Acute exposure to predator odor elicits a robust increase in corticosterone and a decrease in activity without altering proliferation in the adult rat hippocampus

Experimental Neurology 201 (2006) 308 – 315 www.elsevier.com/locate/yexnr

Acute exposure to predator odor elicits a robust increase in corticosterone and a decrease in activity without altering proliferation in the adult rat hippocampus Rosanne M. Thomas a , Janice H. Urban b , Daniel A. Peterson a,⁎ a

Neural Repair and Neurogenesis Laboratory, Department of Neuroscience, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA b Department of Physiology and Biophysics, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA Received 16 January 2006; revised 9 April 2006; accepted 12 April 2006 Available online 5 June 2006

Abstract Stress has long been implicated as a major cause of depression in humans and more recently has been suggested to decrease neurogenesis, which may be a contributing factor to depression development. Animal models of stress may be a relevant tool for investigating links between neurogenesis and depression. This has largely been investigated using chronic stress models in rodents. However, stress may be chronic or experienced in discrete episodes. Acute stress may be particularly relevant to humans experiencing unexpected societal pressures and obligations. Our study examined the effect of acute stress on the proliferative phase of adult hippocampal neurogenesis. Young adult rats were exposed for 20 min to the predator odor TMT, a natural stressor for rodents with significant ethological relevance. BrdU IP injections were concurrent with TMT exposure to assess proliferation effects with animal sacrifice 2 h after BrdU injection. Robust stress responses were evident following TMT exposure as detected by elevated corticosterone (CORT) levels and a significant reduction in exploratory behavior. Exposure to TMT did not alter the number of BrdU-positive cells in the hippocampus despite physiological and behavioral evidence of stress. CORT level elevation has long been accepted as a marker of stress; however, this study indicates that increases in CORT level may not always correlate with diminished neurogenic proliferation. This study further suggests that various stressors may not operate through the same biological substrates resulting in a differential ability to modulate neurogenesis. © 2006 Elsevier Inc. All rights reserved. Keywords: Neurogenesis; Stress; Depression; TMT; Dentate gyrus; Cell cycle

Introduction Modulation of neurogenesis in the dentate gyrus of the adult hippocampus through environmental factors has been reported in a number of experimental models, yet the clinical and functional relevance of these new cells remains elusive (Kempermann et al., 2000; Scharff, 2000). The range of environmental modulation of neurogenesis may reflect the organisms' ability to adapt to novel or changing conditions (Peterson, 2002; Duman et al., 1999, 2000, 2001). While some stimuli increase neurogenesis, other environmental events may lead to a negative adaptive response by reducing neurogenesis (Kempermann, 2002; Schaffer and Gage, 2004). ⁎ Corresponding author. Fax: +1 847 578 8545. E-mail address: [email protected] (D.A. Peterson). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.04.010

Stress has been shown to reduce hippocampal neurogenesis (Gould et al., 1997; Gould and Tanapat, 1999; Santarelli et al., 2003). The hippocampus is sensitive to the environment as evidenced by experience-dependent structural changes and thus is vulnerable to stress-related adverse adaptive modification (Kessler, 1997; McEwen, 1999, 2000). These structural changes in the hippocampus include dendritic atrophy, decreased synaptic plasticity, cell death and diminished neurogenesis (Gould and Tanapat, 1999; Rajkowska, 2000; Duman et al., 2000; Jacobs et al., 2000; D'Sa and Duman, 2002). Depression appears to be a stress-related disorder and evidence for depression-associated structural changes in the hippocampus of adult humans appears similar to that found in animals (Sapolsky, 2001, 2004). In fact, stress has been implicated as a major cause of depression in humans (Garcia, 2002). Recent

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evidence suggests that reduction of neurogenesis in the hippocampus may be a biological substrate of depression (Eriksson and Wallin, 2004; Jacobs et al., 2000; Sheline, 2000; Benninghoff et al., 2002; Gage, 2000; Kempermann et al., 2000). Therefore, animal models of stress may be a relevant tool for investigating links between neurogenesis and depression. Predator odor is a stressor of particular relevance to rodents in their natural setting, as an appropriate response may ensure their survival (Blanchard and Blanchard, 2003). Predator odors elicit responses based on their perceived threat to the rodent rather than in response to a physical stimulation (Blanchard et al., 2001, 2003; Day et al., 2004). These responses appear to be innate, appearing in both pups and in adults exposed for the first time (Wallace and Rosen, 2000). The compound 2,5-dihydro-2,4,5-trimethyl thiazoline (TMT), a sulfur-containing odor isolated from fox feces, has been used to induce stress and produce behavioral responses in both laboratory and wild rodents and thus does not require any conditioning or learning to elicit a response (Soares et al., 2003; Morrow et al., 2000; Wallace and Rosen, 2000, 2001). TMT produces reliable fearful responses such as freezing, decreased exploratory behavior and diminished grooming behavior. It also consistently activates the hypothalamic–pituitary–adrenal (HPA) axis resulting in increased serum corticosterone (CORT) levels (Tanapat et al., 2001; Soares et al., 2003). There are relatively few investigations on the impact of an acute episode of naturally occurring stress on neurogenesis with most studies focusing on chronic stressor effects (Malberg and Duman, 2003; Czeh et al., 2002, 2001). The framework for the stress-based hypothesis of depression has been based on experiments involving chronic stress (Thomas and Peterson, 2003; Kempermann and Kronenberg, 2003). Acute stressors, however, are particularly relevant to human experiences with unexpected societal pressures and obligations. As is the case with predator odors with rats, many human stressful experiences may be episodic and acute in nature. To assess the acute effect of TMT on neurogenic proliferation in the adult rat dentate gyrus, we administered the predator odor TMT and evaluated behavioral responses, activation of the HPA axis and modulation of the proliferation phase of neurogenesis. To investigate the effect of TMT on proliferation separate from survival or differentiation of newly generated cells, animals were simultaneously exposed to BrdU and TMT to examine the effect of TMT on DNA replication occurring at the time of BrdU introduction and sacrificed two hours later. Analysis of cellular proliferation in the hippocampus 2 h following TMT exposure by design-based stereology revealed that proliferation was not altered despite elevation of other indicators of physiological and psychological stress. These results are discussed in the context of possible variability of stress effect on HPA axis activation that may not involve hippocampal circuitry and thus not influence proliferation in the adult dentate gyrus. Methods Animals All animal use followed the NIH Guide for the Care and Use of Laboratory Animals and was approved by the Institutional

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Animal Care and Use Committee (IACUC) of Rosalind Franklin University of Medicine and Science. Animals were housed in a colony room maintained on a 12-h light/dark cycle with constant temperature (21–23°C) and humidity (40–50%). Food and water were freely available. The subjects of this study were naive 6 to 9-week-old adult male Sprague–Dawley rats weighing 200 to 250 g, housed in groups of three. TMT protocol Animals (n = 12) were assigned randomly to one of two groups by cage and all three animals from one cage were assigned to the same test group to avoid exposure of control animals to lingering odor on experimental animals. Prior to odorant exposure, animals were removed from the colony room and placed in a detached, isolated room. Animals were allowed to acclimate to their new surroundings overnight. Group 1 (n = 6) was exposed to TMT with concurrent BrdU (50 mg/kg) intraperitoneal (IP) injection, 2 h later the animals were perfused and sacrificed. Group 2 (n = 6) received exposure to double distilled water (ddH2O) with concurrent BrdU IP injection, and was sacrificed by perfusion 2 h later. The 2-h timeframe for sacrifice after BrdU injection was chosen as this has been reported as the amount of time required for DNA replication to occur (Cameron and McKay, 2001). The testing chamber was a standard rat cage with a thin layer of corncob bedding. Chambers were cleaned with 409™ disinfectant spray, rinsed with 70% ethanol and dried completely between each animal. One day before the test session, rats were habituated to the chamber for 20 min with a dry filter paper disk in a Petri dish in the middle of the chamber. For experimental groups, a large dose of TMT (150 μl, undiluted, Phero Tech Inc., Delta, Canada) was used to assure maximal stress response and to allow comparison with a previous study (Tanapat et al., 2001). A Petri dish with filter paper soaked with TMT or ddH2O was placed in the center of the chamber floor. The chamber was marked into thirds with the middle third containing the Petri dish. The time between placement of TMT or ddH2O in the Petri dish and individual rat introduction to the cage was less than 30 s. Rats remained in the chamber for 20 min and were videotaped for assessment of stressrelated behaviors. Control animal testing was done prior to experimental animals to prevent control animals from being exposed to TMT. Experimental animals were returned to their home cage and remained in the isolation room for the 2 h prior to sacrifice to prevent odor dissipation into the animal colonies. Behavioral scoring Four exploratory behaviors were scored and summated at 5min intervals for the 20 min spent in the experimental chamber. The four behaviors quantified were: (1) number of times the animal crossed the middle of the cage, (2) number of times the animal reared with weight bearing on the two hind legs with or without upper extremity weight bearing on the side wall of the cage, (3) number of times the animal touched the Petri dish that contained the odorant, and (4) number of grooming episodes (licking fur on paws or body). Once the animal engaged in any other behavior, that grooming episode ended. Freezing episodes

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were not quantified directly; however, a reduction in exploratory behavior such as crossing, rearing and touching was indirectly indicative of freezing behavior. Likewise, a reduction in cage crossing or touching the Petri dish positioned in the center of the cage was indicative of anxiety as the animal spent most time along the perimeter of the testing chamber. These behavioral parameters were patterned from the work of Blanchard and Blanchard (2003) and others (Blanchard et al., 2001; Williams, 1999) and are recognized to be diminished with exposure to stress. Determination of corticosterone (CORT) levels Blood samples for CORT determination were obtained from all TMT exposed rats and control rats prior to return to their home cage. The process of tail clip and blood collection took less than 2 min per rat. Each rat was restrained in a clear plastic tube with a diameter narrow enough to prevent the animal from turning around. Tails were exposed outside of the restraining tube, tail clip was performed on the distal 0.5 mm of tail, 0.5 cm3 of blood collected in a small heparinized vial, the tail was sprayed with antiseptic spray and the animal was returned to its home cage. Blood was kept on ice briefly until centrifuged at 1500 rpm × 15 min at −20°C to allow extraction of plasma. Plasma was kept at −80°C until analysis. CORT levels were determined using the Immuno-Chem Double Antibody Corticosterone- 3H Kit for rats and mice (MP Biomedicals, Costa Mesa, CA) as described previously (Meerlo et al., 2002). BrdU labeling Rats received an injection of 5-bromo-2′ deoxyuridine (BrdU: Sigma, St. Louis, MO) i.p. (50 mg/kg) immediately prior to placement in the testing chamber. This single injection labeled a cohort of cells that were undergoing cell cycle S-phase at the time the label was present. BrdU availability was timed to coincide with experimental stress of TMT to measure its impact on cell proliferation only. Histology Two hours after exposure to the experimental paradigm, animals were deeply anesthetized and transcardially perfused for 5 min with ice cold saline followed by ice cold 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. Following perfusion, brains were removed, stored in 4% paraformaldehyde for 24 h then equilibrated with 30% sucrose in 0.1 M phosphate buffer. Serial, 50 μm thick sagittal sections were produced by freezing microtomy and stored in cryoprotectant in 0.1 M phosphate buffer. A 1-in-6 series of sections were taken for analysis. Prior to immunostaining, sections were pretreated to enable nuclear BrdU staining. Tissue was rinsed three times in Tris Buffered Saline (TBS), incubated in formamide/sodium chloride sodium citrate dihydrate (SSC) solution in a water bath at 65°C for 2 h, rinsed in 2× SSC solution, incubated in 2 N hydrochloric acid (HCl) in a water bath at 37°C for 30 min and

then rinsed in 0.1 M borate buffer. Six final rinses were done in TBS at room temperature prior to further tissue processing. For immunoperoxidase detection of cells incorporating BrdU, sections were rinsed in TBS followed by 10 min incubation with 30% hydrogen peroxide (H2O2) to inactivate endogenous peroxides. Following six additional TBS rinses, sections were blocked with 5% donkey serum, permeabilized with 0.25% detergent (TX-100) in TBS for 3 h and then incubated with anti-rat BrdU (1:500; Accurate Chemical and Scientific Corp., Westbury, NY) for 72 h. Following additional rinses and blocking, sections were incubated with a biotinylated donkey anti-rat (1:500; Jackson, Immunoresearch West Grove, PA) for 1 h. Sections were subsequently rinsed and incubated in avidin–biotin solution (ABC/Elite Vectastain kit; Vector Labs, Burlingame, CA) for 1 h. Reaction with 3,3′ diaminobenzidine (DAB: Sigma, St. Louis, MO) solution was carried out for 4 min followed by several TBS rinses. Sections were mounted on slides, dried overnight and dipped in CitriSolv three to five times before being coverslipped in ProTexx (Lerner Labs, Pittsburgh, PA) to maintain maximal section thickness for subsequent stereological analysis. Stereological quantification of BrdU labeling Estimation of BrdU-positive cell number was done using the optical fractionator procedure for design-based stereology (West et al., 1991; Peterson, 1999). Using a 1-in-6 series, approximately 14 equispaced (300 μm) sections were sampled along the entire length of the hippocampus in the left hemisphere of each brain. The dentate gyrus of the hippocampus was outlined using a 10× objective using an Olympus BX51. A systematic sample of the area within the dentate gyrus was made from a random starting point using StereoInvestigator software (MicroBrightField, Inc. Williston VT). Counting of BrdU-positive cells was made at predetermined intervals (x = 100 μm, y = 100 μm) with a counting frame (75 μm × 75 μm) superimposed on the tissue section image. Sections with BrdU-positive cells within the counting frame were imaged using a 60× oil immersion objective with a numerical aperture (NA) of 1.4. Section thickness was measured directly at each site with the optical disector counting frame height (h = 22 μm) set at approximately 75% of mean section thickness. This procedure sampled approximately 1/15th of the total hippocampal dentate gyrus in the hemisphere examined. Statistical analysis Within and between group behavioral differences were analyzed using repeated measures analysis of variance (RMANOVA) to determine if there was an overall significant effect of TMT on counts of four behaviors at four different time points for both the control and the experimental group. GB-STAT (Dynamic Microsystems, Inc. Silver Spring, MD) was utilized for all statistical analyses. A Bonferroni post hoc test was used to evaluate multiple comparison differences when there was a significant RM-ANOVA. Parametric data (cell counts, CORT levels) were analyzed using unpaired t tests to detect differences between control and experimental groups using group mean differences. Results were expressed as the mean ± the standard

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Fig. 1. Behavior was reduced in subjects exposed to TMT. Four measures of activity and exploratory behavior were scored and summated at 5-min intervals to quantify behavioral response to ddH2O or TMT over the 20-min session. (A) Quantification of the number of times the subject crossed the center of the cage containing the treated filter paper showed that there was a highly significant reduction in exploratory behavior within the first 5 min interval when exposed to TMT, and crossing was essentially eliminated in the last half of the session. (B) The number of rearing episodes was also reduced in animals exposed to TMT. This reduction was highly significant relative to control animals and, again, was rarely observed in the last half of the session. (C) Quantification of grooming episodes likewise revealed a significant reduction when exposed to TMT compared to control subjects. (D) Exploratory behavior quantified by number of filter paper touches showed a striking and significant reduction in the TMT-exposed condition, with only one occasion when the TMT-soaked filter paper was touched. Significance between control and experimental conditions indicated by * for P ≤ 0.05 and ** for P ≤ 0.01.

error of the mean (SEM). Pearson r correlation coefficient was calculated to determine if a correlation existed between CORT levels and BrdU-positive cell counts. Significance for all statistical analysis was accepted at a level of P ≤ 0.05. Results Activity was reduced with TMT exposure All animals, TMT exposed and control, exhibited reduced behavior over time with lower scores at the 20-min time point than the 5-min time point. However, robust, definitive behavioral differences were noted between control and TMT exposed animals (Figs. 1A–D graphs). All behaviors, i.e. crossing the cage, rearing, grooming and touching the Petri dish containing the odorant in the middle of the cage, were significantly less in TMTexposed animals than control animals for all time points except for the 5-min and 15-min grooming scores which did not achieve significance. Thus, the TMT-exposed animals exhibited signs indicative of stress with significantly less exploratory behavior than control animals.

Corticosterone levels were elevated with TMT exposure Elevation of plasma corticosterone is generally taken as one indication of a biological response to stress. Mean plasma CORT levels were analyzed in TMT exposed and control animals at the time of removal from the experimental cage (Fig. 2). Plasma CORT levels were significantly higher in TMT-treated animals, [288.25 ± 51.30 ng/ml as compared to control animals with levels of 23.25 ± 8.30 ng/ml (P ≤ 0.01)]. Therefore, TMTexposed animals exhibited levels of CORT indicative of stress. TMT stress exposure at the time of BrdU exposure did not change proliferation of newly generated cells Animals in both groups were examined for newly generated or proliferating cells at the time of exposure to TMT stress. BrdUpositive cells could be detected in the subgranular zone of the dentate gyrus of the hippocampus in both groups (Fig. 3). Exposure to TMT did not decrease the number of proliferating cells in the dentate gyrus. Stereological estimates were performed to estimate the number of newly generated BrdU-positive cells.

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Fig. 2. TMT exposure significantly increased corticosterone levels. Measurement of corticosterone (CORT) levels at the end of the 20 min session revealed that exposure to TMT produced a 12-fold increase in CORT relative to animals exposed to ddH2O (**P ≤ 0.01).

Fig. 4. Stereological quantification of BrdU-positive cells confirmed that exposure to TMT did not alter neurogenic proliferation in the hippocampal dentate gyrus. Systematic sampling throughout the entire hippocampus by design-based stereology confirmed the observation that despite the behavioral and physiological response to TMT exposure, there was no change in the proliferation of cells within this neurogenic region relative to control animals.

Stereological estimates of newly generated cells in the dentate gyrus and subgranular zone of TMT exposed animals were 2087 ± 35 compared to control animals at 2217 ± 237 (P = 0.62). There was no statistical difference in the proliferation rate between these two groups (Fig. 4). Additionally, the Pearson r correlational coefficient value between CORT levels and number of BrdU-positive cells was 0.02, indicating no correlation between CORT levels and number of newly generated cells (P N 0.05). To further substantiate our conclusion, we assessed Ki67 expressing populations relative to BrdU-positive cells. Ki67

is expressed in a wider population of cells at late G1, S, M and G2 phases of cell cycle. We found no difference in the relative number of Ki67-positive cells between control and TMT-exposed animals (data not shown). Discussion This study investigated modulation of neurogenesis by examining the effect of TMT, an acute environmental stressor, on proliferation of new, undifferentiated cells in the dentate gyrus

Fig. 3. Histological detection of BrdU administered during TMT exposure showed no evidence of a change in proliferation. Despite the robust alteration in behavior and CORT levels, examination of BrdU-positive cells (arrows) throughout the dentate gyrus showed no apparent difference in the prevalence of proliferation between animals exposed to ddH2O and TMT in the initial 2 h following exposure. Scale bar = 100 μm for all panels.

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of the adult hippocampus. The present study used a large dose of TMT (150 μl) and achieved a robust stress response. Indeed, animals in our study exhibited highly significant behavioral changes during TMT exposure demonstrating a strong reduction in activity (Fig. 1). For example, 92% of the TMT-exposed animals did not venture near enough to the center of the cage to touch the Petri dish containing the odorant in contrast to control animals, all of which touched the Petri dish for a mean total of 17 times (±5). These behavioral changes were highly significant for three of the four behaviors (crossing, rearing, and touching) immediately upon odorant introduction and remained so throughout the testing session. For example, significantly diminished cage crossing and rearing (P = 0.0045 and 0.0084, respectively) were indicative of a highly significant decrease in exploratory behavior and an increase in freezing. The efficacy of TMT as a predator stressor was further demonstrated by a robust and significant increase in CORT levels (Fig. 2). The novel finding of this study is that the use of TMT as an ethologically relevant stressor did not alter the proliferation rate of new cells in the dentate gyrus of adult rats despite generating appropriate responses to stress including elevated CORT levels and reduced behavior measurements (Fig. 4). These findings suggest that TMT elicited a stress response in rats without a correlational decrease in neurogenic proliferation as has been reported previously (Tanapat et al., 2001; Holmes and Galea, 2002; Falconer and Galea, 2003). Using the same dosage of TMT (150 μl), Tanapat et al. (2001) reported that exposure to TMT decreased the number of proliferating cells in the dentate gyrus at 2 h and that the effect endured up to 1 week after an acute, 1 h exposure. These authors suggested that the decrease in proliferation was mediated by adrenal hormones, since adrenalectomized animals did not show a similar stress-induced reduction in proliferating cells. Similarly, Holmes and Galea (2002) reported that a 30 min exposure to 250 μl of TMT, a higher dosage than in our study, resulted in diminished hippocampal cell proliferation. One interpretation of our result differences may be that there is a threshold dose or duration of TMT exposure required to affect proliferation. We chose a 20 min duration for TMT exposure as longer exposures resulted in the absence of behavioral activity in TMT-exposed animals (data not shown). Furthermore, this duration was consistent with the usual time interval examined in a wide range of previous studies (Blanchard et al., 2003; Day et al., 2004; Dielenberg and McGregor, 2001; Hotsenpiller and Williams, 1997). Likewise, the 150 μl dose of TMT was a high dose that elicited very robust behavioral and CORT changes. This dose was likely appropriate to saturate exposure to TMT as any delivery of TMT above 9.7 μl (150 μM) has produced the maximum number of volatile TMT molecules in the air (Day et al., 2004). Thus, despite minor procedural differences, our results clearly indicated that biological and behavioral stress responses were achieved under our conditions without changes in proliferation. Neurogenesis consists of several distinct phases progressing from cell proliferation through final lineage commitment. Recently, distinction between separate, specific neurogenic events through carefully timed BrdU injections has refined the definition of neurogenesis to distinguish between proliferation and im-

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mediate survival of the newly generated cells (Thomas and Peterson, 2003; Prickaerts et al., 2004). Neurogenic phase timing is specific and sequential. The process of cell division or proliferation results in the birth of a cell that is undifferentiated, with one round of cell cycle S-phase taking approximately 2 h in rats (Cameron and McKay, 2001). Animal sacrifice 24 h after BrdUlabeling allows assessment of early survival of newly generated cells but does not detect the initial proliferative event. Thus, earlier reports using a 24 h time point may describe assessment of proliferation when the event actually examined was early survival (Falconer and Galea, 2003). We examined direct effects of TMT exposure by use of a 2 h duration, which is within the profile of elevated CORT levels following acute stressors (Djordjevic et al., 2003). By examining the 2 h time point, our results indicate that despite the immediate robust biological and behavioral response to TMT, there was no simultaneous alteration of neurogenic proliferation. It is unlikely that our blood collection procedures for CORT analysis exerted a measurable effect on proliferation as other ongoing studies showed no change in neurogenic proliferation between animals with or without blood collection (data not shown). TMT is known to be a natural stressor for rodents with significant ethological relevance (Blanchard and Blanchard, 2003), but our results suggest that it may be an inappropriate stressor for modulation of hippocampal neurogenesis. Apart from the few studies discussed above, there are no other reports of TMT as a natural stressor that directly influences hippocampal cell proliferation. That we found no change in proliferation with TMT exposure is not surprising in light of other studies showing variable responses to TMT using a wide range of physiological and behavioral paradigms (Blanchard and Blanchard, 1989, 2003; Blanchard et al., 2003; Day et al., 2004; Dielenberg and McGregor, 2001). The conditions under which TMT exposure has been presented and results obtained appear to vary widely and may contribute to discrepancies in the outcomes of these studies (Vernet-Maury et al., 1984, 1992; Hotsenpiller and Williams, 1997; Soares et al., 2003). TMT activation of the CNS Stimuli, such as stressors, may not equally evoke activation of CNS pathways. For example, in situ hybridization has been used to determine TMT-activated brain areas by analyzing c-fos mRNA induction as a measure of neural activity. Several studies found elevated c-fos mRNA levels after TMT exposure in various brain structures; namely the amygdala, oval nucleus of the bed nucleus of the stria terminalis and the lateral parabrachial nucleus and medial prefrontal cortex (Day et al., 2004; Morrow et al., 2000; Figueiredo et al., 2003). However, these studies found no response to TMT exposure within the hippocampus. Furthermore, there was no increase in 5-HT metabolism into 5-HIAA in the hippocampus with TMT exposure, which supports a lack of hippocampal involvement in the response to TMT (Soares et al., 2003). These findings raise the possibility that stress evoked by TMT exposure may not involve the hippocampus at all and thus no neurogenic changes would necessarily be observed there.

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CORT levels and neurogenesis Elevation of serum CORT levels has long been accepted as an indication of stress. There is evidence that modulating CORT levels alters some aspects of neurogenesis with or without concomitant stress (Cameron and Gould, 1994; Cameron et al., 1998; Wong and Herbert, 2004). TMT exposure has consistently activated the HPA axis with increased CORT levels, as found in our study. Yet, several studies report that changes in CORT levels may not be entirely predictive of the behavioral measure of stress, but may reflect increases in exploration or arousal. Both TMT and neutral odors have caused similar CORT elevations with or without changes in exploratory behavior (Soares et al., 2003; Morrow et al., 2000; Perrot-Sinal et al., 1999). Thus, it has even been suggested that elevated serum CORT levels do not have a direct correlation to the animal's “stressed” state (Figueiredo et al., 2003). An alternative interpretation is that elevated CORT levels do not negatively regulate proliferation. Our finding of no correlation between CORT levels and BrdU-positive cells supports previous studies investigating the relationship between neurogenic proliferation and CORT levels. For example, artificial stressors such as foot shock have also elicited significantly elevated CORT levels without an effect on neurogenic proliferation (Van der Borght et al., 2005). Direct manipulation of CORT levels has also been recently reported to decrease early cell survival of newly generated cells apart from the proliferative event (Wong and Herbert, 2004). It has further been shown that adult animals adrenalectomized at postnatal day 10 and given chronically low levels of glucocorticoid oral supplementation showed the same rate of neurogenesis at 3 and 12 months old as control animals with higher CORT levels (Brunson et al., 2005). Our results demonstrate that acute TMT exposure elicits robust behavioral and endocrine changes but may not be appropriate as a model to investigate proliferation modulation in the hippocampus of adult rats. Furthermore, it supports the view that elevated CORT levels do not generically suppress neurogenic proliferation. These results argue for caution in the use of CORT levels as the sole measure of stress in studies of neurogenesis. Additionally, while stressed states may modulate neurogenesis, not all types of stressors are equally valid for investigating the relationship between stress, depression and hippocampal neurogenesis. Acknowledgments We thank Dr. Sarah Garber for her critical review of the manuscript and Dr. Tara Teppen for assistance with processing CORT assays. This work supported, in part, by NIH AG020047 to DAP. References Benninghoff, J., Schmitt, A., Mossner, R., Lesch, K.P., 2002. When cells become depressed: focus on neural stem cells in novel treatment strategies against depression. J. Neural Transm. 109, 947–962.

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