ABSTRACT: Many studies have reported on beneficial aspects of running to improve hippocampus-related spatial memory and promote neuroplastic changes like adult hippocampal neurogenesis (AHN). Such research has also revealed molecular factors associated with molecular signaling by BDNF and IGF-I leading to CREB activation. However, the optimum exercise intensity for genome-wide beneficial effects remains unclear. We thus investigate the whole spectrum of hippocampal molecular change by exercise using DNA microarray-based transcript profiling in two different intensity groups. Lactate threshold (LT), at which lactate starts to accumulate in the blood, defined the intensity: mild- (ME, LT). Rats were subjected to 6 weeks of training or sedentary (CONT) regimes. After 6 weeks, spatial memory using Morris Water Maze (MWM), AHN, and hippocampal gene expressions were assessed. The functional relationships among the differentially expressed genes were generated using Ingenuity Pathway Analysis and QIAGEN-based pathway analysis. Results revealed that ME, but not IE, improved the performance on probe trial in MWM and increased newborn mature neurons (BrdU+/NeuN+ cells) compared with CONT. Transcript profiling by comparison with CONT postulated that ME regulated protein synthesis through IRS-dependent pathway (e.g. PI3K-Akt signaling cascade), cholesterol trafficking and perisynaptic environment, while IE induced dysfunction of these process and cell death. The comparison of the expression between each intensity also supported these tendencies. Collectively, our findings indicate mild exercise should be beneficial in the development of both enhanced AHN and spatial memory, and several genes related to protein synthesis and lipid-metabolism may be critical those neuroplastic changes. This study is the first step to identify the potential triggers of exercise-induced cognitive gain. 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).
