Evaluation of a C57BL/6J x 129S1/SvImJ Hybrid Nestin-Thymidine Kinase Transgenic Mouse Model for Studying the Functional Significance of Exercise-Induced Adult Hippocampal Neurogenesis

INTRODUCTION

In many mammals including humans and rodents, new neurons are continuously generated in the subgranular zone (SGZ) of the hippocampal dentate gyrus [1–5]. The new cells integrate into the circuitry and ultimately behave as normal mature granule neurons [6–9]. However, when they are young, they possess unique morphological and electrophysiological properties, including a depolarizing response to GABA neurotransmission as well as decreased threshold for displaying long term potentiation (LTP), suggesting that new neurons may display greater plasticity, or ability to mold to new experiences than older neurons [7, 10, 11].

The processes of neurogenic proliferation, differentiation and survival are highly sensitive to trophic factors among many other chemical features of the microenvironment within the SGZ [12], which in turn are modulated by intrinsic and extrinsic factors such as stress, diet, drugs, genetics and physical activity [13–19]. In particular, aerobic exercise, in the form of voluntary wheel running, has demonstrated robust enhancement of neurogenesis in the hippocampus and increased performance on certain memory tasks in rodent models [20, 21]. In humans, physical activity is also associated with enhanced cognitive performance [22]. Still, while aerobic exercise has been correlated with increased levels of neurogenesis and spatial memory improvement in rodents, causation is difficult to ascertain.

In fact, multiple attempts have been made to discern the causal relationship between levels of hippocampal adult neurogenesis and behavioral performance. Through the use of multiple models and methods, such as focal gamma irradiation, systemic administration of anti-mitotic agents (such as methylazoxymethanol) and genetically engineered mice featuring inducible ablation, a causal relationship between hippocampal adult neurogenesis and behavior has been implied [23–27]. However, more recently, the Flint group published a meta-analysis of all published studies examining the functional role of adult neurogenesis and found no significant effects of ablation of adult neurogenesis on spatial memory [28]. Moreover, through the use of these techniques, research has been performed in order to determine the role of exercise-induced adult neurogenesis on hippocampal-dependent spatial memory. Still, these results are again mixed. For example, some focal irradiation studies report no requirement for exercise-induced hippocampal neurogenesis to mediate observed enhancement in the Morris water maze (MWM) [26, 29], while others show a loss of enhanced performance once new neurons were partially ablated [25]. Thus, an overall discrepancy in the literature exists, even within similar models, leaving the functional role of new neurons in spatial learning and memory unknown in both the sedentary and the exercising animal.

It is possible that the methods and techniques currently used cause side effects which may play a role in the discrepancy in the literature. For example, the irradiation method has limitations as it is known to induce inflammation in the brain that can be relatively long lasting [29]. Further, irradiation can also reduce or impair other undifferentiated or dividing cells in the areas of the brain and head exposed to radiation such as astrocytes, microglia and endothelial cells. It is possible that irradiation induces other defects in the brain, besides neurogenesis, which obliterate benefits that otherwise would be observed from exercise [25, 29].

Recently a transgenic mouse line was developed that allows selective, inducible reduction of adult neurogenesis. The transgene consists of a herpes simplex virus thymidine kinase (HSV-TK) under the control of a modified nestin promoter to restrict expression to neural progenitor cells [30]. The HSV-TK in the nestin positive cells (i.e., neural progenitors) phosphorylates exogenously administered ganciclovir resulting in a nucleotide analog that gets incorporated into newly synthesized DNA, and ultimately leads to termination of DNA elongation and apoptosis of the cell. In the present study, we evaluated performance of the nestin-TK transgene on a hybrid F1 genetic background consisting of C57BL/6J (B6) and 129S1/SvImJ (129) genotypes. The rationale for studying the transgene on a hybrid background is that the F1 offspring are isogenic (i.e., same sex individuals are genetically identical), but also heterozygous at all loci that differ between the two parental strains, and heterozygosity is typically associated with better overall health and behavioral performance.

Another novelty of our study was that instead of administering ganciclovir via surgically implanted minipumps which is the method usually employed with TK models, we administered valganciclovir via the chow. We have found that minipumps are invasive, and can cause skin lesions, or other discomfort, particularly when the animals are running on a running wheel. Valganciclovir gets converted into ganciclovir in the gut before reaching circulation and is preferable because administering via the chow is completely non-invasive [30, 31].

The novel F1 nestin-TK hybrid strain potentially could provide a powerful and minimally invasive model for reducing exercise-induced neurogenesis. However, no previous study has tested or evaluated the extent to which exercise-induced neurogenesis can be reduced using the nestin-TK model in either the B6 transgenic line or F1 hybrid. Therefore the purpose of this study is first to establish that reductions in adult neurogenesis will occur in both the B6 transgenic line and the novel hybrid F1 strain where mice are both housed under standard (sedentary) conditions or with running wheels. Second, we used the F1 nestin-TK strain to directly test whether intact neurogenesis is required for spatial learning and memory on the MWM and rotarod test of motor performance.

MATERIALS AND METHODS

RESULTS

DISCUSSION

The present study assessed the functionality of the nestin-TK mouse model in a novel hybrid C57BL/6J x 129S1/SvImJ background to test the role of newly generated neurons in spatial learning and memory and motor performance on the rotarod. The results demonstrate that intact adult hippocampal neurogenesis is not required for performance on either task. Specifically, valganciclovir chow administration in the F1 nestin-TK animals significantly reduced neurogenesis by approximately one half (Fig. 3B). However, even though the mice displayed reduced neurogenesis, their performance on the tasks was similar to the animals with intact neurogenesis (Figs. 4&5). These findings are the first to demonstrate that new neurons are not required for MWM or rotarod performance in the F1 hybrid mouse strain nor are they required for any of the subtle effects of exercise that were detected in the water maze or rotarod in this strain. Results for rotarod were expected because performance on the rotarod is not dependent on the hippocampus [33]. However, results for the MWM were not necessarily expected as many studies have suggested that new neurons play a critical role in spatial learning and memory, specifically in the MWM [25, 34–37].

Although runners consumed slightly more food and hence received a slightly higher dose of valganciclovir as compared to sedentary animals (Fig. 2D–F), this small difference in dose is unlikely to have contributed significantly to levels of neurogenesis in the animals for two reasons. First, the amount ingested was not significantly correlated with number of BrdU+ cells in the transgenic animals, indicating either that there was a threshold dose that once exceeded results in the same maximal reduction of neurogenesis, or that the differences were too small to be detectable. Second, the transgenic runners displayed higher levels of neurogenesis than transgenic sedentary animals (Fig. 3) even though the runners received a higher dose of valganciclovir. Therefore, differences in neurogenesis between runner and sedentary mice that received valganciclovir cannot be accounted for by the dose.

Unfortunately, we did not observe a clear enhancement in performance on the MWM from exercise in the control-fed hybrid F1 nestin-TK transgenic mice. Previously, we published a study using irradiation to reduce neurogenesis in the C57BL/6J genotype, and found that the enhancement in learning and memory on the MWM was abolished in irradiated animals with reduced neurogenesis [25]. Hence, we were hoping to confirm the finding using the more specific nestin-TK transgenic model since irradiation can induce side effects such as inflammation that itself can affect function and account for the behavioral deficits [38, 39]. However, overall, in the current study, the runners took longer to acquire the task (Fig. 4A), and spent a similar time in the target quadrant during the probe test (Fig. 4D). They displayed significantly more crosses through the target quadrant during the probe test (Fig. 4C), which might be taken as evidence of more accurate spatial memory. However, because of the apparent impaired performance during acquisition and no difference in duration spent in the target quadrant during the probe test, the benefits, if any, on MWM performance from wheel running in the F1 hybrid were small at best.

Many studies across species have shown benefits of exercise on MWM performance or other measures of spatial learning and memory [20, 21, 40–48]. Therefore, the explanation of why the effect was not replicated for the F1 hybrid in our study is not clear. To the best of our knowledge, the standard water maze has never been performed in the B6×129S1 hybrid background. Each strain is known to respond differently to the MWM [49]. Recently we found that the F1 hybrid does in fact display enhanced performance from exercise if tested in the plus version of the water maze, specifically adapted for use in multi-strain comparisons [46]. Hence, the standard water maze parameters used in our study may simply not have been optimal to detect exercise effects in the F1 hybrid. Since the F1 hybrid displayed increased neurogenesis from exercise, it represents another illustration that new neurons are not sufficient to improve learning and memory [50]. On the other hand, results suggest that the F1 hybrid may not be ideal to test the functional significance of new neurons in improved performance on standard versions of the MWM that work well in other strains [21, 40, 44, 45, 51, 52].

We concluded that intact neurogenesis is not required for baseline performance or subtle differences in performance induced from voluntary wheel running exercise in our model. However, it is important to note that our method of reducing neurogenesis did not result in a complete ablation of neurogenesis. Therefore, it is possible that had neurogenesis been completely ablated we would have seen an impact on the behavioral outcomes. Nevertheless, the study shows that intact neurogenesis is not required for learning and that neurogenesis can at least be reduced by 50% without affecting learning on the water maze.

The hybrid B6129SF1/J background mouse strain was selected due to previously documented high levels of running (approximately 10 km/day) and high levels of adult neurogenesis in runners compared to sedentary animals [53]. However, the animals in the current study failed to reach similar high levels of wheel running. One possibility for the slightly reduced running is the weighing of the food, which may have disturbed the animals more than usual. It is also possible that the transgene unintentionally disrupted circuitry related to motivation for running. It is also possible that the difference represents environmental variation related to the mouse handler, season, humidity or other uncontrolled variables between studies. Regardless of the reason, the slightly lower levels of running in the F1 hybrid than expected in our study may have contributed to the lack of a clear improvement on the MWM from wheel running.

Previous methods for administering ganciclovir to nestin-TK mouse and rat strains have typically used osmotic mini-pumps which must be surgically implanted under the skin, and can cause skin lesions (personal observation). The method of administering valganciclovir via the chow is completely non-invasive, and as we demonstrate in this paper works well for reducing neurogenesis in the nestin-TK models. The 50% reduction in neurogenesis in the transgenic mice is similar to what we previously reported using this method [54]. Valganciclovir is the ester prodrug form of ganciclovir that rapidly converts to ganciclovir in the gut via hepatic esterases. The reduction in BrdU+ cells reported in the current study is not as robust as the 90% ablation reported by mini-pump delivery of ganciclovir in the Kernie group [30] or the 98 % reduction exhibited by the Flint group [28]. However, the advantage is that chow-administered valganciclovir does not require anesthesia or mini-pump implantation surgery; factors that may further reduce neurogenesis and cause other side effects that could impair behavioral performance. Hence, the results establish the validity of using valganciclovir via the chow for reducing neurogenesis in the F1 and C57BL/6J nestin-TK models consistent with other studies that have used this method in the B6 background [54]. Further we conclude after having experience with both methods that the chow-administered valganciclovir is better because of the non-invasiveness for evaluating the specific contribution of new neurons in behavior.

Our finding that intact neurogenesis is not required for normal baseline performance on the MWM is consistent with a growing number of studies reporting that neurogenesis reduction has no influence on behavior [28, 29, 54]. The result is consistent with our previous report using irradiation to reduce neurogenesis, because the neurogenesis reduction in that study did not impair baseline performance on the MWM; it only abolished the enhancement in performance from exercise [25]. As mentioned previously, the enhancement in performance could not be addressed in our study because no clear pro-cognitive effect of exercise was observed in the F1 genotype employed here (Fig. 4).

Although a growing number of studies using highly specific methods for reducing neurogenesis such as the nestin- or GFAP-TK mice are finding that new neurons are not required for hippocampus-dependent spatial learning and memory [28, 29, 54, 55], some studies are finding positive results, usually when baseline performance is reduced from injury. For example, work from the Kernie group examining the causal role of newly generated neurons in the recovery of poststroke cognitive function induced an experimental stroke in B6 nestin-TK transgenic mice by occluding the distalmiddle cerebral artery. Transgenic stroke mice administered ganciclovir through osmotic mini-pumps exhibited impaired performance on the Barnes Maze test when compared to saline control or wild-type stroke mice given ganciclovir [39], implicating a causal role between newly generated neurons and cognitive function. Further, nestin-TK animals with neurogenesis ablated, specifically at the time of a traumatic brain injury, are unable to learn spatial memory tasks, including the MWM [30]. Finally, through the use of the nestin-TK, Deng and colleagues [27] were able to specifically reduce the number of adult-born cells at particular maturation stages. In doing so, they demonstrated that, in particular, a reduction in the number of immature neurons led to impaired performance on the MWM.

Although our study and a growing number of others suggest that new neurons are not required for learning or memory on the MWM in otherwise intact animals [26, 28, 29], we are not ready to give up the hypothesis that the new neurons do in fact contribute to learning and memory on this task or any other task that involves the hippocampus. Our revised hypothesis is that while new neurons may not be necessary they may be preferentially involved, similar to the way the hippocampus is not necessary for contextual fear conditioning, but is critically involved [56–58]. In other words, in animals that have neurogenesis reduced, other mechanisms of plasticity such as additional synapse formation may compensate for the loss. Recently it was discovered that suppression of adult neurogenesis through a nestin-TK transgenic model resulted in deficits in LTP, specifically in response to medial perforant path stimulation [59]. Interestingly, LTP recovery was accompanied by an increase in the survival rate of newborn cells that were proliferating just before the ablation and a reduction in inhibitory input to the granule cells of the dentate gyrus [59]. Therefore, it is possible that altered hippocampal connectivity may compensate for the decreased presence of newly generated neurons and allow for the network to function properly.

One way to get around the problem of compensatory effects from reducing neurogenesis is to use an approach that inactivates rather than completely eliminates new neurons. Such a strategy could be implemented using designer receptors exclusively activated by designer drugs (DREADDS) [60] or optogenetic manipulation of new neurons [61] that would allow temporary inactivation of newly generated dentate granule cells during key phases of the learning and memory process. We conclude that transgenic methods for manipulating the number and function of new neurons hold promise for uncovering the functional significance of adult hippocampal neurogenesis in behavior.