The results from the different behavioural paradigms show that in mice, TMZ affects several different parameters, which may be relevant to clinical depression. The most robust of these behavioural findings is the effect of neurogenesis depletion on hyponeophagia in the NSF test seen at different time points. Classically the NSF test is often interpreted to be an indicator of anxiety-like behaviour as well as anhedonia though this study as well as previous studies report that despite clear results in the NSF, neurogenesis depletion does not induce anxiety-like behaviour.26 In fact, a meta-analysis of studies examining anxiety-like effects related to adult neurogenesis show that, in agreement with the current study, decreases in neurogenesis have no effect on anxiety.27 This indicates a deeper complexity of the NSF beyond anxiety and indicates it likely is a measure of other behavioural mechanisms. Indeed, the NSF test is one of the only tests that responds to chronic antidepressant treatment and not acute suggesting that this test engages biological processes, which have parallels to depression and its treatment. Although the nature of these biological processes remains to be confirmed, several recent findings suggest that a central function of adult neurogenesis and even the mechanism central to the NSF test may be in the processing of novel information.26, 28 Whether memory processing is important to the development of depression is currently debatable as exemplified in a recent paper in which changes in hyponeophagia were attributed to memory processing, rather than being related to mood, making thus a clear distinction between memory and mood.26 In contrast, recently it has been proposed that neurogenesis may have a role in stress-related disorders through its function in contextual emotional processing of memories and that deficits in neurogenesis may contribute to depression via negative assessments of novel contexts thus altering perception.29 Accordingly, the memory processing during the NSF may therefore be directly related to mood, rather than being distinct. Therefore, another interpretation is that the robust effects seen in the NSF test indicate that reductions in neurogenesis possibly affect contextual emotional memory processing.

Hyponeophagia is an assessment tool developed for animal models but as described, the mechanistic tangents may be relevant to human depression. Patients undergoing various forms of chemotherapy, similar to TMZ, often present with depressive symptoms suggested to be partly related to decreases in adult neurogenesis.3 The emotional cost of cancer itself undoubtedly has a role in the development of depression in patients. Adult neurogenesis cannot be readily measured in living human individuals but the fact that TMZ has such robust effects on neurogenesis using a dosage and treatment cycle in mice that is comparable to the treatment used clinically suggests that patients treated with TMZ may also have depleted levels of adult neurogenesis. It is therefore possible that chemotherapy-induced reductions in neurogenesis also contribute to depression via neurogenesis-dependent processes such as deficits in contextual emotional processing of memories. The implications of this are that patients being treated with chemotherapy may have behavioural deficits that have parallels to those seen in this study.

Another finding relating TMZ-induced changes to depression are the effects on stress. In this study, mice that have been exposed to an acute forced swim stress display an expected increase in corticosterone. However, mice that have depleted neurogenesis as a result of TMZ have an even greater corticosterone response to stress, which is inversely correlated to the individual levels of neurogenesis. A previous high-profile study by Snyder et al.30 has shown similar findings though this was not correlated to individual levels of neurogenesis. One possible reason this correlation has not been seen before may be the method by which ventral vs dorsal has been sectioned for quantification, which may have led to dorsal regions being quantified as ventral.31 The new method of sectioning described here may allow the proper resolution necessary to observe this correlation. Another factor that may affect both levels of neurogenesis and stress levels are the home-cage environment and light phases during which testing was done. Per local regulations, home cages of all the groups in this study were given shelter and nesting materials, which may be interpreted as slightly enriched environment in comparison with other studies not providing shelter or nesting material. Enrichment is known to increase adult hippocampal neurogenesis32 and could not only potentially mask even more prominent behavioural and neurogenesis changes in the TMZ groups, but may also similarly alter stress responsivity in control groups. Alternatively, it could be argued that the slightly enriched housing is more representative of a natural environment and may result in more interpretable findings. Regarding lighting, both this study and Snyder et al.30 were done during the light phase, whereas experiments done during the dark phase have found the opposite effects that neurogenesis impairs the stress response.33 Snyder et al.30 attribute the increase in stress-induced corticosterone response seen in neurogenesis-depleted animals to an impairment of negative feedback on the hypothalamic–pituitary–adrenal axis. Although this mechanism may be a contributing factor to this observed phenomenon, several challenges in direct response to this idea were raised including a lack of GR receptors in young neurons,34 and the potential for circuitry-related mechanisms, which theoretically may also cause changes in stress regulation proposed by Gould and colleagues.35 Along similar lines of this suggested alternative mechanism,35 the changes in stress perception in the theory described above regarding impaired contextual emotional processing may also affect stress reactivity.29 For example, human measurements of cortisol levels in response to stress show that individuals with deficits in emotional regulation have a higher cortisol reactivity.36 Although there are obvious limitations in rating individual experiences of stress or emotional regulation in animals, corticosterone may similarly be related to emotional regulation and stress reactivity. Therefore, neurogenesis-deficient mice may have deficits in contextual emotional processing causing the observed increase in cortisol to simply be the result of an increased perception of stress. The TMZ-induced changes in corticosterone response again may have important implications for patients treated with chemotherapy as glucocorticoid levels in humans may be similarly affected. Indeed, previous clinical studies indicate dysregulation of the glucocorticoid system in cancer patients treated with different chemotherapy treatments.37, 38, 39 Although hormonal stress response reactivity has not been measured in these studies, one study reported changes in diurnal glucocorticoid rhythm in treated patients that was furthermore associated with depression.39 Measurements of the acute stress response in this study present only a limited aspect of stress-response regulation as a result of neurogenesis depletion. Clearly these results as well as reported clinical deficits in patients indicate that further investigation is warranted and may be of clinical importance.

The results from the sucrose preference indicate that in this study, anhedonia is affected but only transiently. Previous studies examining the effects of depleted neurogenesis on anhedonia show again contrasting results with neurogenesis depletion resulting in both an increase in anhedonia and no effect.30, 33 In the present study, a lack of habituation period, which is however present in the referenced studies, allowed the observance of the transient decrease, which would otherwise have not been observed. This transient period is interesting, nonetheless, as it indicates a neurogenesis depletion-induced difference in behaviour is related to anhedonia. The connection between the observed behaviour and neurogenesis is further supported by the trend seen in the correlation between transient decreased sucrose preference and ventral neurogenesis levels in this study. A similar correlation in whole hippocampus has previously been reported in rats.40 Alternatively, however, the reason for this effect may be similar to the observation in the NSF test and is the response of neurogenesis-depleted mice toward novelty, in this case, the presentation of a new taste. Therefore, the transient decrease in sucrose preference may possibly be a result of transient anhedonia, a deficit in novelty processing or a combination of both.

TMZ as an experimental depletion method is relatively inexpensive and is easily injected, making this pharmacological approach a much more available alternative to decrease adult neurogenesis. Some genetic models of neurogenesis ablations are absolute and permanent, whereas TMZ induces an 80–90% reduction which then slowly recovers. An advantage of this is that there is a general uniformity in the level of depletion across groups, whereas other methods which require precise injections will result in highly variable levels of depletion. In our study, the recovery time allowed a substantial recovery of 6 weeks yet robust behavioural effects were still present while neurogenesis deficits were still present after 9 weeks of recovery. This indicates that total ablation is not necessary to observe biological and behavioural changes and that in fact, such depletion levels are likely more translational and more representative to the clinical scenario. In addition, methods inducing a total ablation may initiate compensation mechanisms, which may lead to misinterpretation of results. However, a specific disadvantage of TMZ depletion is the global nature of the depletion. Some depletion methods allow a spatial specificity targeting only the hippocampus, whereas TMZ also affects the other neurogenic niche as well as other types of cell division. Although TMZ has less side effects than other antimitotic compounds, it still has the potential to affect other tissues. This was in a previous study, however, found to be minimal and a non-issue after sufficient recovery time.7 Previous studies have also demonstrated that TMZ affects certain aspects of cognition, particularly spatial and associative learning.7, 11 Although these types of cognition are unlikely to affect the parameters measured in this study, it cannot be excluded that other types of potential cognitive deficits due to a decrease in adult neurogenesis may have an effect on a depressive-like phenotype. Indeed, cognitive deficits in themselves are considered to be an important aspect of depression.41

As TMZ is the standard treatment for cancers of the brain, thousands of patients are treated with this drug. In addition to its utility as an experimental tool in animal models, studying the effects of TMZ has important translational implications for all patients being treated with chemotherapy. Although it is not possible to experimentally deplete adult neurogenesis in humans, this study gives strong evidence that chemotherapy-induced decreases in adult neurogenesis may affect the stress response and behaviours related to processing novelty, both of which ultimately may contribute to depressive symptoms. In addition to TMZ, the antimitotic action of other cancer drugs has already been demonstrated in animal models to decrease neurogenesis and affect spatial memory,42 though these do not affect neurogenesis-independent functions such as fear conditioning and novel object recognition.43 Although no changes in depressive-like behaviour have been noted in other studies, it is likely that other types of chemotherapy may similarly affect behaviours and biological parameters, which have the potential to contribute to depression. Despite their potential side effects, TMZ and other forms of chemotherapy are indispensable for increasing patient-survival rates. However, future studies, which harness the benefit of doubt of these antimitotic drugs and examine their effects on neurogenesis depletion may yield new insights into neurogenesis function in humans, an area of study that is otherwise highly in need for translational verification. Studying the specific effects of chemotherapy on mood may also initiate important changes in prophylactic and post-chemotherapy treatments, which may increase the quality of life for those affected.