The genetic background of mice has various influences on the efficacy of physical exercise, as well as adult neurogenesis in the hippocampus. In this study, we investigated the basal level of hippocampal neurogenesis, as well as the effects of treadmill exercise on adult hippocampal neurogenesis in 9 mouse strains: 8 very commonly used laboratory inbred mouse strains (C57BL/6, BALB/c, A/J, C3H/HeJ, DBA/1, DBA/2, 129/SvJ, and FVB) and 1 outbred mouse strain (ICR). All 9 strains showed diverse basal levels of cell proliferation, neuroblast differentiation, and integration into granule cells in the sedentary group. C57BL/6 mice showed the highest levels of cell proliferation, neuroblast differentiation, and integration into granule cells at basal levels, and the DBA/2 mice showed the lowest levels. The efficacy of integration into granule cells was maximal in ICR mice. Treadmill exercise increased adult hippocampal neurogenesis in all 9 mouse strains. These results suggest that the genetic background of mice affects hippocampal neurogenesis and C57BL/6 mice are the most useful strain to assess basal levels of cell proliferation and neuroblast differentiation, but not maturation into granule cells. In addition, the DBA/2 strain is not suitable for studying hippocampal neurogenesis.
Adult neurogenesis is a transient process for generating new neurons in the adult mammalian brain, which arise from the subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricles throughout adult life. Newly generated neural stem cells in the dentate gyrus pass through maturation stages, and the surviving neuroblasts migrate into the granular cell layer (GCL), where they finally become mature neurons [
Major extrinsic factors influencing AHN are environmental enrichment, dietary moderation, antidepressant drugs, and exercise conditions [
Strain-dependent genotypes and phenotypes from genetic background have been studied for decades. Using a genome sequencing approach, large differences in genome sequences were found among 17 inbred mouse strains, which could influence phenotypes, gene regulation, and functional variants [
Among inbred mouse strains, markers for cell proliferation and neuroblast differentiation in AHN are expressed differentially in the C57BL/6, ICR, and BALB/c mouse strains [
In the present study, we investigated the basal level of cell proliferation, neuroblast differentiation, and cell survival in eight commonly used inbred mouse strains (C57BL/6, BALB/c, A/J, C3H/HeJ, DBA/1, DBA/2, 129/SvJ, and FVB) and one outbred mouse strain (ICR) to elucidate strain-specific differences in AHN. In addition, we also observed susceptibility to treadmill exercise and its enhancing effects on AHN in these 9 mouse strains to understand the phenotypic variation with genetic background and to determine the best mouse strain for use in AHN studies. Our data confirmed that there is diversity in the basal level of AHN in 9 different mouse strains, and also suggests avenues for future investigations into the mechanism of AHN, as well as the selection of proper mouse strains for genetically engineered mouse models.
Six-week-old male C57BL/6J, A/J, BALB/c, C3H/HeJ, FVB, 129/SvJ, DBA/1, DBA/2, and ICR mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The animals were housed in a specific pathogen-free animal facility at 23°C with 60% humidity, a 12 h/12 h light/dark cycle, with ad libitum access to food and tap water. The handling and care of the animals conformed to guidelines established in compliance with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication number 85-23, 1985, revised 1996) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-120913-1-2). All experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures used in the study.
After a one-week acclimation to laboratory condition, each mouse strain was divided into 2 groups (
Experimental design of the present study. Treadmill exercise was adopted in 7-week-old mice for 1 week, and treadmill exercise was conducted for 4 weeks with 10 m/min speed for 1 h. During the first 3 days of exercise, BrdU was injected twice a day (at 8:00 AM and 8:00 PM) intraperitoneally to label newborn neural stem cells.
Body weight was measured at 10:00 AM every week on Wednesday and at the end of the experiment. Food intake was measured and corrected for spillage by weighing the jars containing food every week between 9:00 and 10:00 AM. Food intake was calculated from the average intake during the 4-week experimental period and expressed as g/mouse/week. Body weight gain was calculated as the difference in body weight between 12 weeks and 8 weeks.
To label newly generated cells in the hippocampus, intraperitoneal injections of 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) were given to all mice twice daily (8:00, 20:00) for three days at the start of the exercise (when the mice were at eight weeks of age). Animals were euthanized at 12 weeks of age, one day after the last exercise (Figure
Animals (
To obtain accurate data for immunohistochemistry, the free-floating sections from all animals were processed carefully under the same conditions. For each animal, tissue sections were selected from between 1.46 mm and 2.46 mm posterior to the bregma by referring to the mouse atlas by Franklin and Paxinos [
For BrdU and NeuN double immunofluorescence, the sections were treated with 2 N HCl for 30 min at 37°C and were incubated with a mixture of mouse anti-NeuN (1 : 1000; Millipore, Temecula, CA, USA) and rat anti-BrdU (1 : 200; Abd Serotec, Bio-Rad Laboratories, Inc., Grand Island, NY, USA) for 2 h at 25°C, followed by overnight incubation at 4°C. After washing with PBS, the sections were subsequently incubated with secondary antibodies, FITC-conjugated goat anti-mouse IgG (1 : 100; Jackson ImmunoResearch, PA, USA), and Cy3-conjugated goat anti-rat IgG (1 : 100; Jackson ImmunoResearch, PA, USA), for 2 h. After that, the sections were mounted on silane-coated slides with DAPI-containing mounting medium (Vector Labs, CA, USA) for nuclei staining.
Two independent masked investigators counted Ki67-, DCX-, BrdU-, or BrdU/NeuN-labeled cells in the dentate gyrus at 400x magnification under a light microscope (BX51, Olympus, Tokyo, Japan). All Ki67-, DCX-, BrdU-, or BrdU/NeuN-labeled cells were counted bilaterally in 10 sections (90
Statistical analysis was performed using SPSS V.20.1 (IBM Corporation, Armonk, NY, USA). Experimental groups were compared using two-way analysis of variance (ANOVA), followed by a least significant difference (LSD) post hoc analysis.
At eight weeks of age, each inbred mouse strain showed similar body weight, except for the outbred ICR strain, which had a significantly higher body weight than the inbred mice (Figure
Body weight at the beginning (8 weeks of age) (a) and end (12 weeks of age) (b) of exercise and its control group (
To observe the proliferation of hippocampal neural progenitor cells in the 9 mouse strains, as well as their susceptibility to the effects of 4 weeks of treadmill exercise, cells in the subgranular zone of the dentate gyrus were stained with the proliferation marker Ki67, and the mean number of Ki67 immunoreactive cells was calculated. In the SED groups, there were different population levels of Ki67-positive cells in the different mouse strains (Figure
Immunohistochemistry for Ki67 in the dentate gyrus of sedentary and exercise mice of 9 different strains (a). GCL, granule cell layer; ML, molecular layer; PoL, polymorphic layer. Scale bar = 100
Summary of NSC proliferation, differentiation, and number of mature neurons across mouse strains, and efficacy of integration into mature neurons from proliferating NSCs and differentiating neuroblasts. (
Strains | CB7BL/6J | A/J | BALB/c | C3H/HeJ | FVB | 129/SvJ | DBA/1 | DBA/2 | ICR | Total increase % | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | |||
Ki-67 | SED | 20.00 | ± 1.58 | 11.60 | ± 0.24 | 8.00 | ± 0.32 | 9.80 | ± 1.46 | 8.40 | ± 0.93 | 10.00 | ± 0.89 | 8.00 | ± 0.71 | 2.40 | ± 0.51 | 9.00 | ± 1.22 | |
Proliferation | EX | 32.20 | ± 2.97 | 18.20 | ± 0.86 | 12.40 | ± 1.03 | 16.40 | ± 2.48 | 13.00 | ± 0.95 | 18.60 | ± 0.93 | 12.60 | ± 1.44 | 3.40 | ± 0.98 | 16.20 | ± 0.86 | |
Increase % | 161.00 |
|
156.90 |
|
155.00 |
|
167.35 |
|
154.76 |
|
186.00 |
|
157.50 |
|
141.67 | ns | 180.00 |
|
162.24 | |
DCX | SED | 122.80 | ± 4.86 | 73.60 | ± 2.91 | 47.20 | ± 2.40 | 73.40 | ± 1.12 | 60.00 | ± 2.77 | 79.20 | ± 5.34 | 45.40 | ± 4.79 | 31.00 | ± 4.18 | 71.60 | ± 5.91 | |
Differentiation | EX | 210.2 | ± 4.89 | 108.6 | ± 2.94 | 68.4 | ± 3.49 | 97.2 | ± 7.44 | 82.2 | ± 6.26 | 127.2 | ± 2.48 | 73.00 | ± 1.10 | 45.20 | ± 4.31 | 97.8 | ± 3.62 | |
Increase % | 171.17 |
|
147.55 |
|
144.92 |
|
132.43 |
|
137.00 |
|
160.61 |
|
160.79 |
|
145.81 |
|
136.59 |
|
148.54 | |
BrdU/NeuN +/+ | SED | 21.20 | ± 1.81 | 8.00 | ± 1.05 | 5.00 | ± 0.55 | 6.40 | ± 1.03 | 6.00 | ± 0.55 | 7.80 | ± 0.73 | 4.40 | ± 1.25 | 2.20 | ± 0.37 | 12.40 | ± 0.51 | |
Maturation & survival | EX | 31.00 | ± 2.18 | 15.60 | ± 1.69 | 10.80 | ± 1.50 | 11.60 | ± 1.21 | 9.00 | ± 0.55 | 12.80 | ± 1.65 | 9.80 | ± 0.58 | 4.00 | ± 0.77 | 18.20 | ± 1.74 | |
Increase % | 146.23 |
|
195.00 |
|
216.00 |
|
181.25 |
|
150.00 |
|
164.10 |
|
222.73 |
|
181.82 | ns | 146.77 |
|
160.39 | |
|
||||||||||||||||||||
CB7BL/6 J | A/J | Balb/c | C3H/HeJ | FVB | 129/Svj | DBA/1 | DBA/2 | ICR | ||||||||||||
Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | |||
|
||||||||||||||||||||
Efficacy of cell maturation | SED-BrdU + NeuN/Ki67 | 106.00 | ± 16.61 | 69.09 | ± 9.16 | 63.17 | ± 7.70 | 72.99 | ± 18.17 | 76.31 | ± 11.45 | 78.14 | ± 3.55 | 56.39 | ± 16.57 | 125.00 | ± 46.10 | 154.08 | ± 32.48 | 85.59 |
EX-BrdU + NeuN/Ki67 | 96.27 | ± 13.39 | 85.32 | ± 7.87 | 91.78 | ± 16.63 | 75.12 | ± 11.02 | 70.51 | ± 5.91 | 69.09 | ± 8.97 | 80.87 | ± 7.48 | 136.43 | ± 20.40 | 113.38 | ± 11.96 | 88.56 | |
EX/SED % | 90.82 | 123.49 | 145.28 | 102.93 | 92.40 | 88.42 | 143.41 | 109.14 | 73.59 | 108.65 | ||||||||||
SED-BrdU + NeuN/DCX | 17.26 | ± 1.34 | 11.04 | ± 1.65 | 10.60 | ± 0.98 | 8.70 | ± 1.39 | 9.96 | ± 0.70 | 9.79 | ± 0.36 | 9.80 | ± 2.84 | 7.80 | ± 1.71 | 17.70 | ± 1.31 | 10.79 | |
EX-BrdU + NeuN/DCX | 14.75 | ± 10.64 | 14.34 | ± 1.48 | 16.10 | ± 2.47 | 12.16 | ± 1.40 | 11.12 | ± 0.82 | 10.06 | ± 1.32 | 14.63 | ± 0.87 | 8.62 | ± 0.87 | 18.78 | ± 2.16 | 12.96 | |
EX/SED % | 85.43 | 129.95 | 151.85 | * | 139.72 | 111.63 | 102.75 | 149.40 | * | 110.52 | 106.13 | 121.57 |
Compared to the SED group, the number of Ki67-positive cells was significantly increased in the dentate gyrus of the EX group in all mouse strains except for the DBA/2 strain (Table
Doublecortin (DCX) immunohistochemistry, which is a marker for differentiated neuroblasts found in the subgranular zone of the dentate gyrus, was used to examine the basal levels of neuroblast differentiation and the effects of 4 weeks of treadmill exercise on the differentiation of hippocampal neural progenitor cells. In the SED group, the mean number of DCX-immunoreactive neuroblasts was different in each mouse strain (Figures
Immunohistochemistry for doublecortin (DCX) in the dentate gyrus of sedentary and exercise mice of 9 different strains (a). GCL, granule cell layer; ML, molecular layer; PoL, polymorphic layer. Scale bar = 100
Exercise significantly increased the number of DCX-immunoreactive neuroblasts in the dentate gyrus of all mouse strains compared to that of the respective strains in the SED group, as shown in Table
In this study, we quantified BrdU and NeuN double-positive cells in the GCL to evaluate integration into mature granule cells in the dentate gyrus, and the mean number of NeuN and BrdU double-positive cells was calculated to compare the effects of strain and exercise on neurogenesis. In the SED group, BrdU and NeuN double-positive cells were the most abundant in the C57BL6 strain (21.20 ± 1.93, Figure
Double immunofluorescence staining for BrdU (red) and NeuN (green) in the dentate gyrus of sedentary and exercise mice of 9 different strains (a). GCL, granule cell layer; ML, molecular layer; PoL, polymorphic layer. Scale bar = 100
The ratio of BrdU and NeuN double-positive cells and DCX-immunoreactive neuroblasts (BrdU + NeuN/DCX) was also calculated and is shown in Table
Our basic objectives were to investigate the differences in cell proliferation, neuroblast differentiation, and integration into mature granule cells in the dentate gyrus and evaluate the efficacy of treadmill exercise on the population of AHN in 9 mouse strains. In this study, we selected 9 mouse strains that are widely used in biomedical research, as well as mouse family tree groups, which were classified with informative single nucleotide polymorphism markers sorted by genetic relationship, resulting in the organization of 102 strains into 7 family tree groups [
In the present study, ICR mice had a higher ratio of BrdU + NeuN/DCX positive cells than other strains. This result suggests that there is a higher efficacy for the integration of neuroblasts into mature neurons in ICR mice as compared to the other 8 inbred mouse strains. This result was consistent with a previous study that showed that proliferation was highest in the C57BL/6 strain, and the survival rate of newborn cells was highest in the ICR strain [
Several lines of evidence show that there are strain-dependent differences in various phenotypes, including hippocampus-dependent learning and memory and long-term potentiation (LTP) in the hippocampus. Electrophysiological and behavioral tests showed strain-dependent differences in LTP and hippocampal-dependent learning memory in C57BL/6, CBA/J, DBA/2J, and 129SvEms mice [
There is evidence that newly generated neurons contribute to learning and memory function, synaptic formation, and integration into the hippocampal network circuit [
In the present study, we could not elucidate the factors that induce differences in neurogenesis among mouse strains. There has been a report, however, that pregnenolone sulfate (PREGS) level is an important factor in differences in neurogenesis that are observed among the strains. In the DBA/2 strain, PREGS levels are significantly lower than in the C57BL/6, BALB/c, ddY, and ICR strains, while dehydroepiandrosterone sulfate (DHEAS) concentrations in the DBA/2 strain were significantly higher than those in other strains [
Neurogenesis in the hippocampus is regulated at each level of cell proliferation, neuroblast differentiation, and survival. Regulation of neurogenesis has been controlled in many paradigms, including physical exercise [
The mechanism by which exercise increases AHN may be explained by systemic improvement and changes in neurotrophins, signaling pathway-related ligands, and receptors. Exercise increases the growth of blood vessels in the hippocampus and blood flow in the dentate gyrus of the hippocampus [
In conclusion, we showed that there are basal strain-dependent differences in AHN, as well as differences in the effectiveness of physical exercise on AHN in 9 mouse strains. In normal mice, AHN was the most abundant in C57BL/6 mice and was the least abundant in DBA/2 mice. However, integration into mature neurons was most effective in ICR mice. The responsiveness to physical exercise was most prominent in the BALB/c strain, and least prominent in the C57BL/6 strain. Choosing the correct inbred mouse strain for transgenic or knockout mouse models for common neurological studies requires significant knowledge of the origin of phenotype for each inbred mouse strain. Furthermore, knowledge about the differences in phenotype between inbred mouse strains may contribute to our understanding of strain-dependent genetic influences on AHN and may therefore aid in choosing the correct approach for generating a suitable animal disease model. Our data showed varying degrees of basal AHN level in 9 mouse strains. Therefore, in the design of AHN studies, it is necessary to take into account the genetic differences related to AHN in each mouse strain. Our study also indicates that the level of physical activity in the study, such as treadmill exercise, should be taken into consideration. Current approaches to reveal the mechanism of AHN use reverse genetics, but the elucidation of clear mechanisms for differences in AHN requires a forward genetics approach with different mouse strains. These results provide information for the selection of appropriate mouse strains and the ideal conditions for AHN experiments in the field of neuroscience.
The authors declare that they have no conflict of interests.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01059314) and by the Korea Mouse Phenotyping Project (NRF-2015M3A9D5A01076747) of the Ministry of Science, ICT, and Future Planning through the National Research Foundation (NRF), Korea. This study was partially supported by the Research Institute for Veterinary Science, Seoul National University.
Table 1: statistical data of proliferative NCSs in 9 mouse strains and the effects of exercise. A red box indicates a significant increase and a blue box indicates a significant reduction as compared to the cross-matched group.