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Neuroscience Research
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Stress by noise produces differential effects on the proliferation rate of radial astrocytes and survival of neuroblasts in the adult subgranular zone
Oscar Gonzalez-Perez a,b, , Oscar Chavez-Casillas a , Fernando Jauregui-Huerta b , Veronica Lopez-Virgen a , Jorge Guzman-Muniz a , Norma Moy-Lopez a , Rocio E. Gonzalez-Castaneda b , Sonia Luquin b
a b

Laboratory of Neuroscience, Facultad de Psicologia, University of Colima, Colima 28040, Mexico Department of Neuroscience, Centro Universitario de Ciencias de la Salud, University of Guadalajara, Guadalajara, Jal 44340, Mexico

a r t i c l e

i n f o

a b s t r a c t
The subgranular zone (SGZ) in the dentate gyrus contains radial astrocytes, known as Type-1 or Type-B cells, which generate neuroblasts (Type-2 cells or Type-D cells) that give rise to granular neurons. Stress increases glucocorticoid levels that target SGZ and modify the proliferation and apoptosis of hippocampal cells. Yet, it is not well-known whether stress differentially affects SGZ progenitors. We investigated the effects of noise-induced stress on the rate of proliferation and apoptosis of the Type-1 cells, Type-2 cells and newly generated granular neurons in the SGZ. We exposed Balb/C mice to noise using a standardized rodents audiogram-tted adaptation of a human noisy environment. We measured corticosterone serum levels at different time points. Animals received BrdU injections for 3 days and sequential sacrices were done to carry out double-immunohistochemical analyses. We found that a 24-h noise exposure did not produce adaptative response in the curve of corticosterone as compared to a 12-h noise exposure. The percentage of BrdU+/GFAP+ cells was signicantly reduced in the stress group as compared to controls. A high proportion of CASP-3+/GFAP+ radial astrocytes were found in the stress group. The percentage of BrdU+/doublecortin+ cells was higher in controls than in the stress group. Interestingly, the apoptosis rate of doublecortin-expressing cells in the stress group was slightly lesser than in controls. Remarkably, we did not nd signicant differences in the number of BrdU+/NeuN+ and CASP-3+/NeuN+ neurons. These data indicate that stress differentially affects the rate of proliferation and apoptosis in SGZ progenitors and suggest a possible compensatory mechanism to keep the net number of granular neurons. 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Article history: Received 12 January 2011 Received in revised form 28 March 2011 Accepted 31 March 2011 Available online 15 April 2011 Keywords: Glucocorticoid Stress Dentate gyrus Neural stem cells Subgranular zone Glial brillary acidic protein

1. Introduction Neurogenesis in vivo has been demonstrated only in discrete regions of the adult brain: the subventricular zone and the subgranular zone (SGZ). The source of new neurons in the adult brain is adult neural stem cells, which are multipotent and selfrenewing throughout life. The SGZ of the dentate gyrus is a proliferative region in the hippocampus that contains neuronal progenitors, which origin granular neurons. Approximately, 250,000 new neurons born in the adult SGZ per month (Cameron and McKay, 2001). The primary progenitors of this region are radial astrocytes, known as Type-1 cells or Type-B cells, that asymmetrically divide to give rise to neuroblasts also called Type-2 or Type-D cells (Seri et al., 2001; Zhao et al., 2008), which differentiate into granular neurons (Seri et al., 2004; Seri et al., 2001).

Corresponding author at: Laboratory of Neuroscience, Facultad de Psicologia, Universidad de Colima, Av. Universidad 333, Colima, COL 28040, Mexico. Tel.: +52 312 316 1091; fax: +52 312 316 1091. E-mail addresses: osglez@gmail.com, osglez@ucol.mx (O. Gonzalez-Perez).

There are a number of factors that may affect the hippocampal neurogenesis, such as: hormones (Gould et al., 1992; Gould and Tanapat, 1999), neurotransmitters (Brezun and Daszuta, 1999; Gould et al., 1994), enriched environments (Ramirez-Rodriguez et al., 2009), exercise (van Praag et al., 1999), alcohol (CadeteLeite et al., 1988; Herrera et al., 2003), seizures (Parent et al., 1997), and others (Garcia-Fuster et al., 2010; Kong et al., 2010; Ramirez-Rodriguez et al., 2009; Yoshinaga et al., 2010). Recent evidence indicates that glucocorticoids reduce the proliferation rate of SGZ precursors (de Kloet et al., 2008; Gould et al., 2000). Stressing conditions activate the HPA axis increasing the serum levels of glucocorticoids, which activate hippocampal glucocorticoid receptor (de Kloet, 2003; De Kloet et al., 1993; de Kloet et al., 1999). Activation of GR under stressing conditions has been associated with a decrease on hippocampal neurogenesis, dendritic atrophy (Magarinos and McEwen, 1995), reduction in cell survival (Joels et al., 2004; Ramos-Remus et al., 2002) and cell adhesion molecules (Sandi, 2003), and loss of excitatory synapses (Zschocke et al., 2005). Nevertheless, little is known about the specic cell type in the dentate gyrus that is predominantly affected by stressors. In this study, we investigated the effects of a noise-induced stress on

0168-0102/$ see front matter 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2011.03.013

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the proliferation/apoptosis rate of the neuronal progenitors in the SGZ. We found that stress reduced the proliferation of the Type-1 and Type-2 cells, and increased the apoptosis of Type-1 cells. Interestingly, these changes do not appear to affect the number of new granular neurons. These ndings suggest the existence of a possible counterregulatory mechanism that compensates the initial low proliferative rate induced by noise-induced stress. 2. Material and methods 2.1. Animals We used 45-day-old Balb/C male mice housed in standard polycarbonate cages (4 animals per cage) and maintained on a 12-h light-dark cycle and were allowed free access to food and tap water. Two groups were assembled (n = 35 per group), a control group was housed under standard biotery conditions and the stress group was exposed to a noisy environment as described below. All animal procedures followed the guidelines of the Committee on Animal Research at the University of Colima. 2.2. Noise exposure

software that delivered acoustic signals at levels of 70 dB for the background noise and from 85 to 103 dB for the noisy events. 2.3. Corticosterone (CORT) assay To quantify serum levels of CORT, mice (n = 3 per time point by each group) were decapitated and their blood was collected in non-heparinized tubes. Serum CORT levels were measured using an enzyme immunoassay kit (Correlate-EIA. Assay Designs Inc., USA), following the step-by-step protocol of manufacturer. In order to avoid circadian variation, blood samples were obtained always between 7:00 and 8:00 A.M. 2.4. Bromodeoxiuridine (BrdU) administration BrdU is an analogous of tymidine that incorporates into DNA during cell division (Falconer and Galea, 2003). A day after the noise-induced stress, we administrated 100 mg/kg i.p. BrdU every 12 h (Cameron and McKay, 2001), which were injected at 7:00 h and 19:00 h for 3 days. To label all progeny derived from the primary SGZ precursors, BrdU was injected from day 2 to day 4 and sacrices were done at day 4, 14 and 21 (Fig. 1) 2.5. Tissue processing

Stress by noise exposure was performed as described previously (Rabat et al., 2004; Rabat et al., 2005) and using the rodents audiogram-tted adaptation of noisy environments donated by Dr. Rabat. Briey, acoustic adaptations of a noisy environment (i.e., the noise of a French aircraft carrier boat) were done using the audio software Wave lab 3.0 (Steinberg, Germany) that enabled to translate all frequencies of the noise from the human audiogram to that of the mouse (Rabat et al., 2004). This allows animals to better detect high (over 8000 Hz) frequencies, which are more relevant to the auditory capacities of rodents (Rabat et al., 2004). Then, mice were exposed to a sound containing random and unpredictable noisy events for 10 days. The intervals of noise oscillated from 18 to 39 s followed by silent intervals ranging from 20 to 165 s. Animals were housed in a sound-isolated acoustic room adapted with professional tweeters (Steren Mexico 80-1088) and connected to an amplier (Mackie M1400; freq. 20 Hz70 kHz; 300 W, 8 ) mixer

Mice were sacriced by an overdose of pentobarbital (100 mg/kg body weight) before transcardial perfusion. For uorescent microscopy (n = 4 per group), mice were perfused with 0.9% NaCl solution at 37 C followed by 4% paraformaldehyde in 0.1 M phosphate buffer, and the brains were post-xed overnight at 4 C in the same xative. 40-m thick coronal sections were cut with a vibratome from 1.70 mm to 2.54 mm Bregma coordinates. Fluorescent immunostainings were performed as described below. 2.6. Immunohistochemistry (IHC) Samples were rinsed (10 min 3) in 0.1 M buffer phosphate buffer saline (PBS). Sections were then incubated in pre-warmed 2 N HCl at 37 C for 30 min. Then, a single wash with 0.1 M borate buffer (pH = 8.4) for 10 min was used to neutralize HCl.

Fig. 1. Differentiation process in the SGZ and the experimental design. On the top: schematic drawing illustrating the differentiation process in the SGZ. Type-1 radial astrocytes self-renew and give rise to Type-2 cells (doublecortin-expressing neuroblasts), which in turn proliferate and differentiate into NeuN-expressing granular neurons. At bottom: experimental time line showing the 3-day injection of BrdU and sequential sacrices at day 4 (A), day 14 (B) and day 21 (4) after noise exposure.

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After blocking in 0.1 M PBS + 10% normal goat serum for 1 h at room temperature, samples were incubated with primary antibodies overnight at 4 C in blocking solution + 0.1% Triton-X. The following primary antibodies were used: mouse IgG anti-GFAP (1:500; Millipore), mouse IgG anti-NeuN (1:500; Millipore), mouse anti-Caspase3 active (1:800; Imgenex IMG-144A), rabbit IgG antidoublecortin (DCX) (1:500; Millipore) and rat anti-BrdU antibody (1:100; Accurate Chemical OBT0030). After that, tissue sections were rinsed three times with 0.1 M PBS, incubated with the appropriate AlexaFluor conjugated secondary antibodies (1:1000; Molecular Probes) dissolved in blocking solution for 60 min at room temperature, and washed three times in 0.1 M PBS. Nuclear counterstaining was done with 4 -6-diamidino-2-phenylindole (DAPI). 2.7. Quantication To quantify the number of double-labeled cells, we analyzed at least ten 40-m sections randomly selected, 120-m apart (n = 4 animals per group). Double-labeling was conrmed and quantied by all matching cellular morphologies with clearly discernible nuclei (DAPI+) and by analyzing non-overlapping high-power (40) elds of view. For imaging, a Leica SP-2 laser scanning confocal microscope was used and optical 0.75 m serial sections were obtained to co-localize signals. For every section, the percentage of co-localization of BrdU+ cells was calculated as the fraction of the number of BrdU+ cells that co-expressed NeuN, DCX or GFAP/the total number of BrdU+ cells per section multiplied by 100. A similar mathematical approach was used to calculate the proportion of CASP-3+ cells for each cell type. Data are expressed as mean standard deviation. For comparisons of means between groups, we used the MannWhitney U test. In all cases, the P < 0.05 value was chosen to establish signicant differences. 3. Results 3.1. Analysis of CORT serum levels To determine the stronger effects of noise on CORT serum levels, we assembled two groups: one group received a 12-h exposure to noise followed by 12-h without noise for 10 days. The other group was exposed to incessantly noise for 24 h per 10 days. Throughout the noise exposure, blood samples were collected by decapitating animals (n = 3 per group) at days 1, 2, 3, 5, 7 and 10, and CORT serum levels were measured. To obtain the basal line of CORT serum levels, we sacrice animals that were not exposed to noise. We found that, in the group of 12-h noise exposure, CORT levels increased at the beginning of the stress induction but, at the day 7, CORT levels started to decrease (Fig. 2). In contrast, the group of 24-h noise exposure showed a progressive and steady increase in the CORT serum levels. This suggests that animals exposed to a continual noise cannot adapt to this stressor. Therefore we chose the 24-h noise exposure as the stress group to continue with the study. 3.2. Effect of stress on radial astrocytes cells in the SGZ To determine the effects of stress on proliferation of astrocytic neuronal precursors in the dentate gyrus, we sacriced animals (n = 4 per group) immediately after the noise exposure at day 4 (Fig. 1) and immunostained brain sections with antiBrdU and anti-GFAP antibodies. The proportion of BrdU+ cells that co-express GFAP was then calculated as described above. We found that the stress group showed a signicant reduction in the percentage of BrdU+ that co-express GFAP+ cells (25 5%; P < 0.05, MannWhitney U test) as compared to the control group (57 8%) (Fig. 3). Since a reduction in the number of immunostained cells may be associated with changes in apoptosis rate, we
Fig. 2. Corticosterone serum levels during environmental noise exposures. Two experimental conditions were assessed: 12-h noise exposure and 24-h noise exposure for 10 days. The group exposed to 12-h noise exposure showed a rapid increase in corticosterone levels that decreased signicantly by day 7. In contrast, the group exposed to incessant noise showed a persistent increase in corticosterone serum levels.

quantied the number of cells that expressed Caspase-3-active (CASP-3). Interestingly, the number of CASP-3+ cells was signicantly higher in the stress group (16 2%; P < 0.05, MannWhitney U test) as compared to controls (4.4 2%). These ndings showed that stress reduced the number of proliferative radial SGZ astrocytes, which coincided with a high apoptosis rate in this cell population. 3.3. Effect of stress on Type-2 cells in the SGZ Next, we investigated whether stress by noise modied the survival and apoptosis of Type-2 cells (SGZ neuroblasts) derived from the Type-1 primary precursors pre-labeled with BrdU at day 4 (Fig. 1). Thus, we sacriced animals (n = 4 per group) immediately after the noise exposure at day 14. The stress group showed a signicant reduction in the percentage of BrdU+/DCX+ cells (56 5%; P < 0.01, MannWhitney U test) as compared to controls (71 8%) (Fig. 4). The proportion of CASP-3+ cells was slightly higher, but not statistically signicant, in the control group (18 3%; P = 0.095, MannWhitney U test) as compared to the stress group (11.8 2%). These ndings indicated that stress reduced the number of BrdU+ Type-2 cells, but did not show statistically signicant differences in apoptosis rate. 3.4. Effect of stress on granular neurons in the SGZ Finally, we investigated whether stress by noise modied the survival and apoptosis of granular neurons in the SGZ derived from the BrdU-labeled Type-1 cells at day 4. Animals were sacriced (n = 4 per group) immediately after the noise exposure at day 21. Interestingly, we did not nd statistically signicant differences between the stress group in the number of BrdU+/NeuN+ cells (59 10%; P = 0.34, MannWhitney U test) as compared to controls (53 7%) (Fig. 5). We did not nd statistically signicant differences in the number of CASP-3+ cells between the studied groups: the control group (7.7 1.8%) and the stress group (6.8 2.1%). Taken together, these results suggest that the noise-induced stress reduces the number of primary and intermediate progenitors in the SGZ, but has no signicant effect on mature granular neurons. 4. Discussion Here we show that a 24-h noise exposure to environmental noise induces a signicant increase in CORT serum levels. With this incessant noise exposure as a stress model, we found that: (1) CORT levels remain increased while the noise is present; (2) the proliferation of radial astrocytes in the SGZ of dentate gyrus is reduced

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Fig. 3. Noise effects on Type-1 radial astrocytes. Immunostaining for GFAP and BrdU (A B) and GFAP/CASP-3 (CD) in controls and the stress group. The percentage of BrdU+ cells that co-expressed GFAP was signicant reduced by the effect of 24-h noise exposure as compared to controls. Asterisk indicates statistically signicant differences (P < 0.05; MannWhitney U test). Scale bars in AB = 50 m; in CD = 15 m.

by stress, which is also associated with an increase in apoptosis in these cells; (3) the survival of Type-2 cells is also affected by stress, but the apoptosis rate is opposite to that found in radial astrocytes; and (4) Mature neuron population is not affected by stress and the apoptosis rate persists slightly reduced in the stress group. Taken together, these ndings indicate that chronic exposure to environmental noise induces a persistent increase in CORT levels and produces differential effects on proliferation/apoptosis in hippocampal progenitors. Hippocampus is an important target for detrimental effects of environmental stressors. Stress reduces the glial proliferation in several hippocampal regions, such as CA1, CA3, dentate gyrus and hilus (Tanaka et al., 1997; Wong et al., 2004; Zhe et al., 2008). Moreover, stress promotes apoptosis of pyramidal neurons in CA1 and CA3 (Li et al., 2010; Zhao et al., 2007). Deleterious effects of stress have been related to an increased activity of the hypothalamicpituitary axis (HPA) system, which increases the serum levels of glucocorticoids (Li et al., 2010; Yehuda, 2009). High glucocorticoid levels suppress neurogenesis and induce dendritic remodeling in CA3 pyramidal neurons (Gould et al., 1992). Additionally, glucocorticoids promote glutamate releasing in CA1 hippocampus and prefrontal cortex (Moghaddam et al., 1994; Stein-Behrens et al., 1994; Venero and Borrell, 1999). Non-transcriptional effects mediated by the mineralocorticoid receptor in hippocampus (de Kloet et al., 2008; Karst et al., 2005; Olijslagers et al., 2008), G protein-coupled receptors (Tasker et al., 2006) and high activity of acetylcholinesterase enzyme have also been involved in stressinduced brain disorders (Sembulingam et al., 2003).

Noise is a well-known model to induce stress (Gesi et al., 2001; Kim et al., 2008; Rabat et al., 2004). In this study, we used a validated model in which a rodents audiogram-tted adaptation of noisy human environments (Rabat et al., 2005). This model significantly increases the levels of CORT (Jauregui-Huerta et al., 2010; Rabat et al., 2004; Rabat et al., 2005). The noise-induced elevation of CORT found in our study was similar to that reported in other studies, which has proven to activate the glucocorticoid receptors and modify the hypothalamus-pituitary axis (Manikandan et al., 2006; Masini et al., 2008). Although, both 12-h and 24-h noise exposure activates the HPA, our ndings indicate that a sustained exposure to 24-h noise for 10 days is a potent stressor. This incessant noise did not allow adaptation as shown by continuous high CORT levels. Thus, we preferred the 24-h noise exposure used in this study over the intermittent noise model described in other studies (JaureguiHuerta et al., 2010; Rabat et al., 2004; Rabat et al., 2005). Noise exposure has been used as a model of sleep deprivation. However, noise by itself is ineffective at reducing cell production in the absence of elevated CORT levels (Mirescu et al., 2006). Rather, high CORT levels appear to be necessary for the inhibition of SGZ neurogenesis (Mirescu et al., 2006). Instead, the effects of stress and sleep deprivation on cognitive performance and brain plasticity appear to be mediated by a reduction in BDNF expression (Hansson et al., 2003; Taishi et al., 2001) and FGF-2 mRNA levels (Molteni et al., 2001), and a decrease the expression of matrix metalloproteinase-9 (Taishi et al., 2001). The persistent activation of glucocorticoid receptors mediated by stress can suppress hippocampal cell proliferation (de Kloet

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Fig. 4. Noise effects on Type-2 cells. Immunostaining for doublecortin-expressing neuroblasts and BrdU (AB) and doublecortin/CASP-3 (CD) in controls and the stress group. The percentage of BrdU+ cells that co-expressed doublecortin was reduced by effect of noise as compared to controls. Asterisk indicate statistically signicant differences (P < 0.05; MannWhitney U test). Scale bars in AB = 30 m; in CD = 10 m.

et al., 2008; Wong and Herbert, 2005) and promote apoptosis by up-regulating the pro-apoptotic gene bax (Cardenas et al., 2002) and suppressing Bcl-2 expression (DeVries et al., 2001). Thus, stress can affect the cell proliferation of hippocampal cells, but the cell phenotype more susceptible to these deleterious effects was not well-known. Our ndings indicate that stress by noise affects the cell proliferation of specic neuronal precursors. Type1 radial astrocytes were the most susceptible SGZ precursors to stress, as shown by reduced proliferation and high apoptosis levels. Effects of stress on neuronal precursors in the SGZ have been described in other studies, but this type of correlation with apoptosis had not been previously studied. In fact, the entire number of newly generated neurons in the SGZ represents a combination of the proliferative rate, the differentiation rate, and net cell survival/apoptosis (Aberg et al., 2000; DErcole et al., 2002). Since Type-1 cells that incorporate BrdU may be also suffering early apoptosis, our ndings suggest that the reduction in the number of GFAP+ BrdU+ cells could be, in fact, a consequence of the increase in apoptosis rate in these proliferating cells (i.e., the higher apoptosis the lesser read-out of proliferation). Remarkably, this opposite ratio apoptosis/proliferation was reversed at a later stage of cell maturation as shown by the analysis of Type-2 cells. We found that the number of BrdU+ Type-2 cells in the stress group was lesser than in the controls, but, a low apoptosis rate was also observed in the stress group when compared to controls. Interestingly, the number of BrdU+ granular neurons did not show signicant differences, whereas the apoptosis rate in the stress group remained slightly reduced. In fact, the given NeuN data suggest a promoting effect of noise on neuronal differentiation or a

compensatory mechanism to protect granular neurons from death, see below. Opposite effects on different cell lineage may represent adaptive actions of glucocorticoids as shown in adrenalectomy models (Nichols et al., 2005), where up-regulation of bax gene promotes apoptosis and the bcl-2 induction provides a compensatory mechanism to protect the cells from death in the SGZ (Cardenas et al., 2002). Such compensatory interactions between cell proliferation and apoptosis have also been found in other multipotent cells and experimental models (Chera et al., 2009; Fan and Bergmann, 2008b; Hawkins et al., 2000; Wells et al., 2006). This compensatory mechanism may involve CASP-3 initiators (Dronc, DrICE or Dcp-1) via the p53, Jun N-terminal kinase, Wnt-3 and Hedgehog signaling (Bergantinos et al., 2010; Fan and Bergmann, 2008a). Nevertheless, the knowledge is still limited about the role of apoptosis in regulating adult neurogenesis in the SGZ, but it has been suggested that up-regulation of transforming growth factor-1 (Bye et al., 2001) and insulin-like growth factor-1 (Aberg et al., 2000; DErcole et al., 2002) may protect against apoptosis. Therefore, it is possible that glucocorticoids trigger compensatory mechanisms, which may mitigate detrimental effects of stress on SGZ progenitors (Fig. 6). Nevertheless, further studies that include adrenalectomized animals and exogenous corticosterone would be needed to fully address this process. In summary, the total number of newly generated neurons in the SGZ is a combination of the proliferative rate, the differentiation rate, and the net cell survival/apoptosis. Present ndings indicate that stress by environmental noise reduces the proliferation rate of radial astrocytes and Type D cells. However, these changes do

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Fig. 5. Noise effects on mature granular neurons. Fluorescent double-immunostaining for NeuN and BrdU in controls and the stress group (AB) and NeuN/CASP-3 (CD) in controls and the stress group. No statistically signicant differences were found in the percentage of BrdU+/NeuN+ cells between groups. P = 0.34; MannWhitney U test). Scale bars in AB = 50 m; in CD = 15 m.

Fig. 6. Hypothetical model of a possible compensatory effect to preserve the cell population in the SGZ. Stress promotes apoptosis rate in Type-1 astrocytes but not in Type-2 neuroblasts. Interestingly, the net number of neurons is not affected by changes in proliferation and apoptosis.

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not appear to affect the net number of granular neurons, which suggests a compensatory interaction between cell proliferation and apoptosis. Yet, further studies are necessary to clarify the cellular and molecular mechanisms involved in this possible interaction. Acknowledgements This work was supported by CONACyTs (Ciencia Basica-2008101476). References
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