Conceived and designed the experiments: SS KY. Performed the experiments: SS KY CH SM YY. Analyzed the data: SS KY HH. Contributed reagents/materials/analysis tools: YY HY. Wrote the paper: SS KY HH.
The authors have declared that no competing interests exist.
The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors. In diabetes and Alzheimer's disease, pathological progression is accelerated by activation of RAGE. However, how RAGE influences gross behavioral activity patterns in basal condition has not been addressed to date. In search for a functional role of RAGE in normal mice, a series of standard behavioral tests were performed on adult RAGE knockout (KO) mice. We observed a solid increase of home cage activity in RAGE KO. In addition, auditory startle response assessment resulted in a higher sensitivity to auditory signal and increased prepulse inhibition in KO mice. There were no significant differences between KO and wild types in behavioral tests for spatial memory and anxiety, as tested by Morris water maze, classical fear conditioning, and elevated plus maze. Our results raise a possibility that systemic therapeutic treatments to occlude RAGE activation may have adverse effects on general activity levels or sensitivity to auditory stimuli.
The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors
Ligands of RAGE include high mobility group box 1 (HMGB1, also known as amphoterin)
RAGE KO mice have been generated by a multiple number of laboratories
Prior to the behavioral experiments, genotyping was performed by PCR (
(A) PCR for ear samples shows RAGE(−/−) mice have a single band at 301 bp, RAGE(+/+) mice have a single band at 380 bp, and RAGE(+/−) mice have both bands, as described in Myint et al.
The mice were assessed for home cage activity. As the room illumination is controlled at 12/12 hour light/dark cycle, the animals' activity was modulated accordingly with more activity during the dark phase (
(A) Average home cage activity records of WT (solid line) and RAGE KO (dashed line) mice are shown for two independent sets of experiments (see main text for more details). The light and dark phases are indicated by white and grey backgrounds, respectively. (B) Group comparison of home cage activity in the light phase. The activity in the light phase is similar and remained constantly low. (C) Group comparison of home cage activity in the dark phase. The activity of KO mice in dark phase is higher than that of WT mice. Note that both WT and KO showed a gradual decrease in activity in the dark during the course of the seven days. For B and C, Set 1 and Set 2 are combined. Data are mean±S.E.M. * p<0.05, ** p<0.01.
In the open field test, both genotypes had similar exploration distance in fifteen minutes (WT vs. KO: 5421.1±833.1 cm vs. KO 5211.4±320.5 cm, p = 0.619 for Set 1; 6283.5±1401.9 cm vs. 5563.6±1189.3 cm, p = 0.232 for Set 2). The mean distance traveled in one minute could not be distinguished by genotype throughout the fifteen minutes of the experiment. The total time spent in the center of the arena was similar between WT and KO in Set 1 (224.7±117.5 s vs. 242.0±70.8 s, p = 0.695, t-test), however KO tended to stay longer in the center position in Set 2 (163.8±41.7 s vs. 281.6±73.5 s, p<0.01, t-test).
In the light-dark box test, the results varied between Set 1 and Set 2 (as summarized in
Both WT and KO displayed comparable behavioral patterns in the elevated plus maze test under 70 lx condition (Set 1) and 40 lx condition (Set 2). The proportion of the time spent in the open arm (WT vs. KO: 14.5±19.6% vs. 24.5±28.1%, p = 0.597 for Set 1; 23.1±15.4% vs. 13.3±14.0%, p = 0.09 for Set 2; Mann-Whitney's U-test,) and the relative frequency of open arm entry (31.0±16.9% vs. 32.1±24.3%, p = 0.971 for Set 1; 31.6±11.0% vs. 24.0±14.0% p = 0.307 for Set 2, Mann-Whitney's U-test) were not significantly different.
Auditory startle response assessment resulted in a higher sensitivity to auditory signal in KO. In both Set 1 and 2, WT were virtually irresponsive to auditory signals up to 90 dB, whereas KO showed response from 85 dB (
(A) KO mice are more sensitive to auditory stimulation (Set 1 & 2 combined). (B) Prepulse inhibition showed the response is more inhibited in KO mice. Abscissa values indicate the volume of prepulse tones. Data are mean±S.E.M. for A and B. * p<0.05, ** p<0.01, *** p<0.001. (C) Four stages of freezing response in the classical fear conditioning test are plotted. The freezing responses at final bin (30 s period, 1 min after the final (second) shock) of the conditioning phase (Conditioning) were not significantly different between WT and KO mice. Both WT and KO show similar freezing responses in the context test (Context). In the cue test, there is a significant difference in the freezing response in the cue test cage
Morris water maze test was done to test animals' spatial learning ability. There was no significant difference in the total distance traveled to find the target during four days of training between the genotypes (two-way ANOVA for genotype, F(1,54), p = 0.962 for Set 1; F(1,54), p = 0.06 for Set 2). Similarly, the probe test did not yield any performance differences in the target ratio measured by stay time (WT vs. KO: 34.3±12.1% vs. 32.4±9.6, p = 0.734 for Set 1; 26.8±19.9% vs. 36.3±13.0%, p = 0.273 for Set 2; Mann-Whitney's U-test) or in the target ratio measured by number of crosses (44.0±15.2% vs. 34.8±16.4%, p = 0.167 for Set 1; 35.3±33.3% vs. 34.9±21.9%, p = 1.00 for Set 2; Mann-Whitney's U-test).
The experimental animals were tested for fear conditioning. During conditioning trials, both WT and KO showed similar freezing response after electric foot shocks (final bin freezing behavior percentage WT vs. KO: 40.7±21.6% vs. 61.2±23.5%, p = 0.070 for Set 1; 29.3±23.1% vs. 34.5±19.2%, p = 0.956 for Set 2, Mann-Whitney's U-test,
Among the series of behavioral tests, the most striking behavioral difference was observed in the home cage activity. RAGE KO mice displayed ∼30% higher activity in darkness on day 1 and persistently higher activity during the seven days of observation. In addition, auditory startle response assessment resulted in a higher sensitivity to auditory signal in KO mice. The higher sensitivity to auditory signal provides an explanation for the increased prepulse inhibition ratio in KO animals and auditory cue-dependent classical fear conditioning. The animals' curiosity or anxiety should be excluded from the subject of the difference, as the open field test and the elevated plus maze test yielded no significantly different scores.
Our results indicate that deletion of RAGE has minimal effects on the animals' spatial learning ability (as tested with Morris water maze and context-dependent classical fear conditioning). Therefore, it appears that RAGE does not have a critical importance in synaptic plasticity of the hippocampus and the associated areas. In fact, long-term potentiation in the entorhinal cortex has been reported to be not affected in RAGE KO mice
As the KO phenotypes were dependent on presentation of sensory stimulus, RAGE may play an active role in sensory organs or the brain. Immunohistochemical localization of RAGE in the brain has remained controversial to date
As activation of RAGE accelerates pathological progression of diabetes or Alzheimer's disease, therapeutic treatments to attenuate activation of RAGE have been suggested
RAGE (−/−) (KO) mice were generated similar to as described in Myint et al.
Tissue samples from the ear were dissolved in a buffer containing (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP-40, 0.45% Tween-20, 0.5 mg/ml proteinase K) at 55°C for overnight. The lysate, dNTP mixture, TaKaRa Ex Taq, Taq buffer and the following three primer were mixed;
A polyclonal anti-RAGE antibody (H-300, Santa Cruz Biotech. Inc.) was coupled to HiTrap NHS-activated HP Columns (GE Healthcare) according to the manufacturer's instructions. Tissue homogenates (1 ml) from lung (0.18 g or 0.2 g) and brain (0.5 g or 0.5 g) of RAGE KO or WT mice, respectively, in tissue lysis buffer of 50 mM Tris-HCl (pH 7.5), 1% TritonX-100, 150 mM NaCl, and proteinase inhibitors (10 KIU/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM benzaminidin, and 1 mM EDTA) were applied to the HiTrap-anti-RAGE antibody column previously equilibrated with the lysis buffer. After washing with a 5 bed volume of the equilibration buffer, bound proteins were eluted with 0.1 M glycine–HCl (pH 2.5). The eluate was precipitated with 10% trichloroacetic acid (TCA) at 4°C for 15 min. The pellet was re-suspended in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue) and boiled at 95°C for 5 min. Proteins in the lysates were resolved by SDS-PAGE (5–20%) and transferred onto a polyvinylidene fluoride membrane (Millipore Corp.). The membranes were incubated with a polyclonal anti-RAGE antibody
The experimental animals were subject to a series of behavioral tests performed according to the schedule described in
Set 1 | ||
Day | Time | Behavioral paradigm |
1 | AM | Introduction to behavioral experiment room |
PM | Home cage activity test started (at 15:00) | |
8 | PM | Home cage activity test finished |
14 | PM | Open field test (15 min, 70 lx) |
15 | PM | Light-Dark box test (10 min) |
19 | PM | Elevated plus maze test (5 min, 70 lx) |
21 | PM | Startle response & PPI test (120 dB) |
22 | PM | Startle response & PPI test (120 dB) |
25 | AM/PM | Water maze test: training day 1 |
26 | AM/PM | Water maze test: training day 2 |
27 | AM/PM | Water maze test: training day 3 |
28 | AM/PM | Water maze test: training day 4 |
29 | PM | Water maze test: probe test |
33 | PM | Fear conditioning test (conditioning trial) |
34 | PM | Fear conditioning test (context trial) |
35 | PM | Fear conditioning test (cued trial) |
Set 2 | ||
Day | Time | Behavioral paradigm |
1 | AM | Introduction to behavioral experiment room |
PM | Home cage activity test started (at 15:00) | |
8 | PM | Home cage activity test finished |
14 | PM | Open field test (15 min, 70 lx) |
20 | PM | Open field test (15 min, 250 lx) |
26 | PM | Light-Dark box test (10 min) |
32 | PM | Elevated plus maze test (5 min, 40 lx) |
39 | PM | Startle response & PPI test (110 dB) |
40 | PM | Startle response & PPI test (110 dB) |
46 | PM | Startle response & PPI test (120 dB) |
47 | PM | Startle response & PPI test (120 dB) |
53 | AM/PM | Water maze test: training day 1 |
54 | AM/PM | Water maze test: training day 2 |
55 | AM/PM | Water maze test: training day 3 |
56 | AM/PM | Water maze test: training day 4 |
57 | PM | Water maze test: probe test |
60 | PM | Fear conditioning test (conditioning trial) |
61 | PM | Fear conditioning test (context trial) |
62 | PM | Fear conditioning test (cued trial) |
Spontaneous activity of mice in their home cage was measured using a 24 channel activity monitoring system (O'Hara, Tokyo, Japan). Cages were individually set into the compartments made of stainless steel in the negative breeding rack (JCL, Tokyo, Japan). A piezoelectric sensor was equipped on the ceiling of each compartment to detect the mouse movements. Activity counts represent the number of active time bin (approximately 0.20–0.25 s each) in which spontaneous activity including locomotor activity, rearing and other voluntary stereotypic movements were detected. Home cage activity was measured for seven consecutive days during which bedding materials were not changed.
Open field test apparatus was placed in a small sound-proof room (185×185×225 cm). The apparatus consisted of four white plastic boxes (50×50×40 cm), two electric fans for ventilation and background noise (35 dB), white LED light source (70 lx at the center of the field) which served as the sole source of illumination during the experiment. For each box, a CCD camera is attached on the ceiling for monitoring mice. Mice were individually introduced at the center of the arena and were allowed to move freely for 15 min. Distance traveled (cm) and % duration of staying at the center area of the field (30% of the field) were adopted as the indices, and they were collected every 1 min.
A light-dark box system was equipped in the same sound-proof room as the open field test. The light box was made of white plastic (20×20×20 cm) and illuminated by LEDs (250 lx at the center of the box) and a CCD camera was equipped on the ceiling, and the dark box was made of black plastic (20×20×20 cm) and an infrared camera was equipped on the ceiling. The light box and dark box was connected by a gate for transition on the center panel between the light box and dark box (5×0.5×3 cm) with a slide door. Mice were individually introduced into the light box, and the door of the tunnel automatically opened after two seconds. Then mice were allowed to move freely for ten min. Total distance traveled, % distance traveled in the light box, % duration staying in the light box, number of the transitions between the light and dark boxes and the latency to first enter the dark box were measured.
An elevated plus maze consisted of a pair of closed arms (25×5×15 cm) and a pair of open arms 25×5×0.3 cm) was placed in the same sound-proof room as the open field test. The floor of each arm was made of white plastic and the walls of the closed arms and ridges of the open arms were made of clear plastic. The closed arms and open arms were arranged orthogonally. The apparatus was elevated 60 cm above the floor and illuminated at 70 lx at the center platform of the maze (5×5 cm). Mice were individually put on the center platform facing to an open arm, and then mice were allowed to move freely in the maze for 5 min. Total distance traveled, % time stayed in the open arms, % number of the open arm entry were measured.
Each mouse was put into a small cage for startle response (30 or 35 mm diameter, 12 cm long) and set on the sensor block in a sound-proof chamber (60×50×67 cm) with dim illumination (10 lx at the center of the sensor block). White noise (65 dB) was presented as background noise. Experimental session began after the mouse was acclimatized to the environment for five min. In the first session, only startle stimuli (SS, 120 dB, 40 ms) were presented for ten times in random inter-trial intervals (ITI, 10–20 s). In the second session, startle response to stimuli at various intensities were assessed. Five rounds of 70 to 120 dB white noise stimuli (in 5 or 10 dB increments, 40 ms) were presented in quasi-random order and random ITI. In the prepulse inhibition (PPI) session, mice experienced five types of trials; no stimulus, SS only, and prepulse (20 ms, lead time 100 ms)-SS pairings with three different prepulse volumes (70 dB, 75 dB, and 80 dB). Each trial repeated ten times in quasi-random order and random ITI. In the final session, only SS were again presented for ten times in random ITI.
A standard Morris' water maze test was performed
Classical fear conditioning test consisted of three parts; a conditioning trial, a context test trial, and a cued test trial. Fear conditioning was carried out in a clear plastic chamber equipped with a stainless steel grid floor (34×26×30 cm) connected to an electric shock generator. A CCD camera was equipped on the ceiling of the chamber. White noise (65 dB) was supplied as an auditory cue (CS). The conditioning trial consisted of a 2 min exploration period followed by two CS-US pairings separated by 1 min each. A US (foot-shock: 0.5 mA, 2 s) was administered at the end of the 30 s CS period. A context test was performed in the same conditioning chamber for three min in the absence of CS. The cued test was performed in an alternative context with different chamber (triangular shape, white color walls, 0–1 lx brightness, solid floor with thin bedding materials). The cued test consisted of a 2 min exploration period to evaluate the nonspecific contextual fear, followed by 2 min CS period (no US) to evaluate the acquired cued fear. Rate of freezing response (immobility excluding respiration and heartbeat) of mice was measured as an index of fear memory.
Behavioral experiments with mouse tracking information were analyzed with custom-modified ImageJ software (O'Hara, Tokyo, Japan). ImageJ is public domain software available from NIH (
Behavioral scores for the light-dark box test.
(0.03 MB DOC)
We are grateful to Dr. Ryusuke Nakagawa for his support and advice. We thank Ms. Kazuko Yahagi for technical assistance.