 s (trial 17). The latency time to reach the platform, the path length and the swimming speed were measured with Video Mot2 (Version 5.45, TSE Systems).
136 137
R O
P
D
93 94
2.3.2. Open field Animals were allowed to explore an empty field (40 × 40 × 24 cm) for 5 min without any disturbing factors. The mouse was placed gently in the center of the field. The program Video Mot2 (Version 5.45) was used to measure patterns of running and rearings as indicators of agility and exploration. The maze was cleaned with 70% 2-propanol between the trials.
2.3.1. Elevated plus maze The elevated plus maze consisted of four cross-shaped arms (28 × 5 cm, height from floor: 60 cm). Two arms, facing each other, were enclosed on three sides by 15.5 cm high walls, the other two arms, also facing each other, were open. The mouse was placed in the center of the plus maze and allowed to explore the maze for 5 min. The time, which the mouse spent in each arm, was measured by the program Video Mot2 (Version 5.45, TSE Systems, Bad Homburg, Germany). An increased proportion of time spent in the open arms indicated reduced anxiety. If the mouse fell off an open arm, the trial was stopped and
131 132 133 134 135
138 139 140 141 142 143 144 145 146 147 148 149
2.3.4. Adhesive removal test Analogous to Freret et al. [15] a piece of tape (0.6 × 0.6 mm) was fixed to the bottom side of both fore paws. Mice were placed in a box (19 × 29 × 19 cm) and the time that they needed to remove the tapes was measured. The test was terminated after 120 s.
150 151
2.4. EEG measurement
155
To implant EEG electrodes, naïve mice were anesthetized with halothane (Sigma, Deisenhofen, Germany) in a mixture of nitrous oxide and oxygen (50:50), and maintained with 2–3% halothane. Six burr holes (1 mm in diameter) were drilled into the skull. The drill holes were located on the left and right posterior and anterior portions of the parietal bones. Stainless steel electrodes were placed on the intact dura and fixed with dental cement (RelyX Luting cement). Mice were monitored constantly until they regained consciousness. Animals were allowed to recover for 14 days. Mice in their home cage were placed into a soundproof chamber and connected to a swivel. Animals were allowed to habituate to the chamber and to the connection for 1 h each on two days prior to the measurements and again half an hour before the measurement started. EEG measurements were done for 1 h on three consecutive days. Impedance of the electrodes was measured and electrodes with an impedance of less than 50 kOhm were used for analysis. EEGs were recorded and analyzed with a Nihon-Kohden System (EEG-1200) and different electrodes were analyzed against a reference electrode. EEGs were digitized at a sampling rate of 500 Hz. Data for analysis were selected by hand from each measurement insofar that only artifact-free periods were used. The average absolute power values were calculated for the following frequency bands: subdelta (0.1–0.5 Hz), delta (0.5–4 Hz), theta (4–8 Hz), alpha (8– 13 Hz), beta (13–38 Hz), gamma (N 38 Hz) and the power of the pairing electrodes was used for further analysis. The sum of all frequency bands was set to 100% for each animal and the percentage was calculated for every frequency. The mean of all three days was calculated and used for group analysis.
156
T
91 92
C
89 90
E
87 88
R
80 81
R
78 79
O
76 77
C
75
N
73 74
U
71 72
excluded from the analysis. The maze was cleaned with 70% 2-propanol 127 between the trials. 128
O
82 83
Recently, we could show in transgenic 5xFAD males, that functional synaptic disconnections and re-organization within cortical columnar microcircuits correlate with pathological Aβ accumulation, rather than with cell death [28]. Behavioral deficits in fear conditioning occur at 6 months of age [21,28,30]. Learning and memory deficits in the Morris water maze were described in several male and female Alzheimer models [12,14,22,45], but not for male 5xFAD mice. In this study we wanted to characterize additional aspects of non-cognitive and cognitive impairments related to spatial memory and electroencephalogram (EEG) changes in this AD model in order to provide a basis for further investigations of mechanisms of AD. Males of the 5xFAD strain were tested in the elevated plus maze, the open field, the adhesive removal test and the Morris water maze test at different stages of age. EEG recordings during alertness revealed network changes before some of the significant behavioral changes.
E
69 70
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
F
2
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
152 153 154
157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
2.5. Sleep recording
185 186
194 195
In order to implant epidural cortical EEG and neck muscle electromyogram (EMG) electrodes for sleep recordings, naïve mice were kept under pentobarbital (40 mg/kg, i.p.) anthesia. After a recovery period of at least one week, animals were habituated to the recording conditions (5 days). Thereafter, EEG and EMG were recorded on a Nihon-Kohden polygraph for 7 h per day over a period of eight days. The EEG and EMG records were evaluated visually as described previously [44]. Thus, each 7-h record was scored as waking (W), slow-wave sleep (SWS) or rapid eye movement (REM) according to 30-s epochs from 9:00 to 16:00 and the following parameters were calculated: SWS-latency, REM-latency, 7-h amounts of W, SWS, REM, TS (total sleep, i.e. SWS + REM), and REM/TS.
196
2.6. Analysis and statistics
197 198
All data are presented as average ± standard error of the mean. The Mann–Whitney Rank Sum Test was used to compare genotypes and Kruskal-Wallis ANOVA was used to compare different ages (Sigma Plot, version 12.3). The RM-ANOVA was used to analyze the curves of EEG sleep recordings and of the Morris water maze (GraphPadPrism 6, version 6.01). Additionally the area under curve (AUC) was analyzed for the learning curves in the Morris water maze. The statistical analyses
O
R O
P D E T
203
209 210
C
201 202
Analogous to Oakley et al. [29] our histological analysis showed the rapid development of plaque burden beginning at 2 months of age in the prefrontal cortex and subiculum and distributing to the amygdala and cortex layer 5 as well as to the cerebellum at 3 months of age and later (at 4 months) to the hippocampus. In the striatum plaques appear at 8 months of age (data not shown). After plaques were distributed to almost all brain regions, 9 month old transgenic mice showed progressive alterations of their phenotype. Contrarily, no changes were observed in 8 month old mice. The animals were still agile and showed normal reflexes. First motor problems, a rigidity of paws and missing reflexes appeared at 8.5 months of age which progress to 9 months of age (Fig. 1A left). Reduced or absent grooming led to ruffled coats. Additionally clasping of the fore- and/or hind paws was evident (Fig. 1B). In 2.9% of transgenic (4 animals of 139) and 3.6% of non-transgenic mice (5 animals of 139) stereotypic behavior, e.g. somersaults, running circles or
E
199 200
208
R
192 193
3.1. Phenotypical characterization
R
190 191
207
N C O
189
3. Results
U
187 188
used p b 0.05 as a basis for the level of significance. Depending on the 204 sample size, Grubb's test or Dixon's Q-test was used to identify and 205 exclude possible outliers. 206
F
184
3
Fig. 1. Comparison of reflex behavior (A) between transgenic (left) and non-transgenic (right) 5xFAD males showed impaired reflexes and typical clasping (B) of fore and hind paws at N9 months. The quality of nest building (C) was decreased in transgenic mice (left) at 9 months of age compared to age matched non-transgenic littermates (right). (D) Quantitative analysis: 9 month old transgenic mice had a reduced nest building compared to age matched non-transgenic littermates (⁎⁎⁎p b 0.001) and 3 month old transgenic mice (#p b 0.05). Non-transgenic 5xFAD mice (FAD wt, white, n = 4 (3 mo); n = 13 (6 mo); n = 18 (9 mo)), transgenic 5xFAD mice (FAD tg, grey; n = 6 (3 mo); n = 10 (6 mo); n = 27 (9 mo)).
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
211 212 213 214 215 216 217 218 219 220 221 222 223
232
3.2. Elevated plus maze
233
The elevated plus maze is a test for anxiety. Normally, mice tend to avoid the open arms and spend more time in the closed arms seeking protection. However, transgenic 9-month old 5xFAD mice spent more time in the open arms (Fig. 2A) compared to non-transgenic littermates (p = 0.031) and to 3 month old transgenic mice (p = 0.002), respectively. Vice versa 9 month old transgenic 5xFAD spent less time in the closed arms (Fig. 2B) compared to wild-type mice (9 months, p = 0.004) and to
245
3.4. Morris water maze
253
E
C
T
E
D
P
The Morris water maze was performed as learning test. We compared the latency time that transgenic and non-transgenic 5xFAD mice needed to reach the platform over all trials. This time tended to be increased for 3 (Fig. 4A) and 6 month (Fig. 4B) old transgenic 5xFAD mice and it was significantly higher for 9 month old transgenic mice (F(1,14) = 5.940; p = 0.0287, Fig. 4C). These results were confirmed by comparing the area under curve (AUC) between the groups (p = 0.026, Fig. 4D). Other tested parameters, e.g. number of visits of the platform or into the target quadrant as well as time spent on the platform or within the target quadrant, did not differ between transgenic and non-transgenic animals (data not shown). Likewise, we did not observe any difference in path length (Fig. 4E), swim speed (Fig. 4F) or quadrant preference (Fig. 4G, H, I) between transgenic and non-transgenic littermates.
R
238 239
The open field test shows the general activity and the explorative behavior of mice. Normal mice explore the field avoiding the center and they rear at the walls as a sign of agility. At 3 and 6 months of age, transgenic 5xFAD mice displayed a tendency for reduced locomotion and less rearing activity. At 9 months of age, they showed significantly reduced locomotion (p = 0.008, Fig. 3A) and significantly less rearing activity (p = 0.002, Fig. 3B) compared to age matched non-transgenic mice.
R
236 237
244
O
234 235
3.3. Open field
C
230
N
228 229
240 241
U
226 227
transgenic mice at 3 months (p = 0.029). Comparing the center time, 9 month old 5xFADs spent less time there (Fig. 2C) in comparison to 9 month old non-transgenic littermates (p = 0.026) and to 3 month old transgenic 5xFAD mice (p = 0.012)
R O
231
jumping in the home cage, was observed. Nest building as a sign of natural motor behavior was impaired in transgenic 5xFAD mice beginning at 9 months of age (Fig. 1C). At this age transgenic 5xFAD mice showed significantly less shredding and composition of the available nesting material compared to 3 month old transgenic mice (p = 0.009) and 9 month old non-transgenic mice (p b 0.001, Fig. 1D). In 3 and 6 month old mice, we did not observe any difference in nest building between transgenic and non-transgenic mice.
F
224 225
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
O
4
Fig. 2. Elevated plus maze test of transgenic (FAD tg, grey, n = 6 (3 months); n = 6 (6 months); n = 13 (9 months)) and non-transgenic (FAD wt, white, n = 6 (3 months); n = 6 (6 months); n = 15 (9 months)) 5xFAD males at different ages. 9 month old transgenic mice spent significantly more time in the open arms (A) and less time in the closed arms (B) or in the middle of the maze (C) compared to age matched non-transgenic mice (*p b 0.05; **p b 0.005) or young transgenic 5xFAD mice (#p b 0.05; ##p b 0.005).
Fig. 3. Open field test of transgenic (FAD tg, grey, n = 9 (3 months); n = 8 (6 months); n = 17 (9 months)) and non-transgenic (FAD wt, white, n = 6 (3 months); n = 6 (6 months); n = 15 (9 months)) 5xFAD males at different ages. In 9 month old transgenic mice the traveled distance (A) and rearing (B) were significantly reduced in comparison to 9 month old non-transgenic mice (⁎p b 0.05; ⁎⁎p b 0.005).
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
242 243
246 247 248 249 250 251 252
254 255 256 257 258 259 260 261 262 263 264 265 266
5
N C O
R
R
E
C
T
E
D
P
R O
O
F
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
Fig. 4. Morris water maze test of transgenic (FAD tg, grey circles, n = 6 (3 months); n = 12 (6 months); n = 7 (9 months)) and non-transgenic (FAD wt, rhombs, n = 8 (3 months); n = 15 (6 months); n = 9 (9 months)) 5xFAD males at different ages. The learning curve of 3 month (A) and 6 month (B) old transgenic and non-transgenic 5xFAD males did not differ significantly. Transgenic mice had an increased latency time to reach the platform at 9 months of age (C) compared to non-transgenic 5xFAD mice. The area under curve (D) confirmed the higher latency of 9 month old transgenic 5xFAD mice (⁎p b 0.05). In the probe trial transgenic 5xFAD mice did not differ in the path length (E), swim speed (F) or quadrant preference (G, H, I) from non-transgenic mice at all ages.
3.5. Adhesive removal test
268 269
272 273
The adhesive removal test was performed to test the sensitivity of the perception at the paws and motor skills in transgenic and non-transgenic 5xFAD mice. 9 month old transgenic mice needed significantly more time to remove the tape from their fore paws than their age matched non-transgenic littermates (p = 0.008, Fig. 5). There were no differences between 3, 6 and 9 month old animals.
274
3.6. EEG recording
275
In most of the behavioral tests a difference between transgenic and non-transgenic animals started with a trend at 6 months. We chose this age as well as 3 month old animals for EEG measurements to find out whether the functional EEG readout is able to diagnose an effect
270 271
276 277 278
U
267
earlier than a behavioral readout. At 3 months of age, the frequency bands of transgenic and non-transgenic mice were indistinguishable from each other, while 6 month old transgenic animals showed a significant increase of the subdelta band and a decrease in all other frequency bands compared to age matched non-transgenic mice (p b 0.05). Significant differences were also observed between 3 and 6 month old transgenic animals—except for the delta frequency band, which was only reduced by trend (Fig. 6).
279 280
3.7. Sleep recording
287
Sleep recording experiments revealed a decrease in REM sleep in 9 month old transgenic FAD mice compared to non-transgenic littermates (Fig. 7D). While no significant %W and %SWS difference could be seen between transgenic and non-transgenic mice, we found that %REM in transgenic mice seemed reduced by trend. This reduction
288 289
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
281 282 283 284 285 286
290 291 292
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
R O
O
F
6
D
P
Fig. 6. Cortical EEG measurement of transgenic (FAD tg, grey, n = 4 (3 months); n = 6 (6 months)) and non-transgenic (FAD wt, white, n = 5 (3 months); n = 4 (6 m months o)) 5xFAD males at different ages. Non-transgenic mice (white) and transgenic mice (grey) were not different at 3 months but showed differences at 6 months (wt-white/ striped); tg-grey/striped) in all frequency bands of subdelta (0.1.0.5 Hz), delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), beta (13–38 Hz) and gamma (N38 Hz; p b 0.05, # difference between 3 and 6 months, ⁎ difference between non transgenic and transgenic mice).
E
investigation of 5xFAD males since in most studies only females of this 301 strain were characterized [2,10,19,46]. 302
E
C
T
4.1. Non cognitive behavior—anxiety and locomotion
293
R
Fig. 4 (continued).
297
4. Discussion
298
None of the known animal models is a perfect representative of the human AD. Current mouse models simply model key aspects of the disease [18]. The aim of this study was the further functional and behavioral
O
C
N
U
299 300
R
296
was statistically significant (F(1,15) = 4.756; p = 0.0455) when the %REM values were calculated in relation to the amount of total sleep (%REM/TS; see Fig. 7D). With regard to SWS- and REM-latencies, we found no differences between transgenic and non-transgenic mice.
294 295
Fig. 5. In the adhesive removal test, 9 month old transgenic 5xFAD males (FAD tg, grey, n = 10 (3 months); n = 8 (6 months); n = 6 (9 months)) needed significantly more time removing the tape from their fore paws than age matched non-transgenic littermates (FAD wt, white, n = 7 (3 months); n = 4 (6 months); n = 5 (9 months), ⁎p b 0.008).
In the past the 5xFAD mouse model was preferred for immunohistochemical and molecular examinations [1,4,8,13,33]. The literature on behavioral abnormalities in the 5xFAD mouse is limited [2,10,46]. Our investigations regarding the behavior of 5xFAD males yielded significantly reduced anxiety paired with a decreased locomotion and exploration in transgenic mice compared to non-transgenic animals at 9 months of age. In females of the same strain the anxiety level was already reduced at six months of age, but locomotion and exploration remained unaltered— even in nine and twelve month old mice [19]. This could imply gender specific behavioral deficits in transgenic 5xFAD mice but also methodical differences across the laboratories. In other models like Tg2576 males [26] and APP23 females [12] anxiety was also decreased, whereas it was increased in 3xTg-AD females [37]. The locomotor activity was also reduced in APP23 mice [22] and in APP/PS1 males [14] while it was increased in several other models [12,26,37,42]. An increase in locomotor activity is interpreted as restlessness and agitation. Decreased locotomotor activity on the other hand, as we observed it in aged 5xFAD males, is linked to other non-cognitive symptoms like apathy and depression [42]. Some of our 5xFAD mice showed severe stereotypes like jumping, circle running and somersaults in their home cage at 9 months of age which were independent on genotype. Continuous jumping against the walls in their home cage was also described in transgenic APP23mice and was defined as species-specific myoclonic jumping. Normally myclonus can be interpreted as pre-epileptic movements resulting from an abnormal neuronal activity in several brain regions, especially in the brain stem and the cerebellum and is observed mostly in late stages of AD [25]. Our phenotypic observation of male 5xFAD mice showed motor deficits, missing reflexes and a rigidity of fore and hind paws beginning at 9 months of age. These results were also found in 5xFAD females [19] and in other transgenic AD mouse models. Normal reflexes are usually characterized by an abduction of all four paws when animals fall on a horizontal surface [25]. Instead our transgenic 5xFAD males clasped their paws, which is due to parenchymal Aβ plaques and an
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337
7
C
T
E
D
P
R O
O
F
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
338 339
E
Fig. 7. Waking (%), slow-wave sleep (%), REM sleep (%) and REM related to total sleep (%REM/TS) in 9 month old FAD tg (n = 8) and FAD wt (n = 9) mice during 8 days (7-h recordings per day; ⁎p b 0.05 (days 1–8)).
362
4.2. Cognitive behavior
363
To investigate learning and memory deficits, the cardinal symptoms of AD, we performed the Morris water maze test. For the first time, we
345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
364
R
343 344
N C O
341 342
U
340
R
361
axonal neuropathology in brain and spinal cord. However this phenotype cannot be detected in all transgenic animals with plaque burden, e.g. 3xTg-AD-females showed no problems of motor coordination [37], for which reason amyloid plaque pathology seems to be no sufficient condition for clasping behavior. However the number of mutants showing abnormal flexion reflexes might be higher than wild-type mice [25]. The increasing impairment of the motor abilities and the increasing apathy in our transgenic 5xFAD males seemed to influence their nest building ability. They were less capable and less motivated than age matched non-transgenic mice to handle their nest building material sufficiently, resulting in very poorly shredded, almost untouched material. Similar results were seen in APP/PS1 males [14]. Moreover, in our study 9 month old male transgenic 5xFAD mice showed deficiencies in sensorimotor abilities compared to age matched littermates e.g. in removing tapes from their fore paws. However, this difference is based on decreased time in non-transgenic mice. Whereas 3 month old non-transgenic 5xFAD mice showed a high explorative behavior in the box, 9 month old mice were calmer and accordingly faster in removing the tapes. In transgenic mice, this age dependent behavior was not observable. In other mouse models such impairments are not necessarily detectable, even at advanced ages [24]. Nonetheless, these abilities seem to play an important role in disturbances of motor control as it occurs in AD [40].
demonstrated significant behavioral deficits in spatial learning in the Morris water maze in 9 month old transgenic 5xFAD-males. Impaired motor disabilities cannot explain the increased latency, as our transgenic 5xFAD males had no difficulties to match the performance of non-transgenic littermates with regard to swim path length and speed. Urano and Tohda [38] showed a similar impairment in female mice at 7 to 9 months. Another study [31] noted deficits in the water maze at 4 and 6 months of age, but there was no specification which gender had been used. Ohno et al. [31] observed a longer search in the target quadrant vs. the opposite quadrant and a closer search of wild-type mice near the correct platform position compared to the corresponding position in the opposite quadrant, whereas we did not find any difference in the probe trial between transgenic and non-transgenic 5xFAD males. In contrast to our findings, they did not observe specific impairments in the learning curve; however, the test conditions imply a different setup of the water maze experiment.
365 366
4.3. EEG
381
Our EEG analysis of the parietal cortex showed clear frequency shifts in transgenic 5xFAD males. Starting at six months of age, we observed an increase of the subdelta band and a decrease of all other analyzed frequency bands. These changes coincide with the impaired functional integrity and the re-organization of cortical columns that we found previously in 5xFAD transgenic mice at 6 months of age [28]. Single cell resolution mapping of neuronal thallium uptake in male 5xFAD mouse revealed that electrical activity of pyramidal cells breaks down throughout the infragranular cortical layer V long before cell death occurs. Laminar investigation of cortical circuit dysfunction with current
382
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
367 368 369 370 371 372 373 374 375 376 377 378 379 380
383 384 385 386 387 388 389 390 391
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422
426
In conclusion our data suggest that
435
Disclosure statement
436
R
O
C
431 432
The authors declare no conflict of interest.
N
429 430
R
433 434
• At 9 months of age 5xFAD males develop non-cognitive deficits in the elevated plus maze and the open field test and they show cognitive impairments when tested in the Morris water maze test. These impairments seem to be gender specific. • In 6 month old mice all EEG bands, except the subdelta band, were shifted to a lower band. • The functional EEG disturbances precede some of the cognitive and non-cognitive behavioral impairments in 5xFAD males.
427 428
Acknowledgments
438
441
This work was supported by the German Federal Ministry of Education and Science (BMBF grant VIP0562-03 V0765). We thank Lydia Löw for her excellent technical assistance and Anja Oelschlegel for constructive comments on the manuscript.
442
References
443 444 445 446 447 448 449 450
U
437
439 440
F
403 404
O
401 402
C
399 400
E
398
R O
5. Conclusion
396 397
P
425
394 395
[4] Bobkova NV, Garbuz DG, Nesterova I, Medvinskaya N, Samokhin A, Alexandrova I, et al. Therapeutic effect of exogenous hsp70 in mouse models of Alzheimer's disease. J Alzheimers Dis 2013;38(2):425–35. [5] Christos GA. Is Alzheimer's disease related to a deficits or malfunction of rapid eye movement (REM) sleep? Med Hypotheses 1993;41(5):435–9. [6] Coben LA, Danziger W, Storandt M. A longitudinal EEG study of mild senile dementia of Alzheimer type: changes at 1 year and at 2.5 years. Electoencephalogr. Clin Neurophysiol 1985;61:101–12. [7] Corbett BF, Leiser SC, Ling HP, Nagy R, Breysse N, Zhang X, et al. Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease. J Neurosci 2013;33(16):7020–6. [8] Crouzin N, Baranger K, Cavalier M, Marchalant Y, Cohen-Solal C, Roman FS, et al. Area-specific alterations of synaptic plasticity in the 5XFAD mouse model of Alzheimer's disease: dissociation between somatosensory cortex and hippocampus. PLoS One 2013;8(9):e74667. [9] Devi L, Ohno M. Genetic reductions of β-site amyloid precursor protein-cleaving enzyme 1 and amyloid-β ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer's disease model mice. Eur J Neurosci 2010; 31(1):110–8. [10] Devi L, Ohno M. Deletion of the elF2α kinase GCN2 fails to rescue the memory decline associated with Alzheimer's disease. PLoS One 2013;8(10):e77335. [11] Diekelmann S, Born J. The memory function of sleep. Nat Rev Neurosci 2010; 11(2):114–26. [12] Dumont M, Strazielle C, Stuafenbiel M, Lalonde R. Spatial learning and exploration of environmental stimuli in 24-month-old female APP23 transgenic mice with the Swedish mutation. Brain Res 2004;1024(1–2):113–21. [13] Eimer WA, Vassar R. Neuron loss in the 5XFAD mouse model of Alzheimer's disease correlates with intraneuronal Aβ42 accumulation and Caspase-3 activation. Mol Neurodegener 2013;8:2. [14] Filali M, Lalonde R, Rivest S. Subchonic memantine administration on spatial learning, exploratory activity, and nest-building in an APP/PS1 mouse model of Alzheimer's disease. Neuropharmacology 2011;60(6):930–6. [15] Freret T, Bouet V, Leconte C, Roussel S, Chazalviel L, Divous D, et al. Behavioral deficits after distal focal cerebral ischemia in mice: usefulness of adhesive removal test. Behav Neurosci 2009;123(1):224–30. [16] Hardy J, Selkoe J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 2002;297(5580):353–6. [17] Houghton PJ, Howes MJ. Natural products and derivatives affecting neurotransmission relevant to Alzheimer's and Parkinsons's disease. Neurosignals 2005; 14(1–2):6–22. [18] Ittner LM, Götz J. Amyloid-β and tau—a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci 2011;12(2):65–72. [19] Jawhar S, Trawicka A, Jenneckens C, Bayer TA, Wirths O. Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease. Neurobiol Aging 2012;33(1):196.e29–40. [20] Jeong J. EEG dynamics in patients with Alzheimer's disease. Clin Neurophysiol 2004;115:1490–505. [21] Kimura R, Ohno M. Impairments in remote memory stabilization precede hippocampal synaptic and cognitive failures in 5XFAD Alzheimer mouse model. Neurobiol Dis 2009;33(2):229–35. [22] Kobayashi DT, Chen S. Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer's disease. Genes Brain Behav 2005; 4(3):173–96. [23] LaFerla FM, Green KN, Oddo S. Intracellular amyloid-β in Alzheimer's disease. Nat Rev Neurosci 2007;8(7):499–509. [24] Lalonde R, Dumont M, Staufenbiel M, Strazielle C. Neurobehavioral characterization of APP23 transgenic mice with the SHIRPA primary screen. Behav Brain Res 2005;157(1):91–8. [25] Lalonde R, Fukuchi K, Strazielle C. Neurologic and motor dysfunctions in APP transgenic mice. Rev Neurosci 2012;23(4):363–79. [26] Lalonde R, Lewis TL, Strazielle C, Kim H, Fukuchi K. Transgenic mice expressing the βAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination. Brain Res 2003;977(1):38–45. [27] Lee JE, Han PL. An update of animal models of Alzheimer disease with a reevaluation of plaque deposition. Exp Neurobiol 2013;22(2):84–95. [28] Lison H, Happel MFK, Schneider F, Baldauf K, Kerbstat S, Seelbinder B, et al. Disrupted cross-laminar cortical processing in β amyloid pathology precedes cell death. Neurobiol Dis 2014 [Vol: Seiten]. [29] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, et al. Intraneuronal β-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci 2006;26(40):10129–40. [30] Ohno M. Failures to reconsolidate memory in a mouse model of Alzheimer's disease. Neurobiol Learn Mem 2009;92(3):455–9. [31] Ohno M, Chang L, Tseng W, Oakley H, Citron M, Klein WL, et al. Temporal memory deficits in Alzheimer's mouse models: rescue by genetic deletion of BACE1. Eur J Neurosci 2006;23(1):251–60. [32] Ohno M, Cole SL, Yasvoina M, Zhao J, Citron M, Berry R, et al. BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice. Neurobiol Dis 2007;26(1):134–45. [33] Ou-Yang MH, Van Nostrand WE. The absence of myelin basic protein promotes neuroinflammation and reduces amyloid beta-protein accumulation in Tg-5xFAD mice. J Neuroinflammation 2013;10(1):134. [34] Palop JJ, Mucke L. Epilepsy and cognitive impairments in Alzheimer disease. Arch Neurol 2009;66(4):435–40.
T
423 424
source density analysis identified an early loss of excitatory synaptic input in infragranular layers, linked to pathological recurrent activations in supragranular layers. These functional changes correlate with deficits in fear conditioning [28]. EEG changes comparable to those that we saw in our 5xFAD mice were also observed in the PLB1 mouse where an increase of the delta frequencies and a decrease of the alpha frequencies in the parietal and prefrontal cortex were found. These changes were accompanied by decreased LTP and reduced paired pulse facilitation at the age of 6 months as well as by impairments in recognition memory and spatial learning at the age of 12 months [35]. Wesson et al. [43] saw that sensory network hyperactivity, found in Tg2576 mice at an early stage contributed to subsequent hyporesponsiveness and dysfunction later in life. Corbett et al. [7] also noted EEG activity changes in association with reduced Morris water maze performances. Although AD mouse models develop spontaneous seizures and frequent epileptiform discharges [34,48], we did not find any seizures in 6 month old 5xFAD mice during our recording periods, when the mice were almost resting but not sleeping. Functional disturbances in the EEG pattern in transgenic 5xFAD mice might be related to the beginning of cognitive impairments at 6 months of age. These changes are comparable to the EEG slowing that is seen in human AD cases. Patients with dementia exhibit a decrease in alpha- and betaactivities and an increase in delta- and theta-activities [6]. While it is feasible that such an EEG shift might be partially related to normal aging, it was also shown that EEG abnormalities reflect directly the anatomical changes and the functional deficits of the cerebral cortex damaged by the disease [20]. Further, our transgenic 5xFAD mice had less rapid eye movement sleep (REM) compared to non-transgenic littermates. These changes coincide with a decline in REM sleep periods seen in patients with Alzheimer and other dementias [3,5,39] as well as in other mouse models of AD [35,47] and they support the hypothesis on the important role of sleep, especially REM sleep, in memory consolidation processes [11,36]
D
392 393
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
E
8
[1] Antonios G, Saiepour N, Bouter Y, Richard BC, Paetau A, Verkkoniemi-Ahola A, et al. N-truncated Abeta starting with position four: early intraneuronal accumulation and rescue of toxicity using NT4x-167, a novel monoclonal antibody. Acta Neuropathol Commun 2013;1(1):56. [2] Aytan N, Choi JK, Carreras I, Kowall NW, Jenkins BG, Debeoglu A. Combination therapy in a transgenic model of Alzheimer's disease. Exp Neurol 2013;250:228–38. [3] Bliwise DL. Sleep disorders in Alzheimer's disease and other dementias. Clin Cornerstone 2004;6(Suppl. 1A):S16–28.
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
451 Q2 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 Q3 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx [35] Platt B, Drever B, Koss D, Stoppelkamp S, Jyoti A, Plano A, et al. Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1. PLoS One 2011;6(11):e27068. [36] Poe GR, Walsh CM, Bjorness TE. Cognitive neuroscience of sleep. Prog Brain Res 2010;185:1–19. [37] Sterniczuk R, Antle MC, LaFerla FM, Dyck RH. Characterization of the 3xTg-AD mouse model of Alzheimer's disease: part 2. Behavioral and cognitive changes. Brain Res 2010;1348:149–55. [38] Urano T, Tohda C. Icariin improves memory impairment in Alzheimer's disease model mice (5xFAD) and attenuates amyloid β-induced neurite atrophy. Phytother Res 2010;24(11):1658–63. [39] Veccierini MF. Sleep disturbances in Alzheimer's disease and other dementias. Psychol Neuropsychiatr Vieil 2010;8(1):15–23. [40] Velasques B, Machado S, Paes F, Cunha M, Sanfim A, Budde H, et al. Sensorimotor integration and psychopathology: motor control abnormalities related to psychiatric disorders. World J Biol Psychiatry 2011;12(8):560–73. [41] Waldau B, Shetty AK. Behavior of neural stem cells in the Alzheimer brain. Cell Mol Life Sci 2008;65(15):2372–84. [42] Walker JM, Fowler SW, Miller DK, Sun AY, Weisman GA, Wood WG, et al. Spatial learning and memory impairment and increased locomotion in a transgenic amyloid
[43]
[44] [45]
[46]
[47]
F
[48]
precursor protein mouse model of Alzheimer's disease. Behav Brain Res 2011; 222(1):169–75. Wesson DW, Borkowski AH, Landreth GE, Nixon RA, Levy E, Wilson DA. Sensory network dysfunction, behavioral impairments, and their reversibility in an Alzheimer's β-amyloidosis mouse model. J Neurosci 2011;31(44):15962–71. Wetzel W, Wagner T, Balschun D. REM sleep enhancement induced by different procedures improves memory retention in rats. Eur J Neurosci 2003;18(9):2611–7. Zhang W, Bai M, Xi Y, Hao J, Liu L, Mao N, et al. Early memory deficits precedes plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction. Free Radic Biol Med 2012;52(8):1443–52. Zhang Z, Liu X, Schroder JP, Chan CB, Song M, Yu SP, et al. 7,8-Dihydroxyflavone prevents synaptic loss and memory deficits in a mouse model of Alzheimer's disease. Neuropsychopharmacology 2013;1-13. Zhang B, Veasey SC, Wood MA, Leng LZ, Kaminski C, Leight S, et al. Impaired rapid eye movement sleep in the Tg2576 APP murine model of Alzheimer's disease with injury to pedunculopontine cholinergic neurons. Am J Pathol 2005;167(5):1361–9. Zuyatdinova S, Gurevicius K, Kutchiashvili N, Bolkvadze T, Nissinen J, Tanila H, et al. Spontaneous epileptiform discharges in a mouse model of Alzheimer's disease are suppressed by antiepileptic drugs that block sodium channels. Epilepsy Res 2011;94:75–85.
O
537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556
9
U
N C O
R
R
E
C
T
E
D
P
R O
577
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576
Contents lists available at ScienceDirect
Physiology & Behavior
1
Behavioral and EEG changes in male 5xFAD mice
2Q1
F. Schneider a, K. Baldauf a,⁎, W. Wetzel b, K.G. Reymann a,b
3 4 5
a
6
H I G H L I G H T S
1 5 1 4 16 17 18 19 20
a r t i c l e
i n f o
21 22 23 24 25 26
Keywords: 5xFAD mice Cognitive deficits Non-cognitive impairments EEG Sleep recording
Transgenic animal models of Alzheimer's disease (AD) are widely used to investigate mechanisms of pathophysiology and cognitive dysfunctions. A model with a very early development of parenchymal plaque load at the age of 2 months is the 5xFAD mouse (Tg6799, Oakley et al. 2006). These 5xFAD mice over-express both human amyloid precursor protein (APP) and human presenilin 1 (PS1). Mice from this line have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of amyloid-β (Aβ). The aim of this study was the behavioral and functional investigations of 5xFAD males because in most studies females of this strain were characterized. In comparison to literature of transgenic 5xFAD females, transgenic 5xFAD males showed decreased anxiety in the elevated plus maze, reduced locomotion and exploration in the open field and disturbances in learning performance in the Morris water maze starting at 9 months of age. Electroencephalogram (EEG) recordings on 6 month old transgenic mice revealed a decrease of delta, theta, alpha, beta and gamma frequency bands whereas the subdelta frequency was increased. EEG recordings during sleep showed a reduction of rapid eye movement sleep in relation to the amount of total sleep. Thus, 5xFAD males develop early functional disturbances and subsequently behavioral deficits and therefore they are a good mouse model for studying Alzheimer's disease. © 2014 Published by Elsevier Inc.
27 28 29 30 31 32 33 34 35 36 37 38 39 40
protein (APP) due to the enzymes β- and γ-secretase [23] and its subsequent aggregation to oligomers, fibrils and extracellular plaques [41]. The cleavage results in a number of isoforms that are between 38 and 43 residues in length. Aβ40 is the most common form, but Aβ42 has a higher tendency to aggregate and is mostly relevant for the pathology of AD [23]. According to the amyloid-cascade-hypothesis by Hardy and Selkoe [16] a point mutation in the APP-, presenilin 1 (PS1)- or presenilin 2 (PS2)- gene causes the accumulation of oligomer Aβpeptides. The imbalance between Aβ production and Aβ clearance leads to hyperphosphorylation of tau and to the formation of intracellular fibrils, which results in synaptic and neuronal loss [16]. In recent years several animal models were generated to represent pathological conditions of AD [27]. One model is the 5xFAD mouse (Tg6799) in which the parenchymal plaque load develops at only 2 months of age [29]. These mice have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of Aβ [32]. The 5xFAD mouse shows hippocampus dependent memory deficits and plaque pathology similar to AD [9,19,29].
51
C
T
E
Article history: Received 17 February 2014 Received in revised form 23 April 2014 Accepted 28 May 2014 Available online xxxx
R O
O
a b s t r a c t
P
Transgenic 5xFAD males showed non-cognitive impairments at 9 months of age. Transgenic 5xFAD males had cognitive impairments at 9 months of age. All EEG bands shifted to a lower band, except the subdelta band at 6 months of age. REM sleep was decreased in 9 month old transgenic 5xFAD males.
D
• • • •
German Centre for Neurodegenerative Diseases (DZNE), D-39120 Magdeburg, Germany Leibniz Institute for Neurobiology, Brenneckestr. 6, D-39118 Magdeburg, Germany
R
R
E
7 8 9 10 11 12 13
b
F
journal homepage: www.elsevier.com/locate/phb
41
47 48 49 50
1. Introduction
Alzheimer's disease (AD) is characterized by an impairment of memory, cognition and spatial orientation [17]. The deposition of amyloid-β (Aβ) peptides as plaques is a hallmark of this disease [16]. Aβ is generated during endoproteolysis of the amyloid precursor
U
44 46
N C O
45 43 42
Abbreviations: Aβ, amyloid-β; AD, Alzheimer's disease; APP, amyloid precursor protein; EEG, electroencephalogramm; EMG, electromyogram; FAD, familiar Alzheimer's disease; PS, presenilin; REM, rapid eye movement sleep; SWS, slow-wave sleep; TS, total sleep; W, waking. ⁎ Corresponding author at: German Centre for Neurodegenerative Diseases (DZNE)-Site Magdeburg, Group Pathophysiology of Dementia, c/o Universitätsklinikum Magdeburg, Leipziger Straße 44, Haus 64, 39120 Magdeburg, Germany. Tel.: +49 391 6724516; fax: +49 391 6724536. E-mail addresses: [email protected] (F. Schneider), [email protected] (K. Baldauf), [email protected] (W. Wetzel), [email protected], [email protected] (K.G. Reymann).
http://dx.doi.org/10.1016/j.physbeh.2014.05.041 0031-9384/© 2014 Published by Elsevier Inc.
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
84
2. Methods
85
2.1. Animals
86
100 101
The 5xFAD mouse model (Tg6799, Jackson Laboratory, Stock 6554) was developed by Oakley et al. [29]. This double transgenic mouse overexpresses APP carrying the Swedish (K670N, M671L), Florida (I716V) and London mutation (V717I) as well as the human PS1 carrying the M14 6 L and L28 6 V mutations. Mutations are expressed under the control of the murine Thy-1-promotor. Mice from this line on the B6SJLF1/J background have high APP expression correlating with high burden and accelerated accumulation of the 42 amino acid species of Aβ. Only males (n = 187) were used in this study and all mice were handled according to German guidelines for animal care. Animals were bred and kept in a temperature controlled room at 20 °C ± 2 °C and a 12/12 h light–dark cycle (light on at 6 a.m.). Food and water were available ad libitum. All animal procedures have been approved by the ethics committee of the German federal state of Sachsen-Anhalt, and are in accordance with the European Communities Council Directive (86/609/EEC).
102
2.2. Phenotypical observations
103
110 111
Transgenic 5xFAD mice and non-transgenic littermates were examined phenotypically for characterization of the 5xFAD strain. Habitus, motor activity, agility, typical behavior and falling reflexes were investigated. To this purpose mice were held on their tail: stretching of paws indicated healthy reflexes, while clasping indicated motoric disturbances. Furthermore nest building was analyzed analogous to Filali et al. [14] by a five point scale: 1 = not noticeably touched, 2 = partially torn up, 3 = mostly shredded but often no identifiable site, 4 = identifiable but flat, 5 = perfect or nearly so.
112
2.3. Behavioral testing
113 114
Mice underwent a consecutive battery of behavioral tests. We started with the elevated plus maze. In the same week the open field test was done. In the second week mice were trained and tested in the Morris water maze. This procedure was maintained for all animals.
95 96 97 98 99
104 105 106 107 108 109
115 116 117 118 119 120 121 122 123 124 125 126
129 130
2.3.3. Morris water maze In a pool (120 cm in diameter, 60 cm deep) filled with opaque water, mice learned to localize a non-visible platform located 0.5 cm under the water surface. Four visible distal landmarks were used as spatial navigation cues. The water temperature was 20 ± 1 °C avoiding hypo- or hyperthermia. In the acquisition phase mice were trained for four days. Four trials were performed each day with an inter trial interval of 30 min (trial 1–16). The starting point was selected randomly for each trial. If a mouse did not reach the platform within 120 s it was placed on the platform for 10 s. On day 5 the probe trial was performed. Thereto the platform was removed and the swim pattern of the mice was recorded for 120 s (trial 17). The latency time to reach the platform, the path length and the swimming speed were measured with Video Mot2 (Version 5.45, TSE Systems).
136 137
R O
P
D
93 94
2.3.2. Open field Animals were allowed to explore an empty field (40 × 40 × 24 cm) for 5 min without any disturbing factors. The mouse was placed gently in the center of the field. The program Video Mot2 (Version 5.45) was used to measure patterns of running and rearings as indicators of agility and exploration. The maze was cleaned with 70% 2-propanol between the trials.
2.3.1. Elevated plus maze The elevated plus maze consisted of four cross-shaped arms (28 × 5 cm, height from floor: 60 cm). Two arms, facing each other, were enclosed on three sides by 15.5 cm high walls, the other two arms, also facing each other, were open. The mouse was placed in the center of the plus maze and allowed to explore the maze for 5 min. The time, which the mouse spent in each arm, was measured by the program Video Mot2 (Version 5.45, TSE Systems, Bad Homburg, Germany). An increased proportion of time spent in the open arms indicated reduced anxiety. If the mouse fell off an open arm, the trial was stopped and
131 132 133 134 135
138 139 140 141 142 143 144 145 146 147 148 149
2.3.4. Adhesive removal test Analogous to Freret et al. [15] a piece of tape (0.6 × 0.6 mm) was fixed to the bottom side of both fore paws. Mice were placed in a box (19 × 29 × 19 cm) and the time that they needed to remove the tapes was measured. The test was terminated after 120 s.
150 151
2.4. EEG measurement
155
To implant EEG electrodes, naïve mice were anesthetized with halothane (Sigma, Deisenhofen, Germany) in a mixture of nitrous oxide and oxygen (50:50), and maintained with 2–3% halothane. Six burr holes (1 mm in diameter) were drilled into the skull. The drill holes were located on the left and right posterior and anterior portions of the parietal bones. Stainless steel electrodes were placed on the intact dura and fixed with dental cement (RelyX Luting cement). Mice were monitored constantly until they regained consciousness. Animals were allowed to recover for 14 days. Mice in their home cage were placed into a soundproof chamber and connected to a swivel. Animals were allowed to habituate to the chamber and to the connection for 1 h each on two days prior to the measurements and again half an hour before the measurement started. EEG measurements were done for 1 h on three consecutive days. Impedance of the electrodes was measured and electrodes with an impedance of less than 50 kOhm were used for analysis. EEGs were recorded and analyzed with a Nihon-Kohden System (EEG-1200) and different electrodes were analyzed against a reference electrode. EEGs were digitized at a sampling rate of 500 Hz. Data for analysis were selected by hand from each measurement insofar that only artifact-free periods were used. The average absolute power values were calculated for the following frequency bands: subdelta (0.1–0.5 Hz), delta (0.5–4 Hz), theta (4–8 Hz), alpha (8– 13 Hz), beta (13–38 Hz), gamma (N 38 Hz) and the power of the pairing electrodes was used for further analysis. The sum of all frequency bands was set to 100% for each animal and the percentage was calculated for every frequency. The mean of all three days was calculated and used for group analysis.
156
T
91 92
C
89 90
E
87 88
R
80 81
R
78 79
O
76 77
C
75
N
73 74
U
71 72
excluded from the analysis. The maze was cleaned with 70% 2-propanol 127 between the trials. 128
O
82 83
Recently, we could show in transgenic 5xFAD males, that functional synaptic disconnections and re-organization within cortical columnar microcircuits correlate with pathological Aβ accumulation, rather than with cell death [28]. Behavioral deficits in fear conditioning occur at 6 months of age [21,28,30]. Learning and memory deficits in the Morris water maze were described in several male and female Alzheimer models [12,14,22,45], but not for male 5xFAD mice. In this study we wanted to characterize additional aspects of non-cognitive and cognitive impairments related to spatial memory and electroencephalogram (EEG) changes in this AD model in order to provide a basis for further investigations of mechanisms of AD. Males of the 5xFAD strain were tested in the elevated plus maze, the open field, the adhesive removal test and the Morris water maze test at different stages of age. EEG recordings during alertness revealed network changes before some of the significant behavioral changes.
E
69 70
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
F
2
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
152 153 154
157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
2.5. Sleep recording
185 186
194 195
In order to implant epidural cortical EEG and neck muscle electromyogram (EMG) electrodes for sleep recordings, naïve mice were kept under pentobarbital (40 mg/kg, i.p.) anthesia. After a recovery period of at least one week, animals were habituated to the recording conditions (5 days). Thereafter, EEG and EMG were recorded on a Nihon-Kohden polygraph for 7 h per day over a period of eight days. The EEG and EMG records were evaluated visually as described previously [44]. Thus, each 7-h record was scored as waking (W), slow-wave sleep (SWS) or rapid eye movement (REM) according to 30-s epochs from 9:00 to 16:00 and the following parameters were calculated: SWS-latency, REM-latency, 7-h amounts of W, SWS, REM, TS (total sleep, i.e. SWS + REM), and REM/TS.
196
2.6. Analysis and statistics
197 198
All data are presented as average ± standard error of the mean. The Mann–Whitney Rank Sum Test was used to compare genotypes and Kruskal-Wallis ANOVA was used to compare different ages (Sigma Plot, version 12.3). The RM-ANOVA was used to analyze the curves of EEG sleep recordings and of the Morris water maze (GraphPadPrism 6, version 6.01). Additionally the area under curve (AUC) was analyzed for the learning curves in the Morris water maze. The statistical analyses
O
R O
P D E T
203
209 210
C
201 202
Analogous to Oakley et al. [29] our histological analysis showed the rapid development of plaque burden beginning at 2 months of age in the prefrontal cortex and subiculum and distributing to the amygdala and cortex layer 5 as well as to the cerebellum at 3 months of age and later (at 4 months) to the hippocampus. In the striatum plaques appear at 8 months of age (data not shown). After plaques were distributed to almost all brain regions, 9 month old transgenic mice showed progressive alterations of their phenotype. Contrarily, no changes were observed in 8 month old mice. The animals were still agile and showed normal reflexes. First motor problems, a rigidity of paws and missing reflexes appeared at 8.5 months of age which progress to 9 months of age (Fig. 1A left). Reduced or absent grooming led to ruffled coats. Additionally clasping of the fore- and/or hind paws was evident (Fig. 1B). In 2.9% of transgenic (4 animals of 139) and 3.6% of non-transgenic mice (5 animals of 139) stereotypic behavior, e.g. somersaults, running circles or
E
199 200
208
R
192 193
3.1. Phenotypical characterization
R
190 191
207
N C O
189
3. Results
U
187 188
used p b 0.05 as a basis for the level of significance. Depending on the 204 sample size, Grubb's test or Dixon's Q-test was used to identify and 205 exclude possible outliers. 206
F
184
3
Fig. 1. Comparison of reflex behavior (A) between transgenic (left) and non-transgenic (right) 5xFAD males showed impaired reflexes and typical clasping (B) of fore and hind paws at N9 months. The quality of nest building (C) was decreased in transgenic mice (left) at 9 months of age compared to age matched non-transgenic littermates (right). (D) Quantitative analysis: 9 month old transgenic mice had a reduced nest building compared to age matched non-transgenic littermates (⁎⁎⁎p b 0.001) and 3 month old transgenic mice (#p b 0.05). Non-transgenic 5xFAD mice (FAD wt, white, n = 4 (3 mo); n = 13 (6 mo); n = 18 (9 mo)), transgenic 5xFAD mice (FAD tg, grey; n = 6 (3 mo); n = 10 (6 mo); n = 27 (9 mo)).
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
211 212 213 214 215 216 217 218 219 220 221 222 223
232
3.2. Elevated plus maze
233
The elevated plus maze is a test for anxiety. Normally, mice tend to avoid the open arms and spend more time in the closed arms seeking protection. However, transgenic 9-month old 5xFAD mice spent more time in the open arms (Fig. 2A) compared to non-transgenic littermates (p = 0.031) and to 3 month old transgenic mice (p = 0.002), respectively. Vice versa 9 month old transgenic 5xFAD spent less time in the closed arms (Fig. 2B) compared to wild-type mice (9 months, p = 0.004) and to
245
3.4. Morris water maze
253
E
C
T
E
D
P
The Morris water maze was performed as learning test. We compared the latency time that transgenic and non-transgenic 5xFAD mice needed to reach the platform over all trials. This time tended to be increased for 3 (Fig. 4A) and 6 month (Fig. 4B) old transgenic 5xFAD mice and it was significantly higher for 9 month old transgenic mice (F(1,14) = 5.940; p = 0.0287, Fig. 4C). These results were confirmed by comparing the area under curve (AUC) between the groups (p = 0.026, Fig. 4D). Other tested parameters, e.g. number of visits of the platform or into the target quadrant as well as time spent on the platform or within the target quadrant, did not differ between transgenic and non-transgenic animals (data not shown). Likewise, we did not observe any difference in path length (Fig. 4E), swim speed (Fig. 4F) or quadrant preference (Fig. 4G, H, I) between transgenic and non-transgenic littermates.
R
238 239
The open field test shows the general activity and the explorative behavior of mice. Normal mice explore the field avoiding the center and they rear at the walls as a sign of agility. At 3 and 6 months of age, transgenic 5xFAD mice displayed a tendency for reduced locomotion and less rearing activity. At 9 months of age, they showed significantly reduced locomotion (p = 0.008, Fig. 3A) and significantly less rearing activity (p = 0.002, Fig. 3B) compared to age matched non-transgenic mice.
R
236 237
244
O
234 235
3.3. Open field
C
230
N
228 229
240 241
U
226 227
transgenic mice at 3 months (p = 0.029). Comparing the center time, 9 month old 5xFADs spent less time there (Fig. 2C) in comparison to 9 month old non-transgenic littermates (p = 0.026) and to 3 month old transgenic 5xFAD mice (p = 0.012)
R O
231
jumping in the home cage, was observed. Nest building as a sign of natural motor behavior was impaired in transgenic 5xFAD mice beginning at 9 months of age (Fig. 1C). At this age transgenic 5xFAD mice showed significantly less shredding and composition of the available nesting material compared to 3 month old transgenic mice (p = 0.009) and 9 month old non-transgenic mice (p b 0.001, Fig. 1D). In 3 and 6 month old mice, we did not observe any difference in nest building between transgenic and non-transgenic mice.
F
224 225
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
O
4
Fig. 2. Elevated plus maze test of transgenic (FAD tg, grey, n = 6 (3 months); n = 6 (6 months); n = 13 (9 months)) and non-transgenic (FAD wt, white, n = 6 (3 months); n = 6 (6 months); n = 15 (9 months)) 5xFAD males at different ages. 9 month old transgenic mice spent significantly more time in the open arms (A) and less time in the closed arms (B) or in the middle of the maze (C) compared to age matched non-transgenic mice (*p b 0.05; **p b 0.005) or young transgenic 5xFAD mice (#p b 0.05; ##p b 0.005).
Fig. 3. Open field test of transgenic (FAD tg, grey, n = 9 (3 months); n = 8 (6 months); n = 17 (9 months)) and non-transgenic (FAD wt, white, n = 6 (3 months); n = 6 (6 months); n = 15 (9 months)) 5xFAD males at different ages. In 9 month old transgenic mice the traveled distance (A) and rearing (B) were significantly reduced in comparison to 9 month old non-transgenic mice (⁎p b 0.05; ⁎⁎p b 0.005).
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
242 243
246 247 248 249 250 251 252
254 255 256 257 258 259 260 261 262 263 264 265 266
5
N C O
R
R
E
C
T
E
D
P
R O
O
F
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
Fig. 4. Morris water maze test of transgenic (FAD tg, grey circles, n = 6 (3 months); n = 12 (6 months); n = 7 (9 months)) and non-transgenic (FAD wt, rhombs, n = 8 (3 months); n = 15 (6 months); n = 9 (9 months)) 5xFAD males at different ages. The learning curve of 3 month (A) and 6 month (B) old transgenic and non-transgenic 5xFAD males did not differ significantly. Transgenic mice had an increased latency time to reach the platform at 9 months of age (C) compared to non-transgenic 5xFAD mice. The area under curve (D) confirmed the higher latency of 9 month old transgenic 5xFAD mice (⁎p b 0.05). In the probe trial transgenic 5xFAD mice did not differ in the path length (E), swim speed (F) or quadrant preference (G, H, I) from non-transgenic mice at all ages.
3.5. Adhesive removal test
268 269
272 273
The adhesive removal test was performed to test the sensitivity of the perception at the paws and motor skills in transgenic and non-transgenic 5xFAD mice. 9 month old transgenic mice needed significantly more time to remove the tape from their fore paws than their age matched non-transgenic littermates (p = 0.008, Fig. 5). There were no differences between 3, 6 and 9 month old animals.
274
3.6. EEG recording
275
In most of the behavioral tests a difference between transgenic and non-transgenic animals started with a trend at 6 months. We chose this age as well as 3 month old animals for EEG measurements to find out whether the functional EEG readout is able to diagnose an effect
270 271
276 277 278
U
267
earlier than a behavioral readout. At 3 months of age, the frequency bands of transgenic and non-transgenic mice were indistinguishable from each other, while 6 month old transgenic animals showed a significant increase of the subdelta band and a decrease in all other frequency bands compared to age matched non-transgenic mice (p b 0.05). Significant differences were also observed between 3 and 6 month old transgenic animals—except for the delta frequency band, which was only reduced by trend (Fig. 6).
279 280
3.7. Sleep recording
287
Sleep recording experiments revealed a decrease in REM sleep in 9 month old transgenic FAD mice compared to non-transgenic littermates (Fig. 7D). While no significant %W and %SWS difference could be seen between transgenic and non-transgenic mice, we found that %REM in transgenic mice seemed reduced by trend. This reduction
288 289
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
281 282 283 284 285 286
290 291 292
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
R O
O
F
6
D
P
Fig. 6. Cortical EEG measurement of transgenic (FAD tg, grey, n = 4 (3 months); n = 6 (6 months)) and non-transgenic (FAD wt, white, n = 5 (3 months); n = 4 (6 m months o)) 5xFAD males at different ages. Non-transgenic mice (white) and transgenic mice (grey) were not different at 3 months but showed differences at 6 months (wt-white/ striped); tg-grey/striped) in all frequency bands of subdelta (0.1.0.5 Hz), delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), beta (13–38 Hz) and gamma (N38 Hz; p b 0.05, # difference between 3 and 6 months, ⁎ difference between non transgenic and transgenic mice).
E
investigation of 5xFAD males since in most studies only females of this 301 strain were characterized [2,10,19,46]. 302
E
C
T
4.1. Non cognitive behavior—anxiety and locomotion
293
R
Fig. 4 (continued).
297
4. Discussion
298
None of the known animal models is a perfect representative of the human AD. Current mouse models simply model key aspects of the disease [18]. The aim of this study was the further functional and behavioral
O
C
N
U
299 300
R
296
was statistically significant (F(1,15) = 4.756; p = 0.0455) when the %REM values were calculated in relation to the amount of total sleep (%REM/TS; see Fig. 7D). With regard to SWS- and REM-latencies, we found no differences between transgenic and non-transgenic mice.
294 295
Fig. 5. In the adhesive removal test, 9 month old transgenic 5xFAD males (FAD tg, grey, n = 10 (3 months); n = 8 (6 months); n = 6 (9 months)) needed significantly more time removing the tape from their fore paws than age matched non-transgenic littermates (FAD wt, white, n = 7 (3 months); n = 4 (6 months); n = 5 (9 months), ⁎p b 0.008).
In the past the 5xFAD mouse model was preferred for immunohistochemical and molecular examinations [1,4,8,13,33]. The literature on behavioral abnormalities in the 5xFAD mouse is limited [2,10,46]. Our investigations regarding the behavior of 5xFAD males yielded significantly reduced anxiety paired with a decreased locomotion and exploration in transgenic mice compared to non-transgenic animals at 9 months of age. In females of the same strain the anxiety level was already reduced at six months of age, but locomotion and exploration remained unaltered— even in nine and twelve month old mice [19]. This could imply gender specific behavioral deficits in transgenic 5xFAD mice but also methodical differences across the laboratories. In other models like Tg2576 males [26] and APP23 females [12] anxiety was also decreased, whereas it was increased in 3xTg-AD females [37]. The locomotor activity was also reduced in APP23 mice [22] and in APP/PS1 males [14] while it was increased in several other models [12,26,37,42]. An increase in locomotor activity is interpreted as restlessness and agitation. Decreased locotomotor activity on the other hand, as we observed it in aged 5xFAD males, is linked to other non-cognitive symptoms like apathy and depression [42]. Some of our 5xFAD mice showed severe stereotypes like jumping, circle running and somersaults in their home cage at 9 months of age which were independent on genotype. Continuous jumping against the walls in their home cage was also described in transgenic APP23mice and was defined as species-specific myoclonic jumping. Normally myclonus can be interpreted as pre-epileptic movements resulting from an abnormal neuronal activity in several brain regions, especially in the brain stem and the cerebellum and is observed mostly in late stages of AD [25]. Our phenotypic observation of male 5xFAD mice showed motor deficits, missing reflexes and a rigidity of fore and hind paws beginning at 9 months of age. These results were also found in 5xFAD females [19] and in other transgenic AD mouse models. Normal reflexes are usually characterized by an abduction of all four paws when animals fall on a horizontal surface [25]. Instead our transgenic 5xFAD males clasped their paws, which is due to parenchymal Aβ plaques and an
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337
7
C
T
E
D
P
R O
O
F
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
338 339
E
Fig. 7. Waking (%), slow-wave sleep (%), REM sleep (%) and REM related to total sleep (%REM/TS) in 9 month old FAD tg (n = 8) and FAD wt (n = 9) mice during 8 days (7-h recordings per day; ⁎p b 0.05 (days 1–8)).
362
4.2. Cognitive behavior
363
To investigate learning and memory deficits, the cardinal symptoms of AD, we performed the Morris water maze test. For the first time, we
345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
364
R
343 344
N C O
341 342
U
340
R
361
axonal neuropathology in brain and spinal cord. However this phenotype cannot be detected in all transgenic animals with plaque burden, e.g. 3xTg-AD-females showed no problems of motor coordination [37], for which reason amyloid plaque pathology seems to be no sufficient condition for clasping behavior. However the number of mutants showing abnormal flexion reflexes might be higher than wild-type mice [25]. The increasing impairment of the motor abilities and the increasing apathy in our transgenic 5xFAD males seemed to influence their nest building ability. They were less capable and less motivated than age matched non-transgenic mice to handle their nest building material sufficiently, resulting in very poorly shredded, almost untouched material. Similar results were seen in APP/PS1 males [14]. Moreover, in our study 9 month old male transgenic 5xFAD mice showed deficiencies in sensorimotor abilities compared to age matched littermates e.g. in removing tapes from their fore paws. However, this difference is based on decreased time in non-transgenic mice. Whereas 3 month old non-transgenic 5xFAD mice showed a high explorative behavior in the box, 9 month old mice were calmer and accordingly faster in removing the tapes. In transgenic mice, this age dependent behavior was not observable. In other mouse models such impairments are not necessarily detectable, even at advanced ages [24]. Nonetheless, these abilities seem to play an important role in disturbances of motor control as it occurs in AD [40].
demonstrated significant behavioral deficits in spatial learning in the Morris water maze in 9 month old transgenic 5xFAD-males. Impaired motor disabilities cannot explain the increased latency, as our transgenic 5xFAD males had no difficulties to match the performance of non-transgenic littermates with regard to swim path length and speed. Urano and Tohda [38] showed a similar impairment in female mice at 7 to 9 months. Another study [31] noted deficits in the water maze at 4 and 6 months of age, but there was no specification which gender had been used. Ohno et al. [31] observed a longer search in the target quadrant vs. the opposite quadrant and a closer search of wild-type mice near the correct platform position compared to the corresponding position in the opposite quadrant, whereas we did not find any difference in the probe trial between transgenic and non-transgenic 5xFAD males. In contrast to our findings, they did not observe specific impairments in the learning curve; however, the test conditions imply a different setup of the water maze experiment.
365 366
4.3. EEG
381
Our EEG analysis of the parietal cortex showed clear frequency shifts in transgenic 5xFAD males. Starting at six months of age, we observed an increase of the subdelta band and a decrease of all other analyzed frequency bands. These changes coincide with the impaired functional integrity and the re-organization of cortical columns that we found previously in 5xFAD transgenic mice at 6 months of age [28]. Single cell resolution mapping of neuronal thallium uptake in male 5xFAD mouse revealed that electrical activity of pyramidal cells breaks down throughout the infragranular cortical layer V long before cell death occurs. Laminar investigation of cortical circuit dysfunction with current
382
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
367 368 369 370 371 372 373 374 375 376 377 378 379 380
383 384 385 386 387 388 389 390 391
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422
426
In conclusion our data suggest that
435
Disclosure statement
436
R
O
C
431 432
The authors declare no conflict of interest.
N
429 430
R
433 434
• At 9 months of age 5xFAD males develop non-cognitive deficits in the elevated plus maze and the open field test and they show cognitive impairments when tested in the Morris water maze test. These impairments seem to be gender specific. • In 6 month old mice all EEG bands, except the subdelta band, were shifted to a lower band. • The functional EEG disturbances precede some of the cognitive and non-cognitive behavioral impairments in 5xFAD males.
427 428
Acknowledgments
438
441
This work was supported by the German Federal Ministry of Education and Science (BMBF grant VIP0562-03 V0765). We thank Lydia Löw for her excellent technical assistance and Anja Oelschlegel for constructive comments on the manuscript.
442
References
443 444 445 446 447 448 449 450
U
437
439 440
F
403 404
O
401 402
C
399 400
E
398
R O
5. Conclusion
396 397
P
425
394 395
[4] Bobkova NV, Garbuz DG, Nesterova I, Medvinskaya N, Samokhin A, Alexandrova I, et al. Therapeutic effect of exogenous hsp70 in mouse models of Alzheimer's disease. J Alzheimers Dis 2013;38(2):425–35. [5] Christos GA. Is Alzheimer's disease related to a deficits or malfunction of rapid eye movement (REM) sleep? Med Hypotheses 1993;41(5):435–9. [6] Coben LA, Danziger W, Storandt M. A longitudinal EEG study of mild senile dementia of Alzheimer type: changes at 1 year and at 2.5 years. Electoencephalogr. Clin Neurophysiol 1985;61:101–12. [7] Corbett BF, Leiser SC, Ling HP, Nagy R, Breysse N, Zhang X, et al. Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease. J Neurosci 2013;33(16):7020–6. [8] Crouzin N, Baranger K, Cavalier M, Marchalant Y, Cohen-Solal C, Roman FS, et al. Area-specific alterations of synaptic plasticity in the 5XFAD mouse model of Alzheimer's disease: dissociation between somatosensory cortex and hippocampus. PLoS One 2013;8(9):e74667. [9] Devi L, Ohno M. Genetic reductions of β-site amyloid precursor protein-cleaving enzyme 1 and amyloid-β ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer's disease model mice. Eur J Neurosci 2010; 31(1):110–8. [10] Devi L, Ohno M. Deletion of the elF2α kinase GCN2 fails to rescue the memory decline associated with Alzheimer's disease. PLoS One 2013;8(10):e77335. [11] Diekelmann S, Born J. The memory function of sleep. Nat Rev Neurosci 2010; 11(2):114–26. [12] Dumont M, Strazielle C, Stuafenbiel M, Lalonde R. Spatial learning and exploration of environmental stimuli in 24-month-old female APP23 transgenic mice with the Swedish mutation. Brain Res 2004;1024(1–2):113–21. [13] Eimer WA, Vassar R. Neuron loss in the 5XFAD mouse model of Alzheimer's disease correlates with intraneuronal Aβ42 accumulation and Caspase-3 activation. Mol Neurodegener 2013;8:2. [14] Filali M, Lalonde R, Rivest S. Subchonic memantine administration on spatial learning, exploratory activity, and nest-building in an APP/PS1 mouse model of Alzheimer's disease. Neuropharmacology 2011;60(6):930–6. [15] Freret T, Bouet V, Leconte C, Roussel S, Chazalviel L, Divous D, et al. Behavioral deficits after distal focal cerebral ischemia in mice: usefulness of adhesive removal test. Behav Neurosci 2009;123(1):224–30. [16] Hardy J, Selkoe J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 2002;297(5580):353–6. [17] Houghton PJ, Howes MJ. Natural products and derivatives affecting neurotransmission relevant to Alzheimer's and Parkinsons's disease. Neurosignals 2005; 14(1–2):6–22. [18] Ittner LM, Götz J. Amyloid-β and tau—a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci 2011;12(2):65–72. [19] Jawhar S, Trawicka A, Jenneckens C, Bayer TA, Wirths O. Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease. Neurobiol Aging 2012;33(1):196.e29–40. [20] Jeong J. EEG dynamics in patients with Alzheimer's disease. Clin Neurophysiol 2004;115:1490–505. [21] Kimura R, Ohno M. Impairments in remote memory stabilization precede hippocampal synaptic and cognitive failures in 5XFAD Alzheimer mouse model. Neurobiol Dis 2009;33(2):229–35. [22] Kobayashi DT, Chen S. Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer's disease. Genes Brain Behav 2005; 4(3):173–96. [23] LaFerla FM, Green KN, Oddo S. Intracellular amyloid-β in Alzheimer's disease. Nat Rev Neurosci 2007;8(7):499–509. [24] Lalonde R, Dumont M, Staufenbiel M, Strazielle C. Neurobehavioral characterization of APP23 transgenic mice with the SHIRPA primary screen. Behav Brain Res 2005;157(1):91–8. [25] Lalonde R, Fukuchi K, Strazielle C. Neurologic and motor dysfunctions in APP transgenic mice. Rev Neurosci 2012;23(4):363–79. [26] Lalonde R, Lewis TL, Strazielle C, Kim H, Fukuchi K. Transgenic mice expressing the βAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination. Brain Res 2003;977(1):38–45. [27] Lee JE, Han PL. An update of animal models of Alzheimer disease with a reevaluation of plaque deposition. Exp Neurobiol 2013;22(2):84–95. [28] Lison H, Happel MFK, Schneider F, Baldauf K, Kerbstat S, Seelbinder B, et al. Disrupted cross-laminar cortical processing in β amyloid pathology precedes cell death. Neurobiol Dis 2014 [Vol: Seiten]. [29] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, et al. Intraneuronal β-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci 2006;26(40):10129–40. [30] Ohno M. Failures to reconsolidate memory in a mouse model of Alzheimer's disease. Neurobiol Learn Mem 2009;92(3):455–9. [31] Ohno M, Chang L, Tseng W, Oakley H, Citron M, Klein WL, et al. Temporal memory deficits in Alzheimer's mouse models: rescue by genetic deletion of BACE1. Eur J Neurosci 2006;23(1):251–60. [32] Ohno M, Cole SL, Yasvoina M, Zhao J, Citron M, Berry R, et al. BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice. Neurobiol Dis 2007;26(1):134–45. [33] Ou-Yang MH, Van Nostrand WE. The absence of myelin basic protein promotes neuroinflammation and reduces amyloid beta-protein accumulation in Tg-5xFAD mice. J Neuroinflammation 2013;10(1):134. [34] Palop JJ, Mucke L. Epilepsy and cognitive impairments in Alzheimer disease. Arch Neurol 2009;66(4):435–40.
T
423 424
source density analysis identified an early loss of excitatory synaptic input in infragranular layers, linked to pathological recurrent activations in supragranular layers. These functional changes correlate with deficits in fear conditioning [28]. EEG changes comparable to those that we saw in our 5xFAD mice were also observed in the PLB1 mouse where an increase of the delta frequencies and a decrease of the alpha frequencies in the parietal and prefrontal cortex were found. These changes were accompanied by decreased LTP and reduced paired pulse facilitation at the age of 6 months as well as by impairments in recognition memory and spatial learning at the age of 12 months [35]. Wesson et al. [43] saw that sensory network hyperactivity, found in Tg2576 mice at an early stage contributed to subsequent hyporesponsiveness and dysfunction later in life. Corbett et al. [7] also noted EEG activity changes in association with reduced Morris water maze performances. Although AD mouse models develop spontaneous seizures and frequent epileptiform discharges [34,48], we did not find any seizures in 6 month old 5xFAD mice during our recording periods, when the mice were almost resting but not sleeping. Functional disturbances in the EEG pattern in transgenic 5xFAD mice might be related to the beginning of cognitive impairments at 6 months of age. These changes are comparable to the EEG slowing that is seen in human AD cases. Patients with dementia exhibit a decrease in alpha- and betaactivities and an increase in delta- and theta-activities [6]. While it is feasible that such an EEG shift might be partially related to normal aging, it was also shown that EEG abnormalities reflect directly the anatomical changes and the functional deficits of the cerebral cortex damaged by the disease [20]. Further, our transgenic 5xFAD mice had less rapid eye movement sleep (REM) compared to non-transgenic littermates. These changes coincide with a decline in REM sleep periods seen in patients with Alzheimer and other dementias [3,5,39] as well as in other mouse models of AD [35,47] and they support the hypothesis on the important role of sleep, especially REM sleep, in memory consolidation processes [11,36]
D
392 393
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx
E
8
[1] Antonios G, Saiepour N, Bouter Y, Richard BC, Paetau A, Verkkoniemi-Ahola A, et al. N-truncated Abeta starting with position four: early intraneuronal accumulation and rescue of toxicity using NT4x-167, a novel monoclonal antibody. Acta Neuropathol Commun 2013;1(1):56. [2] Aytan N, Choi JK, Carreras I, Kowall NW, Jenkins BG, Debeoglu A. Combination therapy in a transgenic model of Alzheimer's disease. Exp Neurol 2013;250:228–38. [3] Bliwise DL. Sleep disorders in Alzheimer's disease and other dementias. Clin Cornerstone 2004;6(Suppl. 1A):S16–28.
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
451 Q2 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 Q3 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536
F. Schneider et al. / Physiology & Behavior xxx (2014) xxx–xxx [35] Platt B, Drever B, Koss D, Stoppelkamp S, Jyoti A, Plano A, et al. Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1. PLoS One 2011;6(11):e27068. [36] Poe GR, Walsh CM, Bjorness TE. Cognitive neuroscience of sleep. Prog Brain Res 2010;185:1–19. [37] Sterniczuk R, Antle MC, LaFerla FM, Dyck RH. Characterization of the 3xTg-AD mouse model of Alzheimer's disease: part 2. Behavioral and cognitive changes. Brain Res 2010;1348:149–55. [38] Urano T, Tohda C. Icariin improves memory impairment in Alzheimer's disease model mice (5xFAD) and attenuates amyloid β-induced neurite atrophy. Phytother Res 2010;24(11):1658–63. [39] Veccierini MF. Sleep disturbances in Alzheimer's disease and other dementias. Psychol Neuropsychiatr Vieil 2010;8(1):15–23. [40] Velasques B, Machado S, Paes F, Cunha M, Sanfim A, Budde H, et al. Sensorimotor integration and psychopathology: motor control abnormalities related to psychiatric disorders. World J Biol Psychiatry 2011;12(8):560–73. [41] Waldau B, Shetty AK. Behavior of neural stem cells in the Alzheimer brain. Cell Mol Life Sci 2008;65(15):2372–84. [42] Walker JM, Fowler SW, Miller DK, Sun AY, Weisman GA, Wood WG, et al. Spatial learning and memory impairment and increased locomotion in a transgenic amyloid
[43]
[44] [45]
[46]
[47]
F
[48]
precursor protein mouse model of Alzheimer's disease. Behav Brain Res 2011; 222(1):169–75. Wesson DW, Borkowski AH, Landreth GE, Nixon RA, Levy E, Wilson DA. Sensory network dysfunction, behavioral impairments, and their reversibility in an Alzheimer's β-amyloidosis mouse model. J Neurosci 2011;31(44):15962–71. Wetzel W, Wagner T, Balschun D. REM sleep enhancement induced by different procedures improves memory retention in rats. Eur J Neurosci 2003;18(9):2611–7. Zhang W, Bai M, Xi Y, Hao J, Liu L, Mao N, et al. Early memory deficits precedes plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction. Free Radic Biol Med 2012;52(8):1443–52. Zhang Z, Liu X, Schroder JP, Chan CB, Song M, Yu SP, et al. 7,8-Dihydroxyflavone prevents synaptic loss and memory deficits in a mouse model of Alzheimer's disease. Neuropsychopharmacology 2013;1-13. Zhang B, Veasey SC, Wood MA, Leng LZ, Kaminski C, Leight S, et al. Impaired rapid eye movement sleep in the Tg2576 APP murine model of Alzheimer's disease with injury to pedunculopontine cholinergic neurons. Am J Pathol 2005;167(5):1361–9. Zuyatdinova S, Gurevicius K, Kutchiashvili N, Bolkvadze T, Nissinen J, Tanila H, et al. Spontaneous epileptiform discharges in a mouse model of Alzheimer's disease are suppressed by antiepileptic drugs that block sodium channels. Epilepsy Res 2011;94:75–85.
O
537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556
9
U
N C O
R
R
E
C
T
E
D
P
R O
577
Please cite this article as: Schneider F, et al, Behavioral and EEG changes in male 5xFAD mice, Physiol Behav (2014), http://dx.doi.org/10.1016/ j.physbeh.2014.05.041
557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576