Activin in the Brain Modulates Anxiety-Related Behavior and Adult Neurogenesis
Estresse, neurônios, doenças, adulto, Brain, Neurogenesis
Activin in the Brain Modulates Anxiety-Related Behavior and Adult Neurogenesis
Hiroshi Ageta,1,2 Akiko Murayama,1,2 Rika Migishima,1 Satoshi Kida,2,4 Kunihiro Tsuchida,5 Minesuke Yokoyama,1,6 and Kaoru Inokuchi1,2,3*
1Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan
2Japan Science and Technology Corporation (JST), CREST, Kawaguchi, Japan
3Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama, Japan
4Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan
5Division for Therapies Against Intractable Diseases, Institute for Comprehensive Medical Science, Fujita Health University, Aichi, Japan
6Brain Research Institute, Niigata University, Niigata, Japan
Seth G. N. Grant, Editor
Wellcome Trust Sanger Institute, United Kingdom
Conceived and designed the experiments: KI HA. Performed the experiments: HA AM RM MY. Analyzed the data: HA. Contributed reagents/materials/analysis tools: SK KT. Wrote the paper: KI HA. Other: Supervised the entire project: KI.
Received August 24, 2007; Accepted February 22, 2008.
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Activin, a member of the transforming growth factor-β superfamily, is an endocrine hormone that regulates differentiation and proliferation of a wide variety of cells. In the brain, activin protects neurons from ischemic damage. In this study, we demonstrate that activin modulates anxiety-related behavior by analyzing ACM4 and FSM transgenic mice in which activin and follistatin (which antagonizes the activin signal), respectively, were overexpressed in a forebrain-specific manner under the control of the αCaMKII promoter. Behavioral analyses revealed that FSM mice exhibited enhanced anxiety compared to wild-type littermates, while ACM4 mice showed reduced anxiety. Importantly, survival of newly formed neurons in the subgranular zone of adult hippocampus was significantly decreased in FSM mice, which was partially rescued in ACM4/FSM double transgenic mice. Our findings demonstrate that the level of activin in the adult brain bi-directionally influences anxiety-related behavior. These results further suggest that decreases in postnatal neurogenesis caused by activin inhibition affect an anxiety-related behavior in adulthood. Activin and its signaling pathway may represent novel therapeutic targets for anxiety disorder as well as ischemic brain injury.
Anxiety disorder represents one of the most common mental illnesses 
. Recently, disturbance in adult hippocampal neurogenesis was proposed to underlie anxiety-like behavior in rodents 
; however, molecular mechanisms that link hippocampal neurogenesis to anxiety disorder remains poorly understood.
Activin, a member of the transforming growth factor-β superfamily, is an endocrine hormone that regulates differentiation and proliferation of a wide variety of cells 
. In the brain, activin receptor ActRII is highly expressed in forebrain region 
, and its scaffold protein ARIP/S-SCAM is also localized in synaptic region 
. Furthermore, activin protects neurons from ischemic damage 
, and its expression is upregulated by neuronal activity 
. Recently, we showed that activin modulates dendritic spin morphology that is important for synaptic plasticity in the hippocampus 
In this study, we generated and analyzed transgenic mice in which activin function in the forebrain is either suppressed or enhanced. We found that the activin activity in the adult forebrain influences locomotor activity, anxiety-related behavior, and hippocampal neurogenesis.
We explored the role activin plays in anxiety-related behavior using a transgenic mouse model that overexpresses activin or follistatin, an activin-inhibitory protein, in a forebrain-specific manner. Disturbance of activin signal during the developmental stage causes a lethal phenotype in mammals 
. To achieve postnatal, forebrain-specific expression, the αCaMKII promoter was used to drive expression of a transgene (Fig. 1A
. We microinjected activin and follistatin transgenes into 549 and 1183 fertilized eggs, and obtained 42 and 55 weaned mice, respectively. From these, two lines of activin transgene-integrated mice (designated ACM3 and ACM4) and one line of follistatin transgene-integrated mice (designated FSM) were obtained. Transgene-integrated mice were generated in 1% of microinjected fertilized eggs 
. This low efficacy may have been caused by unexpected transgene expression in various tissues during the embryonic stage, because the activin signal is important for normal development. In contrast to previously generated activin- or follistatin-transgenic mice 
, these heterozygous transgenic mice were fertile and bred healthily, and their body weight (data not shown) and muscular strength were normal when compared to their wild-type littermates (Figure S1
). Since ACM3 mice showed phenotypes similar to ACM4 mice in behavioral experiments, we hereafter describe the phenotypes of ACM4 and FSM mice.
Generation of transgenic mice and expression analysis of the transgene.
hybridization analyses of brain sections revealed that transgene expression was restricted to the forebrain such as the hippocampus and neocortex in the adult brain (Fig. 1B
). ELISA analyses also showed forebrain-specific expression of activin and follistatin in ACM4 and FSM adult mice, respectively (Fig. 1C
). Low level of endogenous activin was detected in the hippocampus and neocortex in wild-type mice. Follistatin level in FSM was enough to antagonize this level of activin activity 
. Follistatin was not detected in the infant hippocampus of FSM mice (Fig. 1C
). RT-PCR revealed that follistatin- and activin-transgene were not expressed in peripheral tissues including, heart, lung, spleen, liver and kidney (Figure S2
). Nissel staining showed no apparent structural abnormality in the hippocampus of each transgenic mice (Figure S3
Open field tests were performed on transgenic mice to investigate locomotor activity (Fig. 2
). FSM mice showed a decrease in time spent in locomotion and rearing when compared with wild-type littermates. In contrast, ACM4 mice showed a significant increase in rearing time. There was no genotype effect in the walking speed (Fig. 2B
) and the total pathlength (Fig. 2C
), indicating that walking ability of FSM and ACM4 mice was normal. These results indicate that the level of functional activin in the brain is related to general locomotor activity in a novel environment.
Activin protein levels in the brain influence locomotor activity.
In open field tests, the amount of time spent in the center of the field is strongly correlated with an animal’s level of anxiety, a characteristic called risk-taking behavior 
. FSM mice showed decreased performance in risk-taking behavior (Fig. 2D
), while ACM4 mice showed increased performance. To further assess these differences, a light and dark choice test and elevated plus-maze test were conducted. In the light and dark test, ACM4 mice accessed the lighted compartment significantly more often than wild-type littermates (Fig. 3A
), however, FSM mice spent significantly more time in the dark compartment as compared to wild-type littermates. In the elevated plus-maze test, ACM4 mice spent significantly more time in the open arms of the testing apparatus than did wild-type and FSM mice (Fig. 3B
). FSM mice showed no significant change in phenotype for this test.
Activin protein levels in the brain modulate anxiety-related behavior.
We next designed and performed an original behavioral test to measure anxiety levels (Fig. 3C
), based on the observation that mice generally prefer novel objects encountered in a familiar place 
. In this test, mice were placed in a closed box on the first day to become familiar with the box. On the second day mice were placed in the same box to which a cylinder with two entrances (novel area) had been added. FSM mice spent significantly less time accessing the novel area as compared to wild-type mice, while the total distance they traveled during the test was normal. This suggests that higher anxiety in FSM mice resulted in lower access to the novel area. Taken together, the level of functional activin in the brain modulates anxiety-related behavior. Finally, no depressive behavior was observed in FSM mice in the forced swimming test (Figure S4
Adult neurogenesis is the production of new neurons in areas of the adult brain including the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampus 
. This formation of new neurons plays a number of physiological roles including damaged neuron replacement
, memory formation 
and response to stress 
. Moreover, some reports have recently shown that neurogenesis is involved in depression 
We therefore examined adult neurogenesis in hippocampal SGZ of FSM and ACM4 mice (Fig. 4
) using 5-bromodeoxyuridine (BrdU)-labeling experiments. Transgenic mice were injected with BrdU (75 mg/kg body weight) three times per day for three consecutive days. Mice were sacrificed either 24 h or 4 weeks after the final injection day. BrdU is incorporated into genomic DNA by cells at S-phase, therefore, by staining with a neuronal marker (NeuN) and an anti-BrdU antibody, newly generated neurons were easily detected. A significant difference between FSM and ACM4 mice was observed in the number of SGZ BrdU-positive cells after 24 h (Fig. 4A, B
). No significant difference, however, was observed between transgenic mice and wild-type littermates, indicating that the number of neuronal progenitor cells and the rate of BrdU incorporation into progenitor cells in transgenic mice were essentially normal. At the 4-week stage, however, the number of BrdU- and NeuN-double positive cells in FSM mice was markedly decreased (Fig. 4C
). This reduction was partially rescued by crossing with ACM4 (Fig. 4D
). These results indicated that the level of activin in the brain is crucial for the maturation and maintenance of newly generated neurons.
Activin signal is essential for survival of newly generated neurons.
The decrease in BrdU- and NeuN-double positive cells at the 4-week stage may be attributed to a decrease in the survival rate of newly formed neurons or a decrease in the rate for neuronal differentiation of new cells. Therefore, the change in the number of BrdU- and NeuN-double positive cells following BrdU injections (Fig. 4E
) was monitored at various developmental stages. The number of BrdU- and NeuN-double positive cells was normal at the 1-week stage in FSM mice, suggesting a normal differentiation rate. However, a marked decrease was observed in the number of BrdU- and NeuN-double positive cells at 2- and 3-week stages compared with wild-type littermates. Therefore, in FSM mice, the survival of newly generated neurons is significantly decreased. This indicates that activin signal is essential for the maintenance of newly generated neurons. Activin overexpression did not enhance the number of BrdU- and NeuN-double positive cells at 4 weeks, suggesting that activin overexpression is not sufficient for enhancement of adult neurogenesis (Fig. 4C
Taken together, FSM and ACM4 mice showed opposite phenotypes in behavior. Furthermore, decrease in neurogenesis in FSM mice was partially rescued in FSM/ACM4 double transgenic mice. These results strongly suggest that the observed effects of overexpression, either follistatin or activin, are not positional transgene effects such as insertional mutations.
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