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New neurons are continuously born in the hippocampus of several mammalian species throughout adulthood. Adult neurogenesis represents a natural model for understanding how to grow and incorporate new nerve cells into preexisting circuits in the brain. Finding molecules or biological pathways that increase neurogenesis has broad potential for regenerative medicine. One strategy is to identify mouse strains that display large vs. small increases in neurogenesis in response to wheel running so that the strains can be contrasted to find common genes or biological pathways associated with enhanced neuron formation. Therefore, mice from 12 different isogenic strains were housed with or without running wheels for 43 days to measure the genetic regulation of exercise-induced neurogenesis. During the first 10 days mice received daily injections of 5-bromo-2′-deoxyuridine (BrdU) to label dividing cells. Neurogenesis was measured as the total number of BrdU cells co-expressing NeuN mature neuronal marker in the hippocampal granule cell layer by immunohistochemistry. Exercise increased neurogenesis in all strains, but the magnitude significantly depended on genotype. Strain means for distance run on wheels, but not distance traveled in cages without wheels, were significantly correlated with strain mean level of neurogenesis. Furthermore, certain strains displayed greater neurogenesis than others for a fixed level of running. Strain means for neurogenesis under sedentary conditions were not correlated with neurogenesis under runner conditions suggesting that different genes influence baseline vs. exercise-induced neurogenesis. Genetic contributions to exercise-induced hippocampal neurogenesis suggest that it may be possible to identify genes and pathways associated with enhanced neuroplastic responses to exercise.
Artificially replicating the microenvironment in the dentate gyrus responsible for increasing neurogenesis will be extremely difficult. The process involves activation of granule neurons (Deisseroth et al. 2004), extracellular changes in neurochemistry (Bequet et al. 2001; Neeper et al. 1995), changes in gene expression (Neeper et al. 1996) and changes in vascular density (Clark et al. 2009; Van der Borght et al. 2009). An alternative approach is to identify specific molecules or biological pathways that increase sensitivity to a natural stimulator of neurogenesis such as aerobic exercise (van Praag et al. 1999b). Exercise increases the rate of neurogenesis, resulting in increased total numbers of granule neurons and volume of the entire granule cell layer (Clark et al. 2008, 2009; Rhodes et al. 2003). If biological pathways could be manipulated to increase exercise-induced neurogenesis, then it might be possible to accelerate or enhance the integration of new neurons into hippocampal circuits by combining a therapeutic manipulation with an exercise intervention.
One strategy is to identify strains of mice that display extremely high vs. extremely low levels of neurogenesis in response to exercise (Rhodes et al. 2003). Such strains can then be contrasted to discover common genetic and neurobiological differences that contribute to differential sensitivity. Previous studies have characterized strain differences in baseline levels of adult hippocampal neurogenesis when the animals are housed under sedentary conditions (Kempermann & Gage 2002a,b; Kempermann et al. 1997, 2006). However, recent evidence suggests that different genetic pathways may influence baseline sedentary levels of neurogenesis as compared to exercise-induced neurogenesis (Thuret et al. 2009). Moreover, the magnitude of changes in neurogenesis from exercise exceeds natural genetic variation in neurogenesis under sedentary conditions (Rhodes et al. 2003). Hence, an analysis of genetic variation in exercise-induced neurogenesis contributes unique information toward understanding how to effectively grow and incorporate new nerve cells into brain circuits.
Prior to this report, most of what we knew about exercise-induced neurogenesis in mice was conducted in a single mouse strain, C57BL/6J and recently a few others (Bednarczyk et al. 2009; Clark et al. 2009; Rhodes et al. 2003; Thuret et al. 2009). Therefore, the goal of this study was to compare exercise-induced adult hippocampal neurogenesis in 12 different genetically divergent mouse strains. Results identify specific genotypes that display high levels of exercise-induced neurogenesis and other genotypes that display low levels of neurogenesis in response to exercise. Future studies could contrast these mouse strains to identify molecular or neurobiological factors common to the high responding strains and uncommon in the low strains that enhance the growth of new nerve cells in the adult hippocampus.
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- Materials and methods
Our results illustrate the robustness of the effects of wheel running on increasing adult hippocampal neurogenesis in mice and the importance of background genetics for determining the magnitude of the neuroplastic response. The significant gene by environment interaction for the effects of running on neurogenesis across strains implies that it should be possible to find plasticity genes or genes that predispose large vs. small enhancement in neurogenesis from running. The list of reasonable candidates from the literature is large and includes, but is not limited to, genes that regulate signaling from trophic factors such as brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and growth factors such as insulin-like growth factor 1, fibroblast growth factor 2 and vascular endothelial growth factor; genes that are expressed during neuron development (e.g. nestin, sox2, NeuroD, TLX, Cdk5, doublecortin and Tis21) and genes that regulate cellular metabolism (e.g. leptin signaling and glucose transport) (Aberg et al. 2000; Fabel et al. 2003; Favaro et al. 2009; Frielingsdorf et al. 2007; Fukuda et al. 2003; Garza et al. 2008; Johnson et al. 2003; Lagace et al. 2008; Membrez et al. 2006; Palmer et al. 1995; Rossi et al. 2006; Seki 2002; Zhang et al. 2008).
An important discovery is that C57BL/6J, the most frequent mouse strain previously used to characterize effects of wheel running on adult hippocampal neurogenesis, was the least responsive strain to have a running wheel. Running increased neurogenesis in C57BL/6J by 1.6-fold, a level consistent with other reports (Clark et al. 2008, 2009, 2010; van Praag et al. 1999a,b), whereas all other strains displayed at least a doubling of neurogenesis, and several displayed four- to fivefold increases (see Fig. 2). The low-fold response in C57BL/6J relative to other strains was in part due to a relatively high baseline level of neurogenesis and in part to a low level of running. The high baseline level of neurogenesis under sedentary conditions (Fig. 2) is consistent with previous reports (Kempermann & Gage 2002a,b; Kempermann et al. 1997, 2006). Moreover, consistent with literature, C57BL/6J also ran less than other strains (Fig. 1b) (Lightfoot et al. 2004) and average daily distance run was significantly correlated with neurogenesis (Figs. 3b and 4c). In the ancova, C57BL/6J displayed average levels of neurogenesis relative to other strains for a fixed level of running. Hence, the C57BL/6J genotype may not be the ideal model organism to study exercise-induced neurogenesis because the change in neurogenesis from running is relatively small in comparison with other strains, and therefore, statistical power to find correlated responses is limited.
Another important discovery was that genetic variation in neurogenesis under standard housing conditions was unrelated to running levels of neurogenesis (Fig. 4d). The lack of genetic correlation suggests that different genes influence variation in adult hippocampal neurogenesis under sedentary vs. runner conditions. Previous work has characterized genetic variation in levels of adult hippocampal neurogenesis among laboratory strains of mice under standard conditions similar to our sedentary treatment, without running wheels or enrichment (Kempermann & Gage 2002a,b; Kempermann et al. 1997). Recent work has begun mapping genes that influence variation in levels of neurogenesis under these standard sedentary conditions in genetic reference populations (e.g. BXD and AXB) (Kempermann et al. 2006; Philip et al. 2010). Our findings suggest that the genes and pathways discovered in these studies will likely be different from those that increase neurogenesis in response to running. If the goal is to identify genes and biological pathways that enhance neurogenesis, it will be useful to include aerobic exercise in the analysis because results show that the greatest increases in neurogenesis are produced from combining the appropriate genes with the appropriate environmental conditions.
To the best of our knowledge, this is the first study to examine the relationship between physical activity in the home cage without a running wheel and number of new neurons in the hippocampus. Strains differed in how physically active they were in cages without wheels (Fig. 1a). However, physical activity was not correlated with adult hippocampal neurogenesis in animals housed in cages without wheels (Fig. 3a). This contrasts markedly with results for animals housed with running wheels. Individual level of running was significantly correlated with adult hippocampal neurogenesis (Fig. 3b), a result that has been reported before for individuals within strains (Allen et al. 2001; Bednarczyk et al. 2009; Clark et al. 2009; Rhodes et al. 2003). We interpret this finding to imply that physical activity must reach a certain threshold level that taxes aerobic capacity before it can increase neurogenesis in a quantitative fashion (Chaddock et al. 2010; Erickson et al. 2009; Pereira et al. 2007).
Adult hippocampal neurogenesis is a multistage process that occurs over a period of weeks in rodents (van Praag et al. 2002). It requires stem cell proliferation, survival of daughter cells, differentiation into neurons and integration into circuits. In this study we measured net neurogenesis resulting from the entire process occurring over 6 weeks because in the end, the survival and integration of new neurons is what matters for regenerative medicine. We did not attempt to separate the contribution of proliferation vs. survival as was performed in early studies (Kempermann & Gage 2002a,b; van Praag et al. 1999b). Theoretically, exercise could increase neurogenesis by increasing proliferation, survival or differentiation. Although early reports suggested that wheel running increases neurogenesis primarily by increasing proliferation (Olson et al. 2006; van Praag et al. 1999b), more recent studies on C57BL/6J show that the effect of increased proliferation from wheel running is small and transient and that the major factor sustaining increased neurogenesis from wheel running is increased survival and differentiation (Clark et al. 2010; Fuss et al. 2009; Kronenberg et al. 2006; Snyder et al. 2009). Future work is needed to determine whether survival and differentiation are the major factors contributing to increased neurogenesis from exercise in the other genotypes.
The strains of mice compared in our study were all isogenic, meaning that same sex individuals within a strain were genetically identical. We used this approach to separate genetic from environmental contributions to phenotypic variation. When individuals from a strain are all genetically identical, variation within strains can only be attributed to environment, whereas variation between strains can be attributed to genetic influences, assuming contributions of maternal effects and early rearing environment to between-strain variation are minimal (Crabbe et al. 1990; Rhodes et al. 2007). Using this strategy, we found evidence for significant genetic influences on physical activity, neurogenesis and volume of the granule layer of the dentate gyrus (DG). Broad sense heritability estimates, representing R2 values from one-way anovas with strain as the factor, are summarized in Table 2. The results suggest that it should be possible to find genes or specific locations in the DNA where changes occur between strains that explain the heritable variation in the phenotypes (Kempermann et al. 2006).
Table 2. Heritability estimates
| ||Distance||DG volume||Neurogenesis|
An interesting finding was that the pattern of inheritance for physical activity and neurogenesis in the F1 hybrids was different under sedentary and runner conditions. Wheel running behavior and running-induced neurogenesis showed significant overdominance or heterosis. The hybrids ran more (Fig. 1b) and grew more new neurons when exposed to running wheels (Fig. 2c) as compared to either of their parental strains (all post hoc P < 0.05). This result suggests that heterozygosity (having alternative alleles at a single locus) increases exercise-induced adult hippocampal neurogenesis above and beyond the additive contributions of each allele as measured separately in the homozygous state. The implication is that combinations of alternative gene transcripts or expression patterns produce the greatest increases in wheel running and exercise-induced neurogenesis. In contrast, home cage activity (Fig. 1a) and sedentary levels of neurogenesis (Fig. 2c) showed additive or dominant effects. The F1 hybrids were intermediate to the parental strains or not significantly different from C57BL/6J.
We identified two low and four high responding strains that could be contrasted in a future research program to discover genes and biological correlates of enhanced or suppressed adult hippocampal neurogenesis. The following two strains displayed low levels of exercise-induced neurogenesis relative to the other strains and low levels for a fixed amount of running as indicated by ancova: 129S1/SvImJ and DBA/2J. The following four strains displayed high levels of neurogenesis for a fixed amount of running and high levels overall: AKR/J, C57BL/10J, BALB/cByJ and NOD/ShiLtJ. These particular strains deserve follow-up examination for correlated features at multiple levels of biological organization from molecules to behavior. For example, it would be useful to find changes in gene expression in the hippocampus from running that are common in the high responding strains and uncommon in the low responding strains. Such genes would represent candidates for increasing sensitivity to exercise-induced neurogenesis that could be directly tested using knockin or knockdown approaches such as transgenic mice carrying multiple copies of the gene or RNA interference, respectively. Moreover, if the same strains were tested for effects of exercise on learning and memory tasks, it would be possible to determine whether the strains that display the greatest levels of exercise-induced adult hippocampal neurogenesis also display the greatest enhancement in behavioral performance from exercise. Such data would bring useful information to the debate about the functional significance of exercise-induced neurogenesis in behavioral performance (Kempermann 2002; van Praag 2009; Schinder & Gage 2004). Analysis of exercise-induced neurogenesis in more strains including BXD Recombinant Inbred lines would substantially increase the power for testing these and other related hypotheses (Kempermann et al. 2006).
Artificially replicating the microenvironment in the brain responsible for increasing neurogenesis for therapeutics may be difficult. But, it may be possible to identify genes or specific molecules that increase sensitivity to a natural stimulator of plasticity in the adult mammalian brain, e.g. aerobic physical activity. Finding genes and biological pathways that differentiate high and low responders represents a promising approach. In the future, it might be possible to manipulate a specific pathway in the brain that in combination with exercise could enhance the growth and incorporation of new nerve cells into the adult hippocampus.