Auditory stimuli

Six musical pieces were used in the experimental study. One was Mozart’s piano sonata K.448, which is usually used in studies of the Mozart effect. The second piece was the retrograde version of Mozart’s K.448 (retrograde Mozart), which was obtained by arranging the component notes of Mozart’s K.448 in a reversed sequence that maintained the same physical properties as the original except for the melodic patterns. The third piece was the rhythm of Mozart’s K.448, which preserved only the rhythm component (the pitch was changed to be the same throughout the piece). The fourth was the pitch component of Mozart K.448 (the rhythm was removed by distributing the notes uniformly in a bar). Figure 1 shows the material of the four music stimuli used in the experiments. The fifth and sixth stimuli were the retrograde versions of the third and fourth stimuli (omitted in Fig. 1).

Animals and treatments

All experiments were performed according to the experimental guidelines of the University of Electronic Science and Technology of China (UESTC) and were approved by the ethics committee of the UESTC. Sprague Dawley (SD) rats born from time-mated dams (Animal Research Institute of Sichuan Province, China) at UESTC were used in the experiments. The rats were maintained on a 12-h-light/12-h-dark cycle (light on at 08:00 h) at a controlled ambient temperature (22 ± 2 °C). Food and water were made available ad libitum.

The environmental sound level of the control group was 65 dB (ambient noise) and the sound level of the musical group was 65–75 dB. The musical stimuli were played repeatedly for 12 h from 8:00 p.m. to 8:00 a.m. daily so that the rats would not be disturbed in their sleep. All acoustic interventions began on PND 1 and continued through PND 98.

Morris water maze test

The hidden platform water maze task23,24 was used to study the learning and memory ability of the rats. The pool (130 cm diameter ×50 cm high) was filled with water (26 ± 1 °C) to a depth of 25 cm and the water was made opaque with a mixture of white, thick, non-toxic milk. A circular Plexiglas platform (10 cm diameter), onto which the rat could escape, was positioned in the centre of the target quadrant. One day before each testing period, each rat was placed in the pool for 2 min as a pretraining session. The rat was then allowed to climb onto the platform where it could rest for 20 s. This procedure was repeated three times to habituate the rat to the training environment. On days 1–4 of testing, the rats performed four trials and each trial began with the rat being placed in the pool and released facing the sidewall at one of four positions (the boundaries of the four quadrants, labelled Left, Right, Target and Opposite). The time between immersion into the pool and escape onto the platform was recorded to provide a measure of spatial reference memory for each trial (four trails a day, total 16 trials for each rat).

The day after the last latency trial was completed (day 5), the platform was removed and the rats were placed in the water maze on the opposite side of the target quadrant from where the platform had previously been located. The rats were then allowed to explore the pool for 60 s (probe trials). Spatial memory ability was measured as the duration of time spent in the target quadrant (% total time, chance level = 25%).

The behavioural assessment was conducted at three time points: PND 28, PND 56 and PND 98, which were selected based on developmental stages in rats.

Human behavioural experiment

To demonstrate that Mozart and retrograde Mozart music would produce similar effects on humans as in rats, the performed cognitive effect of humans were performed. We employed three groups of undergraduate university students without formal music training (10 males and 10 females per group), ranging in age from 18 years to 24 years (mean = 20.6 years) and each group had 20 subjects. This study was approved by the ethics committee of the University of Electronic Science and Technology of China (UESTC) and performed according to the experimental guidelines of the UESTC. Every subject provided written consent to participate in this experiment. The tests included a paper-and-pencil maze test and the paper folding and cutting test, the use of which has been well established in previous Mozart studies. Before the formal testing, 30 subjects (different from those in the formal experiment) were pretested to divide all of the test items into two sets according to the degree of difficulty. The experimental protocols were the following: on the first day, 60 subjects participated in two cognitive tests: the pencil-and-paper maze test and the paper folding and cutting test. Based on the experimental results, the 60 students were divided into three groups with approximately equivalent abilities and each group had 20 subjects. From the second day, one of the three groups (in the same fixed classroom) listened to half an hour of Mozart music every morning (MG), continuing to the seventh day (for a total of 6 days of listening). The second group (at the same time and in the same classroom as the MG) listened to half an hour of retrograde Mozart music (RMG), continuing to the seventh day (for a total of 6 days of listening). The music was played through earphones and subjects were blind to the type of music in both the MG and RMG; thus, they did not know what music they were going to hear. The third group of subjects remained silent in the same classroom for the same amount of time, also continuing until the seventh day (6 days of sitting in silence). On the eighth day, all three groups performed these two tasks (with different test items from the first items) at the same time as the first experiment.

Neurogenesis and immunohistochemical procedures

Five rats from the MG, the RMG and the CG were used to detect the neurogenesis and BDNF/TrkB level. These rats had been intraperitoneally injected with 50 mg/kg 5-bromo-2’-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) three times, at 4-h intervals one week before.

The rats were anesthetized and transcardially perfused with fixative (4% paraformaldehyde) at the end of each behaviour experiment (PND 28, PND 56 and PND 98). The brains were then removed and placed in the same fixative for 24 h before being transferred into a 30% sucrose solution for cryoprotection. Samples were serially sectioned in the coronal plane at 30 μm using a freezing microtome (Leica, Nussloch, Germany).

The free floating sections were processed for immunohistochemistry (IHC) as previously described25. Briefly, for antigen retrieval, sections were incubated with 0.25% trypsin in phosphate-buffered saline (PBS) for 5 min. Following extensive washes in PBS, the sections were blocked with 10% goat serum solution for 1 h. Primary antibodies were applied overnight at 4 °C. The sections were then washed three times with PBS and incubated for 1 h with a species-specific secondary antibody. Subsequently, the sections were extensively washed again and placed on Superfrost Plus slides. After mounting, the sections were observed and photographed using a Leica microscope equipped with a Spot® digital camera.

For double BrdU/DCX immunofluorescence labelling, sections were permeabilized by incubation with 0.5% Triton X-100 in PBS for 15 min and denatured in 1 N HCl at 37 °C for 30 min. Then, the sections were washed three times in 100 mM sodium borate (pH 8.5). The remaining experimental steps were carried out as described above. However, to exclude the possibility of false labelling, primary antibodies produced in different species were co-incubated and secondary antibodies were tested for cross reactivity in a double labelling experiment.

The following antibodies and final dilutions were used. The primary antibodies included: rabbit anti-BDNF (1:200, Santa); rabbit anti-TrkB (1:200, Abcam); mouse anti-BrdU (1:300, Cell Signalling Technology, Inc.); and goat anti-DCX (1:300, Cell Signalling Technology, Inc.). For corresponding secondary antibodies, goat anti-rabbit (1:200, Southern Biotech) donkey anti-mouse (1:200, Abcam) and donkey anti-goat (1:200, Southern Biotech) were used.

Statistical analysis

For the behavioural tests, the escape latency during the hidden- platform acquisition training was analysed with a repeated measures analysis of variance (ANOVA) with a general linear model. Group differences in the duration of time spent in quadrants were analysed with a two-way ANOVA.

For the IHC, the area of the selected region was measured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The mean density of protein expressed in the area was measured automatically. Statistical analysis was performed with a one-way ANOVA.

All values are expressed as the mean ± S.E.M. (standard error of the mean) and the statistical analyses were performed using SPSS (version 16.0). Post hoc comparisons were performed using the Least-significant difference (LSD) method, where applicable, to provide more detail about the differences among groups. The level of statistical significance was set at P < 0.05.