Cent. Eur. J. Biol. • 5(6) • 2010 • 757-764 DOI: 10.2478/s11535-010-0062-9

Central European Journal of Biology

Early stress affects neurogenesis in the rat rostral migratory stream

Research Article Kamila Lievajová1,*, Marcela Martončíková1, Juraj Blaško1, Judita Orendáčová1, Viera Almašiová2, Enikő Račeková1

1Institute of Neurobiology, Slovak Academy of Sciences, 04001 Košice, Slovak Republic 2University of Veterinary Medicine, 04181 Košice, Slovak Republic Received 17 March 2010; Accepted 01 June 2010

Abstract: Stressful experience during the early postnatal period may influence processes associated with neurogenesis (i.e. proliferation, cell death, appearance of astrocytes or cell differentiation) in the neonatal rat rostral migratory stream (RMS). To induce stress, pups were subjected to maternal deprivation daily for three hours, starting from the first postnatal day till the seventh postnatal day. Immunohistochemical methods were used to visualize proliferating cells and astrocytes; dying cells and nitrergic cells were visualized using histochemical staining. Quantitative analysis showed that maternal deprivation decreased the number of proliferating cells and significantly increased the number of dying cells in the RMS. Maternal deprivation did not influence the appearance of astrocytes in the RMS, but caused premature differentiation of nitrergic cells. In control rats, nitrergic cells can be observed in the RMS as early as the tenth postnatal day. In maternally deprived pups, these cells were detected as early as the seventh postnatal day. The observed earlier appearance of nitrergic cells in the RMS was associated with altered proliferation and increased cell dying and this observation supports the hypothesis that nitric oxide has an anti-proliferative role in the RMS. Our study demonstrates that maternal deprivation represents a stressful condition with a profound impact on early postnatal neurogenesis.

Keywords: Rat • Rostral migratory stream • Maternal deprivation • Neurogenesis © Versita Sp. z o.o.

1. Introduction Recent studies indicate that the mechanisms promoting neurogenesis in the RMS of neonatal and In mammals, neurogenesis occurs mainly during adult animals may be fundamentally different. During the embryonic and early postnatal stages. However, first three postnatal weeks, remarkable changes in the in the adult mammalian brain there are at least morphology, cellular composition, proliferation activity, two areas that generate new neurons throughout apoptosis and the pattern of the RMS cell migration are life: the olfactory bulb (OB) and the dentate gyrus evident. For instance, the prenatal and early postnatal of the hippocampus [1,2]. The germinal zone SVZ contains an open olfactory ventricle, which closes responsible for neurogenesis in the OB is the after birth in rodents, giving rise to the RMS [6]. The subventricular zone (SVZ), which lies adjacent to RMS constitutes the ‘‘highway’’ for neuronal progenitors the lateral wall of the telencephalic ventricle. SVZ- en route to the OB. In adult rats, the ventricular cavity derived precursors migrate tangentially along the is mentioned only as vestigial [7] or virtual olfactory rostral migratory stream (RMS) to the OB, where ventricle [8]. The early postnatal RMS is U-shaped they switch from tangential to radial migration and because the elbow between the vertical and horizontal differentiate into local interneurons within the granule arms is not as sharp compared to later postnatal stages. and periglomerular layers [3,4]. Interneurons of the The typical „L“-shaped column, characteristic of the granular and periglomerular layers modulate the adult rat RMS, develops in two week-old rats [9]. activity of major output neurons in the OB and play The main differences between neonatal and adult essential roles in olfactory information processing [5]. SVZ/RMS are associated with cell migration and

* E-mail: [email protected] 757 Early stress affects neurogenesis in the rat rostral migratory stream

organization of glial cells. In the early postnatal period, deprivation, during the critical period of the first postnatal the radial glial cells within the SVZ, identified as the week affects proliferation of cells, cell death, appearance embryonic neural stem cells, give rise to astrocytes, of astrocytes or cell differentiation in the rat RMS. located at the borders of the SVZ, oligodendrocytes and olfactory interneurons [10-12]. During the first postnatal week, although a great number of small glial processes 2. Experimental Procedures are detectable in the SVZ, the migrating neuroblasts form large, uniform and widely intercommunicating 2.1 Maternal Deprivation masses. Most of these cells are in contact with one Wistar albino rats were used in this study. Experimental another, and only occasional interposition with glial protocols were approved by the Institutional Ethical processes can be observed. This arrangement is very Committee, in accordance with current Slovak Republic different from the typical chain organization of adult legislation. SVZ, in which large portions of neuroblast cell surface To induce stress in neonatal rats, we used a well- regularly forms contact with astrocytes [13,14]. It described model of maternal deprivation. Rat pups from has been demonstrated that in the newborn animals, postnatal day 1 (P1) to P7 were separated from the neuronal progenitors travel from the SVZ to the OB dam daily for 180 min. Dams were first removed and along the RMS in absence of the astrocytic tube [15]. placed in an adjacent cage. Litters were then transferred Another notable difference between early and late to a plastic container and placed in an incubator at the postnatal neurogenesis seems to be related to their temperature consistent with nest measurements (34°C). distinct function in the OB network. Early postnatal After the separation period, the pups were returned to neurogenesis, considered from postnatal day 3 to their home cage, where they were reunited with the dam. postnatal day 7, endows the neonate bulbar circuit Control rats were reared under the same conditions with a population of newborn granule cells that differs except maternal deprivation. morphologically and functionally from those produced during the adulthood [16]. Some studies havefocused on 2.2 Bromodeoxyuridine adiministration and finding the agents responsible for differences between tissue processing early and late postnatal neurogenesis. It was discovered On the last experimental day, maternally deprived that molecules expressed within the RMS can directly animals (n=15) and control animals (n=15) of the affect the processes of neurogenesis. Recent studies same age (P7) were intraperitoneally injected with suggest that nitric oxide (NO), a versatile diffusible bromodeoxyuridine (BrdU; 50 mg/kg body weight; signaling molecule, produced by nitrergic cells, has Fluka) in order to label dividing cells. Two hours after significant regulatory action on adult neurogenesis BrdU administration the rats were deeply anesthetized [17]. Morphological examination from our laboratory with a mixture of xylazine and ketamine and perfused has revealed that a number of RMS cells contain the transcardially with a solution of 4% paraformaldehyde in neuronal isoform of NO synthase (nNOS) [18]. We 0.1 M phosphate buffer (PB). Brains in the skulls were have also shown that the presence of NO producing fixed in the skulls overnight. The following day, brains cells within the RMS is evident after the first postnatal were removed and postfixed overnight in the same week [19]. Most recently it has been confirmed that the fixative and transferred into cryoprotective solution: regulatory action of NO on neurogenesis is acquired 30% sucrose in 0.1 M phosphate-buffered saline after the seventh postnatal day. This is most likely a (PBS). 40 mm thick sagittal serial sections were cut on consequence of cytoarchitectonic changes that take the cryostat and stored in dishes with 0.1 M PBS. The place, leading to adult SVZ organization [20]. In addition sections were processed for immunohistochemical and to the RMS, the expression of nNOS is developmentally histochemical labeling. regulated in the olfactory system. During the first two weeks of age, nNOS expression disappears in the 2.3 BrdU immunohistochemistry olfactory receptor neurons and progressively increases In order to identify proliferating cells, sections from the in periglomerular cells in the OB [21]. right hemisphere of both control (n=6) and maternally Experimental results from studies examining deprived rats (n=6) were washed in 0.1 M PBS, treated neurogenesis during early postnatal stage suggest that with 2 N HCl at 60°C for 30 min to fragment DNA, and the most important changes in neonatal neurogenesis then neutralized in 0.1 M borate buffer (pH 8.4). The coincide with the end of the first postnatal week. Thus, sections were incubated for 30 min in 20% methanol

the objective of the present study was to investigate containing 0.3% H2O2 to suppress endogenous whether stressful experience, such as maternal peroxidase activity. After blocking in normal serum

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the sections were exposed to 1:500 diluted BrdU mounted on gelatin slides, air-dried overnight, cleared (Accurate, NY) primary monoclonal antibody overnight with xylene and cover-slipped with Entellan. at room temperature. Sections were then incubated with biotinylated goat anti-rat IgG (dilution 1:200), the 2.7 Quantitative analyses secondary antibody, and AB complex (Vector, CA). For The number of BrdU, FJ-C and NADPH-d-positive visualization of BrdU-immunoreactive cells, DAB was cells in the RMS of experimental and control rats was used as a sensitive chromogen. examined. Only sections in which the whole RMS was visible were evaluated (eight sections for each animal). 2.4 Fluoro-Jade C histochemistry The microphotographs of individual sections were taken To detect dying cells, we used a fluorochrome, Fluoro- using a light microscope (Olympus BX51) with computer Jade C (FJ C), which has been confirmed to detect dying support (camera system DP50). neurons regardless of the cause of cell death. Sections, Proliferating cells were counted in three specific obtained from the right hemisphere of control (n=6) and parts of the RMS (the vertical arm, the elbow and maternally deprived rats (n=6), were mounted on 2% the horizontal arm) using the method for unbiased gelatin coated slides and air dried at 50°C for 30 min. estimation of cell density (Disector software, version The slides were then immersed in absolute alcohol 2.0) [23]. The outcomes were expressed as the number for 3 min, 70% alcohol for 1 min and 1 min in distilled of BrdU-positive cells per mm3 (Figure 1). water. Slides were transferred to a 0.06% potassium permanganate solution for 12 min and washed in distilled water for 2 min. The staining solution was prepared from 0.01% FJ-C stock solution made by adding 10 mg of dye powder to 100 ml of staining solution. 1 ml of stock solution was added to 99 ml of 0.1% acetic acid vehicle. After 1 hour in the staining solution, the slides were rinsed for 1 min in distilled water. Three consecutive washes were performed. The dried slides were cleared in xylen and cover-slipped with DPX. 2.5 Glial fibrillary acidic protein immunohistochemistry Figure 1. Schematic drawing of the P7 rat forebrain. Dashed lines To recognize astrocytes in the RMS, demarcate individual parts of the RMS: (I) the vertical immunohostochemical staining for glial fibrillary acidic arm; (II) the elbow; (III) the horizontal arm. LV - lateral ventricle; OB - olfactory bulb; RMS - rostral migratory protein (GFAP) was used. Sections from the control stream; SVZ - subventricular zone. (n=3) and experimental rats (n=3) were rinsed three times for 10 min in 0.1 M PBS , blocked with 5% normal To evaluate the number of dying cells we used the goat serum at the room temperature and then incubated Olympus Reflected Fluorescence system U-RFL-T, the overnight at 4°C with primary antibody mouse anti-GFAP Olympus BX51 and the digital camera DP50. FJ-C and (1:500; ICN Biomedicals Inc.). The binding of anti-GFAP NADPH-d-positive cells were counted with supporting antibody was detected by fluorescein-conjugated goat UTHSCSA Image Tool version 3.0 (University of Texas, anti-mouse IgGf (1:200; Jackson ImmunoResearch) for USA). The number of labeled cells was expressed as 2 h at room temperature. After thorough rinsing in PBS, number of cells per section. the sections were cover-slipped with Fluoormont and Differences in the number of proliferating cells or visualized under a fluorescence microscope. dying cells between control and experimental animals were analyzed by the one-way ANOVA test, Tukey- 2.6 NADPH-diaphorase histochemistry Kramer and Student t- tests. The values were expressed NADPH-diaphorase (NADPH-d) reaction was performed as means ± S.E.M. by incubating the free-floating sections, obtained from the left hemisphere of both control and maternally deprived rats (n=6 in both groups), in 0.1 M PB solution 3. Results (pH 7.4) containing 0.4 mg/ml Nitroblue tetrazolium, 0.3% Triton X-100, 5 mg/ml malic acid, 4 mg/ml 3.1 Cell proliferation magnesium chloride and 0.8 mg/ml NADPH-d at 37°C Our immunohistochemical findings showed that for 1 h [22]. The sections were then rinsed in 0.1 M PB, repeated maternal deprivation (7 days for 3 hours

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daily) caused considerable changes in the number of most distinct in the RMS caudal part, specifically in proliferating cells within the RMS. Previously we have the vertical arm. In this part of the migratory stream, demonstrated that there are physiological changes the number of proliferating cells is the highest under in mitotic activity in the RMS during the first postnatal physiological conditions (Figure 3). month. The lowest number of proliferating cells has been noted at day P7 [9]. In maternally deprived rats of 3.2 Cell death the same age, the low density of dividing cells was even Maternal deprivation induced striking changes also in more striking (Figure 2). the amount of dying cells (Figure 4). In order to label Taking into account the high density of proliferating dying cells we used a fluorochrome, Fluoro-Jade C, cells and the length of the migratory pathway (several which detects dying neurons regardless of the cause millimeters), we counted the labeled cells in three of cell death [24,25]. One week of maternal deprivation successive areas of the RMS in a caudo-rostral direction: caused a significant increase in the number of Fluoro in the vertical arm, elbow and horizontal arm separately Jade C positive cells in whole RMS compared with (Figure 3). The result of quantitative analysis of BrdU- control animals (Figure 5). positive cells confirmed this morphological observation and showed a significant decrease in the number of 3.3 Cell differentiation proliferating cells in all three parts of the RMS compared In addition to alterations in cell proliferation and cell with the control animals of the same age. Interestingly, death, maternally deprived animals displayed distinct the decrease in the number of BrdU-positive cells was changes in the appearance of NO producing cells.

Figure 2. Photomicrographs of sagittal brain sections treated by immunohistochemic staining for BrdU-positive cells in the RMS of P7 control (A) and maternally deprived rats (B). The BrdU-positive cells are visible by brown stained nuclei (arrows). Marked reduction of proliferating cells in the RMS of maternally deprived rats (B) can be seen. Scale bars=100 μm.

Figure 3. Quantitative analysis of BrdU-positive cells in the RMS of control and maternally deprivated rats. Graph shows the number of BrdU-positive cells in the vertical arm (VA), elbow (E) and horizontal arm (HA). Maternal deprivation decreased the number of proliferating cells in all parts of the RMS. Statistical significance of differences between experimental and control group: *P<0.05.

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Figure 4. Representative photomicrographs of Fluoro-Jade C positive cells (arrows) in the RMS vertical arm of P7 control (A) and maternally deprived rats (B). Note the increased number of Fluoro Jade C positive cells after one week of maternal deprivation. Scale bars=50 μm. sa staining. N - nucleus. Bar = 50 μm (A) and 25 μm (B).

Figure 5. Quantitative analysis of Fluoro-Jade C positive cells in the Figure 6. Representative photomicrograph of NADPH-d staining RMS of control and maternally deprived rats shows highly in the RMS vertical arm of maternally deprived rat. significant increase of labeled cells following maternal The NADPH-d-positive cells have bipolar spindle-shaped deprivation. Statistical significance of differences body with short fibers (arrows). The RMS is demarcated between experimental and control groups: **P<0.01. by dotted lines. Scale bar=100 μm.

Differentiated nitrergic cells in the RMS are evident from neonatal animals has received less attention. Both the tenth postnatal day in control rats [19]. However, adult and neonatal neurogenesis can be influenced in maternally deprived pups nitrergic cells appear as by a wide range of exogenous factors such as early as the seventh postnatal day. These NADPH-d- ionizing radiation, electromagnetic field [27,28], positive cells were bipolar, had short fibers and other odor deprivation [29], or enriched environment [30]. morphological characteristics that are typical for Recent studies have investigated factors that regulate NADPH-d-positive cells of ten days old rats (Figure 6). neurogenesis with special emphasis on effects of GFAP immunohistochemistry has revealed an stress. Given that extensive neurogenesis occurs absence of GFAP-positive cells in the RMS of maternally during the early postnatal period, it is not surprising deprived pups. A small concentration of GFAP-positive that adverse early life events affect structural brain cells was located at the ventral border of the elbow. development. Changes in neurogenesis after Similar findings were obtained in P7 control animals, postnatal application of stress have been reported which is in agreement with previous findings that show in the hippocampus [31,32]. Although mitotically that GFAP expression appears in restricted areas of the active cells are present in the RMS in adult rodents RMS after the first postnatal week [26]. [33], little is known about how stress affects ongoing neurogenesis specifically in this region. The present study investigates whether stress induced by maternal 4. Discussion deprivation during the first postnatal week has an effect on neurogenesis in the rat RMS. Numerous studies examining postnatal neurogenesis It has been shown that maternal deprivation focus on adult neurogenesis, while neurogenesis in during the first two-three weeks of life can induce

761 Early stress affects neurogenesis in the rat rostral migratory stream

long-term changes in endocrine, behavioral and brain deprivation did not influence the appearance of development [34]. It has been previously demonstrated astrocytes in the migratory pathway suggests that the that maternal deprivation affects hippocampal structural mechanisms regulating cell migration during early life plasticity in a sex-dependent manner [35]. In addition, are less vulnerable than mechanisms involved in the ischemia stress in the brain has been shown to activate process of migration in adults. the proliferation of neural stem cells in the neonatal In addition to neuronal progenitors and neuroblasts, SVZ and promote neural stem cells differentiation into the RMS also contains differentiated neurons [13]. oligodendrocytes [36]. This includes a subpopulation of NO producing cells. In the present study, maternal deprivation resulted In intact rats, the first NADPH-d-exhibiting cells in a significant decrease in the number of proliferating appear in the RMS at postnatal day 10 [19] and persist cells as compared with control rats. We previously throughout the entire life of the animal [18]. Here, we demonstrated the physiological dynamics of mitotic demonstrated that nitrergic cells respond to stress by activity in the RMS during the first postnatal month. The accelerating their maturation, thus appearing in the lowest number of proliferating cells was observed in P7 RMS as early as the seventh postnatal day. It has been animals [9]. In maternally deprived rats of the same age, previously demonstrated that NO acts as an important the low density of dividing cells was even more striking. negative regulator of cell proliferation in the adult Changes in the number of BrdU-positive cells may result mammalian brain [41,42]. A similar anti-proliferative from altered cell proliferation and differences in survival. effect of NO has been reported in neuronal precursors The number of dying cells is several times higher in isolated from the embryonic [43] and neonatal [44] neurogenic areas as compared with any other area of brain. The observed early appearance of nitrergic the brain. This is a typical observation in the RMS under cells in the RMS indicates an association between physiological conditions [37]. We show that short-term NO production and decreased proliferation induced maternal deprivation caused significant increases in the by maternal deprivation. number of dying cells within the RMS as compared with In conclusion, our findings suggest that even control rats. A comparison of the extent of decrease in short periods of early stressful experience can proliferation with the extent of cell dying, using the same markedly influence cell proliferation, cell death and method of quantification, would be of interest. the appearance of differentiated nitrergic neurons in During development, radial glial cells contribute the neonatal rat RMS. Further studies are needed to to neuronal migration and neurogenesis. During the identify the mechanisms by which environmental factors first 10 days after birth, these cells begin to transform influence the processes of neurogenesis. into astrocytes. They increase their branching in the SVZ and change the orientation of their processes from radial to tangential along the RMS [38]. Several Acknowledgements investigations describe altered migration and increased GFAP immunostaining after various form of injury This study was supported by the following projects: such as status epilepticus [39] or transection lesions VEGA 2/0147/09; 2/0058/08. of the migratory stream [40]. Our finding that maternal

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