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| Status |
Public on Apr 05, 2014 |
| Title |
Transcriptomic profile of mild exercise-enhanced adult hippocampal neurogenesis: Comparison with the effects of intense exercise |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by array
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| Summary |
Mild exercise (ME) with an intensity below the lactate threshold (LT) is sufficient to enhance hippocampal function, while intense exercise (IE) above the LT negates such benefits. However, the question as to why ME more effectively enhances hippocampal function than does IE remains to be clarified. Here, we investigated adult hippocampal neurogenesis (AHN) as a mechanism of ME-induced cognitive improvement, and comprehensively delineated the transcriptomic profile of the hippocampus, using a rat whole-genome microarray approach through comparison with IE. Immunohistochemical results showed that less intense exercise (ME) is better suited to improve AHN, especially in regards to the survival and maturation of newborn neurons. DNA microarray analysis revealed that ME regulated more genes than did IE (ME: 604 genes, IE: 415 genes), and only 44 genes were modified with both exercise intensities. The identified molecular components did not comprise well-known factors related to exercise-induced AHN, such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF1), probably due to the timing of hippocampal tissue collection after the last training session and the technical feature of microarray. Rather, network analysis of the microarray data using Ingenuity Pathway Analysis algorithms revealed that the ME-influenced genes were principally related to lipid metabolism, protein synthesis and inflammatory response, which are recognized as associated with hippocampal neuroadaptations including AHN. In contrast, IE-influenced genes linked to immune response, a negative regulatory system of AHN and hippocampal function, were identified. Collectively, these results support our hypothesis that AHN could explain why ME enhances hippocampal function, and provide the ME-specific gene list that contain some potential regulators of this positive regulation. The list will become a foundation to elucidate the molecular pathway involving the ME-induced cognitive gain.
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| Overall design |
Eleven-week-old male Wistar rats (220-270 g; SLC, Saitama, Japan) were housed in polycarbonate steel cages with a 12-h light/dark schedule (lights on at 7:00 a.m.) and given ad libitum access to food and water. A total of 98 rats were used in this study to assess the influence of exercise intensity on physiological states (training effect of skeletal muscle and stress level) (n= 22), MWM (n= 32), AHN (n= 22), and global gene expression profiles of hippocampal by microarray (n= 22). All animal care and experimental procedures were performed in accordance with protocols approved by the University of Tsukuba Animal Experiment Committee, based on the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH publication, revised 1996). All efforts were made to minimize animal suffering and to reduce the number of animals used in this study. To adapt the rearing environment and remove any unintended stress effects, animals underwent a week of preliminary rearing to ambient conditions in groups housing conditions (2-3 rats/cage). After the preliminary rearing, the animals were randomized into three groups based on the LT of treadmill running: Sedentary control (CONT, rest on treadmill), Mild Exercise (ME, below LT, 15 m/min) or Intense Exercise (IE, above LT, 40 m/min). Exercise group was habituated to run on a treadmill (KN-73, Natsume Ltd., Tokyo, Japan) for 1 (for ME) or 4 (for IE) weeks. Within a given test period, rats were able to run on the above-mentioned running speeds. After this running habituation, rats were entered into the exercise training session for 6 weeks in total. The running duration was 60 min/day and frequency was 5 times/week. Exercise training was performed between 19:00 and 22:00 (during dark phase). Rat’s body weight and physical condition were monitored until the end of training. If rats could not perform well running on each determined speed and showed poor health, those were eliminated from the study. Based on the criteria, a total of 13 rats were eliminated (bad runner - 4 rats; poor health - 7 rats). Twenty-four or thirty-six hours after the last training, rats were processed to the next step. In the microarray group, the whole brain of each animal was rapidly removed, and hippocampus was separated on ice according to the method of Glowinski and Iversen (1966), with minor modifications. Hippocampi were immediately flash-frozen in liquid nitrogen. The deep-frozen hippocampi were transferred to a pre-chilled (in liquid nitrogen) mortar and pestle, and ground to a very fine powder with liquid nitrogen. The powdered samples were stored in aliquots at -80ºC till used for further analysis. Total RNA was extracted from each sample powder (~50 mg) using the QIAGEN RNeasy Mini Kit (QIAGEN, Maryland, USA). To verify the quality of this RNA, the yield and purity were determined spectrophotometrically (NanoPhotomaterTM, IMPLEN, Munich, Germany) and visually confirmed using formaldehyde gel electrophoresis. We used the sample with a ratio of spectrophotometric absorbance at 260 nm to that at 230 (A260/A230) or 280 nm (A260/A280) above 1.8. An equal amount of RNA (1000 ng) from the five rats, randomly picked up from each exercise group (n = 5), was pooled and used for microarray analysis. In this study, we performed three combinations of comparison analysis of gene expression change. First, we compared the difference between CONT and ME (combination 1) or IE (combination 2) to confirm the exercise-intensity-dependent gene expression change. Subsequently, we made a comparison of ME with IE (combination 3) to elucidate a ME-based difference of IE inducible gene. In all combinations, total RNA (1000 ng) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., CA, USA). Fluorescently labeled targets of control as well as treated samples were hybridized to the same microarray slide with 60-mer probes (4 x 44K (41,090 gene probes), rat whole genome, Agilent). A flip labelling (dye-swap or reverse labelling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA. Hybridization and wash processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray scanner. For the detection of significantly differentially expressed genes between control and exercise samples each slide image was processed by Agilent Feature Extraction software (version 9.5.3.1).
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| Contributor(s) |
Inoue K, Okamoto M, Shibato J, Lee MC, Matsui T, Rakwal R, Soya H |
| Citation(s) |
26061528 |
| Submission date |
Apr 05, 2013 |
| Last update date |
Dec 21, 2016 |
| Contact name |
RANDEEP RAKWAL |
| E-mail(s) |
plantproteomics@gmail.com
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| Phone |
+81-(0)90-1853-7875
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| Organization name |
University of Tsukuba
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| Department |
Institute of Health and Sport Sciences
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| Lab |
GSI 403
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| Street address |
1-1-1 Tennodai
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| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8574 |
| Country |
Japan |
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| Platforms (1) |
| GPL4135 |
Agilent-014879 Whole Rat Genome Microarray 4x44K G4131F (Feature Number version) |
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| Samples (6)
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| GSM1115926 |
Control Sedentary (Cy3) x Treatment ME (Cy5) |
| GSM1115927 |
Control Sedentary (Cy5) x Treatment ME (Cy3) |
| GSM1115928 |
Control Sedentary (Cy3) x Treatment IE (Cy5) |
| GSM1115929 |
Control Sedentary (Cy5) x Treatment IE (Cy3) |
| GSM1115930 |
Control ME (Cy3) x Treatment IE (Cy5) |
| GSM1115931 |
Control ME (Cy5) x Treatment IE (Cy3) |
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| Relations |
| BioProject |
PRJNA196416 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE45813_RAW.tar |
74.9 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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