The detection of learning-related regulation of adult hippocampal neurogenesis might be confounded by physical activity and stress
In the present study, we intended to examine the effect of learning the Morris water maze on adult hippocampal neurogenesis in mice. Specifically, we examined hippocampal cell populations that had gone through S-phase of the cell cycle 5–6 days prior to the first training session.
This is the period where most of the regulation aiming at cell survival occurs. The time-point of morphometric analysis was chosen because BrdU-labelled hippocampal cell populations reach a quantitatively stable level quickly after cell divisions, and therefore, we assume that regulation of survival that occurs during this vulnerable period should be readily detectable. It is likely that regulation occurs also at later stages of neuronal development than addressed in the present study. However, the calretinin stage likely comprises the phase of highest synaptic plasticity in the course of neuronal development in the adult dentate gyrus (Brandt et al. 2003; Kempermann et al. 2004a). Given the comparatively lower ranges of regulation measurable on the level of cell numbers and marker expression at the late stages of development, these late effects become increasingly difficult to detect. The finding of increased synaptic plasticity and increasing maturation in the first few weeks (Jessberger & Kempermann 2003; van Praag et al. 2002; Snyder et al. 2001; Wang et al. 2000), however, suggests that other parameters might be successfully addressable during this period.
In the present study, we found a robust decrease of measures related to adult hippocampal neurogenesis (BrdU-positive precursor cells and calretinin-positive immature neurons in the dentate gyrus) by both training in the cued and hidden version of the Morris water maze as compared to animals that had been housed under standard laboratory conditions during the entire experiment (Fig. 2a,f). In addition, we report a differential regulation of early (type 2 cells) hippocampal progenitors by HID and VIS, whereas such a differential effect was not detectable on the level of later hippocampal progenitors (type 3 cells, calretinin-positive cells; Fig. 2d,f). However, given the relatively small differences in terms of absolute cell numbers, the differential effect on differentiation phenotypes, although statistically significant, should not be over-interpreted.
Several aspects of Morris water maze training other than hippocampus-dependent spatial learning do or might have effects on adult hippocampal neurogenesis and could therefore confound the assessment of spatial learning-related processes on hippocampal neurogenesis.
First, water maze training and pretraining procedures included a variety of potential stressors; the mice were handled and before training received several intraperitoneal injections of BrdU. Water maze training itself, particularly during the initial period, constitutes a major source of stress. The stress response to water maze training as assessed behaviourally (vocalizations, struggling) and physiologically (serum glucocorticoids) is rather acute, levels out quickly and is more pronounce in females than in males (Beiko et al. 2004). Pre-exposure to water maze stressors prior to training markedly reduced the stress response during training (Beiko et al. 2004). Stress has been reported to be a powerful negative regulator of adult hippocampal neurogenesis, largely mediated by glucocorticoid release from the adrenal gland upon activation of the hypothalamic-pituitary-adrenal axis, but the relationship is not linear (Fuchs et al. 2001; Gould et al. 1997; Heine et al. 2004). In order to adapt our mice to the water maze and reduce stress levels associated with training, we pre-exposed the animals to the water maze setting prior to BrdU injections and water maze training. Pre-exposure reversed the negative regulatory effect of Morris water maze training on adult hippocampal neurogenesis observed in experiment 1, where no pre-exposure had been conducted (Fig. 2g). Thus, we suggest that stress associated with water maze training might be an important confounder, when studying the effect of spatial learning on hippocampal neurogenesis in mice.
Second, Morris water maze training is associated with locomotion, and physical activity robustly increases hippocampal neurogenesis (van Praag et al. 1999a, 1999b). Our findings may suggest that physical activity is a relevant confounder: First, the finding that HID had more BrdU-positive cells than VIS (Fig. 2a) was paralleled by the observation that HID were physically more active during Morris water maze training than VIS. Furthermore, HID increased the number of early neuronal progenitors (type 2 cells), but not late neuronal progenitors (type 3 cells, calretinin-positive cells), as compared to VIS (Fig. 2d,f; but note caveat stated above), which corresponded to the pattern in which physical activity has been reported to affect adult hippocampal neurogenesis (Kronenberg et al. 2003). How the pronounced effect of physical exercise on type 2 cells is achieved mechanistically is unknown at present. Given the important regulatory roles of VEGF (Fabel et al. 2003) and IGF-1 (Trejo et al. 2001) in exercise-induced increases of hippocampal neurogenesis, however, specificity might be implemented at sites of action of these growth factors. Finally, animals trained on the hidden version did not differ from a time-yoked control group (that had a similar degree of physical activity on water maze exposure) with regard to BrdU-positive cells in the dentate gyrus (Fig. 2g). We assume that physical activity during swimming has a similar upregulating effect than voluntary wheel running, the paradigm that has been used to address activity-dependent regulation of adult neurogenesis (van Praag et al. 1999b). However, voluntary swimming paradigms cannot be implemented for mice, and our present data indicate that the normally used protocols for time-yoked controls in the water maze task seem to be confounded in this regard.
Third, besides spatial learning, learning the Morris water maze involves non-spatial learning strategies. It is unknown if and to what degree such strategies might impact on hippocampal neurogenesis, and thus, they have to be taken into account as potential confounders as well.
Based on our present data, we hypothesize that the observed effects of Morris water maze training on adult hippocampal neurogenesis in mice (experiment 1) might be largely explainable by physical activity and stress related to the procedure and may thus confound the recognition of potential effects of spatial learning on adult hippocampal neurogenesis under these conditions. Our data do not rule out that a direct effect of learning the water maze on adult hippocampal neurogenesis in mice occurs. However, our data demonstrate that if such effect exists, it may be masked by confounding parameters such as physical activity and stress. Because these two factors with their well-documented strong influence on adult hippocampal neurogenesis act in opposite directions, the potentially small direct survival-promoting effect of learning on the survival of new neurons will often be lost in other less-specific influences of the learning situation.
The Morris water maze might thus not be an optimal tool to investigate possible learning-dependent effects on adult hippocampal neurogenesis in mice. Previous studies on the effects of Morris water maze training on adult hippocampal neurogenesis had yielded heterogeneous results. Gould et al. (1999) found that adult hippocampal neurogenesis was specifically increased by the hidden version of the water maze, thus suggesting a specific regulation of neurogenesis by learning-related processes. There are differences in the study design, which might contribute to the discrepant results; Gould et al. used male Sprague Dawley rats, whereas we studied female C57BL/6 mice. Second, in terms of absolute time, in the present study, the animals were trained at a shorter absolute interval after the BrdU application than in the Gould study. However, the interval in our study was chosen exactly based on the rationale underlying the choice of interval length in the Gould study and was meant to reflect the species differences in the duration of neuronal maturation. Our previous data have shown that the downward sloping survival curve of the newly generated cells is the steepest in the interval we chose here (Kempermann et al. 2004a). We assume that the early postmitotic phase, during which the new neurons make first mature functional contacts (Hastings & Gould 1999; Jessberger & Kempermann 2003; Markakis & Gage 1999) exactly reflects the vulnerable period of hippocampal neurogenesis (Brandt et al. 2003; Kempermann et al. 2004a). Third, a few technical differences in the training protocols existed. For example, on day 4 of the water maze procedure, Gould et al. continued to train the animals, whereas in the present study (following the Lipp–Wolfer protocol; Wolfer et al. 1998), probe trials were given with the rationale to obtain an additional measure to judge spatial learning.
Ambrogini et al. (2000) reported an increase of hippocampal neurogenesis in rats, specifically in the spatial learner group as well. However, in that study only, cell densities and ratios were compared and no absolute numbers of BrdU-positive cells taken into consideration, so that the small observed difference might be spurious. Also, factors that may have differentially affected the volume of tissue constituents could confound this result. In a more recent report, Ambrogini et al. (2004) reported a decrease in the density of BrdU-positive cells in the rat dentate gyrus, specifically in the group trained in the hidden version of the Morris water maze as compared to the cued trained and control group. The second study by Ambrogini et al. more closely resembled the one by Gould et al. with regard to timing of BrdU administration and training than the first one. If this observation holds, it might not be species differences alone that explain the differences between our present and past data (van Praag et al. 1999b) and the results by Gould et al. (1999) and the initial claim by Ambrogini et al. (2000).
Döbrössy et al. described a differential effect of spatial learning in the Morris water maze on proliferating cells in the rat dentate gyrus. The cells that were generated during the early phase of learning appeared to be more susceptible to elimination, whereas those that were born during the later phase of learning were induced to proliferate more (Döbrössy et al. 2003). Besides species differences, the different temporal relationship of BrdU-injections and Morris water maze training in this study and the one by Döbrössy et al. provides a possible first explanation why we might not have seen similar differential effects of spatial learning on adult hippocampal neurogenesis, although our approach included the distinction of early (type 2) and late (type 3) precursor cells and early postmitotic neurons (stage of calretinin expression).
All mentioned studies have examined the effect of Morris water maze training on adult hippocampal neurogenesis in rats, while in the present study and our previous study (van Praag et al. 1999b), C57BL/6 mice were used. The rat studies, however, used time-yoked control groups as well, so that they controlled for some aspects of Morris water maze training not directly related to spatial learning such as physical activity and stress (Gould et al. 1999; van Praag et al. 1999b). Consequently, the discrepant results of this and previous studies might reflect species differences with regard to regulation of adult hippocampal neurogenesis by different aspects of Morris water maze training. In addition, a species-specific differential stress response associated with the exposure to a water pool in rats and mice appears particularly likely, given that rats swim in their natural habitat, whereas mice do not. Rats outperform mice in several maze tasks in water, but not on dry land (Whishaw & Tomie 1996). Furthermore, the fact that investigators of previous studies used male animals, while in the present study, females were used, may be particularly relevant with regard to stress responses induced by the experimental procedure, since females display more pronounced acute stress responses to water maze training (Beiko et al. 2004).
Other behavioural models involving hippocampal function such as trace eyeblink conditioning have been linked to direct effects on adult neurogenesis (Gould et al. 1999; Leuner et al. 2004), but it remains to be shown that this applies to complex cognitive tasks as well. At least, environmental paradigms that are ethologically more relevant than the task used in the present study have found an upregulation of hippocampal neurogenesis in mice (Kempermann et al. 1997), rats (Nilsson et al. 1999) and birds (Barnea & Nottebohm 1994, 1996). Although the possibility that these effects are caused by factors other than learning can not be excluded, the cognitive demands required in the enriched environment are different from those in the water maze. In any case, ethologically more relevant tasks (Gerlai & Clayton 1999) will have to be developed in order to assess the immediate functional contributions of adult neurogenesis.
Aspects of hippocampal neurogenesis not dealt with in the present study may of course be highly relevant for learning-related processes in the hippocampus. Physiological regulation with functional implications might potentially occur beyond the level of cell proliferation and the determination of cell survival. For example, adult-generated neurons are particularly susceptible to activity-dependent synaptic plasticity (Schmidt-Hieber et al. 2004; Snyder et al. 2001; Wang et al. 2000). As a consequence, the addition of new hippocampal neurons may contribute to learning-related processes, without being quantitatively regulated by learning itself.