Functional granule neurons are generated in the hippocampus throughout life via a multistep process that begins with glial fibrillary acidic protein (GFAP)-expressing radial cell precursors8, 9
. To investigate the role of adult neurogenesis in hippocampal function, we created mice that express herpes simplex virus thymidine kinase (TK) under control of the GFAP promoter (TK mice; ). TK renders mitotic cells sensitive to the antiviral drug valganciclovir but spares post-mitotic cells10
. Stellate astrocytes and radial neuronal precursor cells both express GFAP, and both cell types express TK in the transgenic mice. However, the number of astrocytes is unaltered by treatment with valganciclovir in TK mice (), consistent with a lack of cell proliferation in astrocytes in the adult brain11
. In contrast, immature neurons expressing doublecortin (DCX) were virtually eliminated in the dentate gyrus of v-TK mice (, Supplementary Fig. 1
). One-day-old neuronal progenitors, identified with DCX and the cell cycle marker bromodeoxyuridine (BrdU), were reduced in v-TK mice by ≥99% relative to control conditions (). Transgene expression alone and valganciclovir treatment of WT mice had no effect (). Valganciclovir-treated TK (v-TK) mice showed weight gain comparable to v-WT mice (Supplementary Fig. 2
), and histolopathological examination found no abnormalities in the small intestine or submucosal or myenteric plexuses (not shown), indicating that the gastrointestinal effects described in another strain expressing TK under the GFAP promoter8
are absent in this line of mice. Taken together, these data indicate that v-TK mice have a specific loss of adult neurogenesis without detectable effects on astrocytes or general health.
Figure 1 GFAP-TK mice show specific inhibition of adult neurogenesis. a) Confocal image of endogenous GFAP and transgenic TK expression in a radial precursor cell in the dentate gyrus (arrow). b) Confocal photographs of valganciclovir-treated mice show GFAP+ astrocytes (more ...)
The hippocampus provides negative control of the HPA axis6, 7
, but the circuitry involved is not well understood. Since adult hippocampal neurogenesis is highly sensitive to stress and glucocorticoids3
, we hypothesized that adult neurogenesis may be important for hippocampal regulation of the HPA axis. We therefore examined serum levels of corticosterone, the predominant rodent glucocorticoid, in several conditions that activate the HPA axis. Neurogenesis-deficient v-TK and control v-WT mice had equivalent levels of corticosterone at the onset of both the light and the dark phase (Supplementary Fig. 3
), indicating that adult-born neurons are not required for normal circadian fluctuation of glucocorticoids. v-WT and v-TK mice also had similar corticosterone levels after exposure to a novel environment (Supplementary Fig. 3
), a mild stressor, consistent with previous findings12
To test the response to, and recovery from, a moderate psychological stressor, we subjected mice to 30 minutes of restraint and measured corticosterone 0, 30, or 60 min later (). v-TK mice and v-WT mice had similar levels of corticosterone immediately after termination of the stressor. However, v-TK mice had elevated corticosterone relative to v-WT mice 30 min after stress, suggesting impaired negative feedback control of glucocorticoid release similar to that observed in mice with complete loss of glucocorticoid receptors in the forebrain13
. To investigate whether the hypersecretion of glucocorticoids habituates, mice were subjected to daily restraint for 16 additional days, and corticosterone was measured on the final day. As on the first day of restraint, v-TK mice had elevated corticosterone relative to v-WT mice (). Control TK mice, which were never treated with valganciclovir and therefore had normal levels of neurogenesis, had levels of corticosterone identical to untreated WT mice 30 min post-restraint (). Therefore, corticosterone hypersecretion in neurogenesis-deficient mice is not caused by nonspecific or insertion site effects of the TK transgene.
Figure 2 The glucocorticoid response to stress is increased in neurogenesis-deficient mice. a) Restraint, a moderate psychogenic stressor, resulted in higher corticosterone in neurogenesis-deficient v-TK mice than in v-WT mice 30 min after the end of stress. b) (more ...)
The experiments above indicate that adult neurogenesis regulates the endocrine stress response, but they do not pinpoint the brain region involved. Hippocampal damage primarily alters the response to psychological stressors such as restraint, which produce fear without causing a direct threat to well-being, but does not typically affect responses to physical stressors, such as hypoxia, hemorrhage, inflammation, or anesthesia7
. Consistent with HPA regulation at the level of the hippocampus, v-WT and v-TK mice showed comparable elevations in corticosterone after exposure to isoflurane anesthesia (Supplementary Fig. 4
). Additionally, in agreement with the lack of global HPA dysregulation, we found no evidence for reduced cell birth in the hypothalamic paraventricular nucleus of v-TK mice (Supplementary Fig. 5
To directly test the contribution of adult neurogenesis in other regions, we exploited the spatial specificity of X-irradiation to reduce hippocampal neurogenesis while sparing neurogenesis in the subventricular zone (SVZ)12, 14
, a source of GFAP+ neuronal precursors added to the olfactory bulb throughout adulthood8, 15
. Irradiated mice had elevated corticosterone during recovery from restraint stress (), replicating the initial finding with an independent ablation method. In addition, there was no relationship between the extent of SVZ neurogenesis inhibition and the corticosterone response in irradiated mice (). Therefore, the most parsimonious interpretation of our results is that inhibition of adult neurogenesis in the dentate gyrus leads to hypersecretion of glucocorticoids in response to stress.
Figure 3 Increased stress response is not due to reduced neurogenesis in the subventricular zone. a) Increased corticosterone response 30 min after restraint was confirmed in mice in which neurogenesis was reduced by irradiation (irradiation effect F1,50=2.0, (more ...)
A functional link between new neurons and anxiety/depression has been suggested by the demonstration that some antidepressant effects on behavior are blocked by irradiation12, 16-18
. However, normal anxiety- and depressive-like behavior in animals lacking neurogenesis has led to speculation that impaired neurogenesis does not directly contribute to the etiology of depression in adulthood4, 5
. Our findings above suggest that stress may be a key unexplored factor linking adult-born neurons to anxiety- and depression-like behaviors.
To probe a potential interaction between stress, neurogenesis and depression, we first employed the dexamethasone suppression test, which is commonly used to test HPA axis feedback in depressed patients. A subgroup of depressed patients, and mice that display depressive behaviors, show impaired inhibition of endogenous glucocorticoids by the synthetic glucocorticoid dexamethasone19, 20
. We found that dexamethasone effectively suppressed the restraint-induced rise in corticosterone to near basal levels in v-WT mice (). However, the level of corticosterone in dexamethasone-injected v-TK mice was significantly higher than that in v-WT mice, consistent with a depressive-like phenotype.
We next examined whether adult neurogenesis regulates the behavioral response to stress in the novelty-suppressed feeding (NSF) test, which shows robust effects of antidepressants that are blocked by irradiation12, 18
. In this test, food-deprived mice are introduced to a novel open field containing a food pellet at its center and the latency to begin feeding is recorded12
. When tested in the NSF paradigm under normal conditions, v-WT and v-TK mice showed similar feeding latencies (), indicating similar levels of approach-avoidance behavior. Restraint stress just prior to testing, however, significantly increased the feeding latency in v-TK mice while having no effect on v-WT mice. Moreover, by the end of the 10 min test only 53% of stressed v-TK mice had fed compared to 92% of stressed v-WT mice (). Mice from all groups consumed food upon returning to their home cage, indicating that decreased motivation to eat was not responsible for change in behavior (Supplementary Fig. 6
). Thus, adult neurogenesis does not alter behavior under baseline conditions in this test, consistent with previous observations,12, 18
but buffers the effects of stress on feeding behavior.
Figure 4 Mice lacking neurogenesis show increased anxiety/depression-like behaviors. a) In the novelty-suppressed feeding (NSF) test, v-TK mice showed increased latency to feed in a novel environment following restraint stress but not under control conditions (more ...)
Because the NSF test is associated with both anxiety- and depressive-like behavior, we investigated the interaction of neurogenesis and stress in additional behavioral tests. In the elevated plus maze, a test of anxiety-like behavior, there was an anxiogenic effect of stress but no significant difference between v-WT and v-TK mice (Supplementary Fig. 7
). We next tested depressive-like behavior, using the forced swim test, in which rodents are placed in an inescapable cylinder of water and immobility is used as a measure of behavioral despair21
. Under control conditions neurogenesis-deficient v-TK mice became immobile more rapidly and for a greater duration than v-WT mice (), consistent with a depressive phenotype. In v-WT mice, restraint stress reduced the latency to become immobile and increased total immobility to the level of v-TK mice. Thus, neurogenesis-deficient mice displayed a depressive phenotype at baseline, which could be induced in intact mice by acute stress. Consistent with previous reports16, 18, 22
, v-WT and v-TK mice showed similar levels of immobility during later stages of the test, when high (potentially ceiling) levels of behavioral despair are observed21
(Supplementary Fig. 8
Anhedonia is a hallmark symptom of depression and, in rodents, presents as a decrease in preference for a sucrose solution compared to water23
. To assess whether adult neurogenesis is required for hedonic behaviors, we habituated v-WT and v-TK mice to freely-available water and 1% sucrose for 3 days. Both groups similarly preferred sucrose (Supplementary Fig. 9a
). Then, following water and sucrose deprivation, the bottles were reintroduced and preference was measured during a 10 min test. To introduce an aspect of reward-based decision making23
, bottle locations were switched for the test. While v-WT mice showed a preference for sucrose as before, neurogenesis-deficient v-TK mice showed no sucrose preference during the test (). The decreased sucrose preference in v-TK mice was observed not only in the 10-min test but also during the following night (). Overall consumption levels were not different, indicating that preference differences did not result from altered thirst or motivation to drink (Supplementary Fig. 9a-d
). No differences were observed in WT and control TK mice that were not treated with valganciclovir, indicating that anhedonic behavior does not result from TK expression alone (Supplementary Fig. 9e-g
). The loss of sucrose preference in v-TK mice was observed whether or not the mice were restrained prior to testing, perhaps reflecting a basic difference between reward-related behaviors tested in this paradigm and the stress response behaviors tested in despair and avoidance paradigms. Taken together, our behavioral results suggest that adult neurogenesis buffers the effect of prior stress in the NSF test, buffers the effect of inescapable stress in the forced swim test, and enhances reward-seeking behavior independent of stress in the sucrose preference test.
Elucidating the strong but poorly-understood association between stress and depression is critical for development of more effective treatments1, 2
. The hippocampus has long been known to regulate the HPA axis6, 7
, and the importance of the hippocampus for emotional behavior is emerging24
. Our data show that adult-born hippocampal granule neurons dynamically regulate stress reactivity at both the endocrine and behavioral levels. A direct role for adult neurogenesis in depression-like behavior was observed in three behavioral paradigms that are commonly-used to assess antidepressant efficacy and characterize the development of a depressive phenotype in response to chronic stress12, 18, 25
. In addition to its effects on emotional behavior, stress is an important modulator of learning and memory.26
Our results therefore also suggest that buffering of stress responses by new neurons may play a role in learning and memory under novel or aversive conditions, in addition to any more direct function of young neurons in encoding of information. Since the production of new granule neurons is itself strongly regulated by stress and glucocorticoids,3
this system forms a loop through which stress, by inhibiting adult neurogenesis, could lead to enhanced responsiveness to future stress. This type of programming could be adaptive, predisposing animals to behave in ways best suited to the severity of their particular environments27
. However, maladaptive progression of such a feed-forward loop could potentially lead to increased stress responsiveness and depressive behaviors that persist even in the absence of stressful events.