Autism is a neurodevelopmental disorder and this disorder shows impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests. The effect of music on short-term memory in the view of cell proliferation in the hippocampus was evaluated using valproic acid-induced autistic rat pups. Animal model of autism was made by subcutaneous injection of 400-mg/kg valproic acid into the rat pups on the postnatal day 14. The rat pups in the music-applied groups were exposed to the 65-dB comfortable classic music for 1 hr once a day, starting postnatal day 15 and continued until postnatal day 28. In the present results, short-term memory was deteriorated by autism induction. The numbers of 5-bromo-2′-deoxyridine (BrdU)-positive, Ki-67-positive, and doublecortin (DCX)-positive cells in the hippocampal dentate gyrus were decreased by autism induction. Brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) expressions in the hippocampus were also suppressed in the autistic rat pups. Music application alleviated short-term memory deficits with enhancing the numbers of BrdU-positive, Ki-67-positive, and DCX-positive cells in the autistic rat pups. Music application also enhanced BDNF and TrkB expressions in the autistic rat pups. The present study show that application of music enhanced hippocampal cell proliferation and alleviated short-term memory impairment through stimulating BDNF-TrkB signaling in the autistic rat pups. Music can be suggested as the therapeutic strategy to overcome the autism-induced memory deficits.
Autism is a neurodevelopmental disorder and this disorder shows impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests (
Prenatal exposure to teratogenic agents has been suggested as the pathogenic etiology of autism (
Hippocampus is an important brain area for the learning process and memory capability, and neuropathological alterations of hippocampus probably account for early symptoms of neuropsychiatric diseases (
For the detection of newly formed neurons, 5-bromo-2′-deoxyridine (BrdU) immunohistochemistry and Ki-67 immunohistochemistry have widely been used (
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, and performs its action through binding to its receptor, tyrosine kinase B (TrkB). BDNF-TrkB interaction promotes neuronal survival and differentiation, increases synaptic plasticity, and improves learning ability and memory function (
Music therapy has been used for developmentally delayed children, mentally ill adults, atrisk youth, chronic disease patients, geriatric patients, and patients in psychotherapy. It is also recommended as a treatment modality for children with autism (
In the present study, we evaluated the effect of music application on short-term memory in the view of cell proliferation in the hippocampus using valproic acid-induced autistic rat pups. Step-dawn avoidance task was conducted for the evaluation of short-term memory. Immunohistochemistry for BrdU, Ki-67, and DCX in the hippocampal dentate gyrus was performed for the calculation of cell proliferation. BDNF and TrkB expressions in the hippocampus were evaluated by Western blot analysis.
Male Sprague-Dawley rat pups (25±5 g, 2 weeks old) were used, and the experimental procedures were performed in accordance with the animal care guidelines of the Korean Academy of Medical Sciences and also with the National Institutes of Health. The animals were divided into 4 groups (n=8 in each group): Control group, music-applied group, autism-induced group, autism-induced and music-applied group. One hr before the exposure to music, all rat pups received 50-mg/kg BrdU (Sigma Chemical Co., St. Louis, MO, USA) intraperitoneally, once a day, from postnatal day 15 to postnatal day 19.
To make autism-like animal model, 400-mg/kg valproic acid (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), dissolved in saline at a concentration of 0.1 mL/kg, was subcutaneously injected into the rat pups on the postnatal day 14, as the previous method (
The rat pups in the music-applied groups were exposed to the 65-dB comfortable classic music for 1 hr once a day, starting postnatal day 15 and continued until postnatal day 28, as the previous method (
Latency in the step-down avoidance task was used for the evaluation of short-term memory, as the previous method (
The rat pups were sacrificed on the postnatal day 30, immediately after step-down avoidance task. The animals were anesthetized using Zoletil 50 (10 mg/kg, intraperitoneally; Vibac Laboratories, Carros, France), transcardially perfused with 50-mM phosphate-buffered saline (PBS), and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in 100-mM phosphate buffer (pH, 7.4). The brains were dissected and postfixed in the same fixative overnight and transferred into a 30% sucrose solution for cryoprotection. Forty-micrometer-thick coronal sections were made using a freezing microtome (Leica, Nussloch, Germany).
Immunohistochemistry for BrdU was performed, as the previous method (
After BrdU-specific staining, differentiation of BrdU-positive cells was determined on the same section using a mouse antineuronal nucleic antibody (1:300; Chemicon International, Temecula, CA, USA). For staining, the sections were incubated in a reaction mixture consisting of 0.02% DAB and 0.03% H2O2 for 5 min. The sections were finally mounted onto gelatin-coated slides. The slides were air dried overnight at room temperature, and coverslips were mounted with Permount (Fisher Scientific, New Jersey, NJ, USA).
Immunohistochemistry for Ki-67 was performed, as the previous method (
DCX immunohistochemistry was performed, as the previous method (
BDNF and TrkB expressions were determined by Western blot analysis (
The numbers of BrdU-positive, Ki-67-positive, and DCX-positive cells in the hippocampal dentate gyrus were counted hemilaterally using Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA) attached to a light microscope (Olympus, Tokyo, Japan). These numbers were expressed as the numbers of cells per mm2 of cross-sectional area of the hippocampal dentate gyrus. For confirming the expressions of BDNF and TrkB, the detected bands were calculated densitometrically using Molecular Analyst, ver. 1.4.1 (Bio-Rad). Statistical analysis was performed using one-way analysis of variance followed by Duncan
The present results show that short-term memory was deteriorated by induction of autism and that application of music alleviated autism-induced impairment of short-term memory. In the normal rat pups, music application did not exert any effects on the short-term memory (
The present results show that the number of BrdU-positive cells was decreased by induction of autism and that application of music increased the number of BrdU-positive cells in the autism-induced rat pups. In the normal rat pups, music application did not exert any effects on the number of BrdU-positive cells (
The present results show that the number of Ki-67-positive cells was decreased by induction of autism and that application of music increased the number of Ki-67-positive cells in the autism-induced rat pups. In the normal rat pups, music application increased the number of Ki-67-positive cells (
The present results show that the number of DCX-positive cells was decreased by induction of autism and that application of music increased the number of DCX-positive cells in the autism-induced rat pups. In the normal rat pups, music application increased the number of DCX-positive cells (
The present results show that expressions of BDNF and TrkB were suppressed by induction of autism and that application of music enhanced BDNF and TrkB expressions in the autism-induced rat pups. In the normal rat pups, music application increased BDNF and TrkB expressions (
Autistic spectrum disorder is one of the pervasive neurodevelopmental disorders and accompanies with progressive deficits in learning ability, memory capability, and cognitive function (
Many therapeutic strategies were tried to improve communication and social interaction in patients with autistic spectrum disorder, however, the evidences of their effectiveness are not sufficient (
Music is also known to improve spatial learning ability through increasing neurogenesis (
DCX is widely used as the marker reflecting the level of neurogenesis and its modulation (
BDNF is implicated in the neuroprotective action and memory consolidation. Over-expression of BDNF and its receptor TrkB enhanced memory function through increasing neurogenesis, whereas inhibition of BDNF and TrkB caused memory impairment through suppressing neurogenesis (
Our study shows that music exerted enhancing effect on hippocampal cell proliferation in the autistic rats. Application of music also exerted alleviating effect on autism-induced short-term memory impairment through stimulating BDNF-TrkB signaling.
Based on the present study, suppression of cell proliferation in the hippocampus might be one of the etiologic factors causing autism-induced memory impairments. Music can be suggested as the therapeutic strategy to overcome the autism-induced memory deficits. These effects of music could be ascribed to the enhancing effect of music on cell proliferation in the hippocampus through BDNF-TrkB signaling pathway.
CONFLICT OF INTEREST
No potential conflict of interest relevant to this article was reported.
Effect of music application on latency in the step-down avoidance task. The data are represented as the mean±standard error of the mean. A, control group; B, music-applied group; C, autism-induced group; D, autism-induced and music-applied group. *
Effect of music application on the number of 5-bromo-2′-deoxyridine (BrdU)-positive cells in the hippocampal dentate gyrus. Upper panel: Photomicrographs of BrdU-positive cells in the hippocampal dentate gyrus (immunohistochemistry). The scale bar represents 50 μm. Lower panel: The number of BrdU-positive cells in each group. The data are represented as the mean±standard error of the mean. A, control group; B, music-applied group; C, autism-induced group; and D, autism-induced and music-applied group. *
Effect of music application on the number of Ki-67-positive cells in the hippocampal dentate gyrus. Upper panel: Photomicrographs of Ki-67-positive cells in the hippocampal dentate gyrus (immunohistochemistry). The scale bar represents 50 μm. Lower panel: The number of Ki-67-positive cells in each group. The data are represented as the mean±standard error of the mean. A, control group; B, music-applied group; C, autism-induced group; and D, autism-induced and music-applied group. *
Effect of music application on the number of doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. Upper panel: Photomicrographs of DCX-positive cells in the hippocampal dentate gyrus (immunohistochemistry). The scale bar represents 50 μm. Lower panel: The number of DCX-positive cells in each group. The data are represented as the mean±standard error of the mean. A, control group; B, music-applied group; C, autism-induced group; and D, autism-induced and music-applied group. *
Effect of music application on the expressions of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) in the hippocampus. The data are represented as the mean±standard error of the mean. A, control group; B, control and music-applied group; C, autism-induced group; and D, autism-induced and music-applied group. *