Our results validate the anxiogenic effect of elevated neurogenesis after voluntary running. Here we were able to show that running-induced anxiety does not occur after hippocampus-focalized irradiation, demonstrating the essential contribution of neurogenesis. In contrast, hippocampal BDNF levels were not altered by irradiation and thus did not correlate with the development of an anxious phenotype. Interestingly, we observed a difference in hippocampal c-Fos activation in sham-treated runners following exposure to an environment soliciting anxiety, suggesting an additional functional correlate of the increased anxiety. Together these findings are in accordance with the concept that the hippocampus is a core structure in the genesis of anxiety 
. It is indeed an accepted notion that decreased hippocampal activity following lesions or applications of various anxiolytics results in decreased anxiety behavior 
. However, it has been less investigated whether elevated hippocampal activity coincides with increased anxiety.
The functional incorporation of adult-born neurons into hippocampal circuits increases hippocampal activity 
. Animals with higher hippocampal neurogenesis perform better in tests that are highly sensitive for hippocampal function in learning and memory 
, while decreased hippocampal neurogenesis results in poorer performances 
, albeit a recent report challenges this view 
. Together, these findings indicate a strong relation of hippocampal neurogenesis with increased hippocampal activity and cognitive performance.
The connection between anxiety and increased neurogenesis as a modulator of hippocampal functioning is less clear. In an earlier report we found a positive correlation for hippocampal neurogenesis and anxiety in voluntary runners 
. In the present study we corroborated this finding and validated it by blocking hippocampal neurogenesis. Sham-treated runners showed increased anxiety after long-term voluntary running in 3 different ethologically-based tests. In contrast, irradiated runners exhibited similar anxiety behavior compared to irradiated sedentary mice. Thus we conclude that increased neurogenesis is a crucial mechanism by which anxiety is induced in voluntarily running mice. The anxiogenic effect of voluntary running was also described in rats recently 
. The finding that improved cognition and high trait anxiety both occur in rodents with high neurogenesis might be counterintuitive at a first glance. But it should be borne in mind that anxiety is a highly cognitive state and not in general a detrimental emotion 
. Anxiety has a profound evolutionary significance for the survival of individuals. Therefore increased anxiety should not be generally interpreted as a model for psychiatric pathology, e.g. wild mice display increased anxiety behavior in response to open areas compared to inbred laboratory strains 
. Wild wood mice have moreover much higher neurogenesis rates in the dentate gyrus 
Previous reports demonstrated that ablation as well as increase of hippocampal neurogenesis may result in increased anxiety 
, suggesting that intermediate levels of neurogenesis are related to low trait anxiety. In our present study, we observed such a U
-shaped relation for neurogenesis and anxiety in the dark-light box test. Irradiated mice showed increased anxiety and reduced neurogenesis compared to sham-treated sedentary controls, while sham-treated runners exhibited increased anxiety along with increased neurogenesis compared to sedentary controls. In contrast, neither the open field nor the O-maze test revealed an anxiogenic effect of irradiation. Of note, however, irradiation did not ablate neurogenesis completely in our experimental setting, thus the decrease in neurogenesis was milder compared to genetic approaches 
. Consistently, in Klf
-9-null mice decreased differentiation and plasticity of adult born neurons elicit anxiety which is also only detectable in a dark-light-paradigm and not in other anxiety-related tests 
. These data suggest that the dark-light-paradigm may be more sensitive to deficits in hippocampal neurogenesis compared to other anxiety-related tests. Thus, our data show that physiological levels of neurogenesis may be important for anxiety processing and deviations from this range can result in increased anxiety behavior. Nevertheless, in some previous reports neither ablation nor increase of neurogenesis did affect anxiety-related behavior 
. In line with these reports, in two anxiety tests (open field and O-maze) irradiated mice performed comparably to sham-treated sedentary controls. Profound differences in neurogenesis in irradiated mice compared to sham-irradiated sedentary controls had therefore no influence on anxiety behavior in these tests. Irradiation did moreover not alter the running activity of mice during 4 weeks of voluntary wheel running. This is of particular importance, since general locomotor activity differences between mice could explain alterations in anxiety tests. To rule out a general hypo-locomotion in running mice, we performed a home cage activity measurement in another cohort of mice. These measurements revealed no significant difference between sedentary and running mice (), thus neither wheel running nor irradiation affected basal locomotion.
While irradiation resulted in reductions in the number of proliferating cells, we observed no influence of long-term voluntary running on the number of Ki67
-positive cells, confirming thus our recent observation 
. Though previous studies have reported a stimulating effect of running on proliferation under comparable conditions 
, our data are in line with a recent study reporting that proliferation is only increased at the onset of wheel running and returns to baseline levels after 21 days of running 
To investigate the influence of running and irradiation on hippocampal activation we further performed c-Fos staining of the hippocampal subregions. The dentate gyrus receives its main excitatory input from the perforant pathway. Different inputs mediating anxiety converge upon the neurons of the dentate gyrus, which in turn project with their axons to the CA3 region 
. Adult born neurons in the dentate gyrus form synapses with hilar and CA3 neurons around the age of 2 weeks 
influencing the activity of the input regions of the trisynaptic circuit. Following the hypothesis that altered hippocampal activity affects anxiety, we were aiming to find a functional cellular correlate in a situation of anxiety. Expression of the c-Fos gene is a robust correlate for neuronal activity 
. Previous studies reported lower numbers of anxiety-induced c-Fos positive cells in rodents with higher trait anxiety 
. Consistently, we found less c-Fos cells in ventral CA1 following an anxiogenic stimulus in sham-treated runners (), which exhibit both more anxiety and neurogenesis (–). Moreover sham-treated runners failed to alter ventral CA1 activity when exposed to anxiety, while all other groups increased c-Fos expression. It is noteworthy, that the ventral CA1 region of Ammon's horn is the main output of the trisynaptic circuit to regions crucial for anxiety and neurogenesis processing 
. At a first glance, these results seem to be at odd with the hypothesis of increased hippocampal activity due to increased neurogenesis in sham-treated runners. But one has to keep in mind that c-Fos expression does not give insight into the quality of activity, namely whether cells are excitatory or inhibitory. Approximately 70% of the target cells of mossy fibers of adult born neurons are inhibitory interneurons 
. Mossy fibers have contacts with interneurons en passant
before they reach the pyramidal cells of CA3. Acsády and colleagues (1998) averaged that granule cells in general have about 50 times more connections to inhibitory cells. Such anatomical connections might account for the reason why increased activity of granule cells is generally associated with reduction of pyramidal cell activity in CA3 
, highlighting the intricate orchestration of the hippocampal circuitry.
Besides our findings of c-Fos expression under anxiety-inducing conditions, we observed a strong effect of regular voluntary running on dentate gyrus c-Fos activity in cage controls. Animals equipped with a wheel had 6 times more c-Fos cells in the dentate gyrus than sedentary controls, irrespective of irradiation. High dentate gyrus activity might be one underpinning of the increased neurogenesis in this neurogenic niche. Particularly, since cell activity also regulates ambient neurotransmitter levels which give first input to newborn neurons before synaptic innervations 
. It is tempting to speculate that the continuous hyperactivation along with increased neurogenesis could have negative effects on the functioning of the hippocampal network. A resulting reduction in excitatory drive of neurons in the hippocampal subfield could be one compensational mechanism to adjust this activity gain. In this context, reduced c-Fos levels in animals with higher trait anxiety could be a consequence of synaptic scaling 
In conclusion, our data demonstrate that an excessive increase of neurogenesis has potential negative consequences as indicated in previous reports 
. These data thus challenge the simplistic view that more newborn neurons are always better for mental health. However, many brain changes might have occurred following exercise and irradiation which affected neurogenesis in an indirect fashion leading to anxiety. In previous findings hippocampal BDNF levels were correlated with anxiety 
. By blocking neurogenesis and thereby altering anxiety without any changes in BDNF levels, we could demonstrate that BDNF failed to correlate with anxiety. Further investigations are nevertheless still needed to determine the mechanism through which alterations in adult hippocampal neurogenesis induce anxiety.