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, celldeath,appearanceofastrocytesorcelldifferentiation)intheneonatalratrostralmigratorystream(RMS).Toinducestress, pups were subjected to maternal deprivation daily for three hours, starting from the first postnatal day till the seventh postnatal day.Immunohistochemicalmethodswereusedtovisualizeproliferatingcellsandastrocytes;dying cellsandnitrergiccellswere visualizedusinghistochemicalstaining.Quantitativeanalysisshowedthatmaternaldeprivationdecreasedthenumberofproliferating 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 postnatalday. TheobservedearlierappearanceofnitrergiccellsintheRMSwasassociatedwithalteredproliferationandincreased cell dying and this observation supports the hypothesis that nitric oxide has an anti-proliferative role in the RMS. Our study demonstratesthatmaternaldeprivationrepresentsastressfulconditionwithaprofoundimpactonearlypostnatalneurogenesis. Keywords: Rat • Rostral migratory stream • Maternal deprivation • Neurogenesis ©VersitaSp.zo.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 758 K. Lievajová et al. 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